Characterization of Leptin Signaling in the Developing

CHARACTERIZATION OF LEPTIN SIGNALING IN THE DEVELOPING

ZEBRAFISH (Danio rerio) USING MOLECULAR, PHYSIOLOGICAL, AND

BIOINFORMATIC APPROACHES

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

Of the Requirements for the Degree

Doctor of Philosophy

Mark Dalman

December, 2014 CHARACTERIZATION OF LEPTIN SIGNALING IN THE DEVELOPING

ZEBRAFISH (Danio rerio) USING MOLECULAR, PHYSIOLOGICAL, AND

BIOINFORMATIC APPROACHES

Mark Dalman

Dissertation

Approved: Accepted:

______Advisor Committee Member Dr. Richard L. Londraville Dr. Qin Liu

______Committee Member Committee Member Dr. Zhong-Hui Duan Dr. Brian Bagatto

______Committee Member Dean of the College Dr. Ahmed Mustafa Dr. Chand Midha

______Department Chair Interim Dean of the Graduate School Dr. Monte Turner Dr. Rex Ramsier

______Date

ABSTRACT

In this dissertation, I tested the hypothesis that leptin A in zebrafish (D. rerio) plays a similar role to mammalian leptin in regulating metabolic rate and immune function, whereas leptins’s effects on the zebrafish transcriptome may be distinct. Leptin is now identified in all major vertebrate lineages, but its role in controlling food intake, development, metabolic rate, and fat storage is best studied in mammals. In that group, leptin has pleiotropic effects including those on angiogenesis, bone formation, reproductive status, immune function, and energy expenditure. A homozygous mutation

(ob-/ ob-) for leptin is the most common model for leptin study in mammals. The use of leptin-null mutants in non-mammal models is not common. We recently developed a leptin knockdown model in zebrafish and applied a comparative approach to studying some well-characterized mammalian leptin functions in this new system.

I tested the impact of leptin expression on metabolism in the developing zebrafish embryo. Leptin knockdown reduced oxygen consumption most prominently during early development (24-48 hours post fertilization, hpf) whereas carbonic acid production was most significantly attenuated later in development (48-72 hpf). Cardiac output was significantly reduced in embryos with reduced leptin expression (leptin morphants); all of these effects could be rescued by co-injection of recombinant fish leptin.

The second part of my research focused on the innate immune response. When presented with a bacterial challenge, leptin morphants had reduced macrophage respiratory burst activity and bacterial load clearance was unaffected 12 hours post

iii infection (hpi). By 36 hpi, leptin morphants had significantly increased bacterial burden and reduced survival compared to control embryos.

I then focused on the transcriptomic effects of reduced leptin A expression in the developing zebrafish embryo. Microarray analysis identified sensory and development pathways as the most significantly enriched in embryos with leptin expression (at a variance with mammalian adult microarray studies). Citrate synthase, 3-hydroxy acyl-

CoA dehydrogenase, and carnitine palimitoyltransferase enzyme assays confirmed the general pattern of reduced aerobic respiration transcripts in leptin morphants.

Furthermore, confirmation of microarray by enzyme assays found leptin morphants to have reduced enzymes in fatty acid oxidation and general aerobic respiration. The microarray study was complemented by an analysis of techniques used to filter microarray data. I found that choice of the selection criteria used during analysis can significantly impact data interpretation. I proposed that simultaneous use of two types of cutoffs (significance and fold change) was a ‘best practice’ in microarray analysis.

These studies are among the first to quantify effects of leptin knockdown in the developing zebrafish embryo. Leptin function in nonmammals is conserved with mammalian leptin function in the dimensions of metabolic rate and immune function; its effects on the transcriptome (sensory and developmental pathways) differ from similar studies in mammals. This may reflect an adult bias in mammalian leptin studies.

DEDICATION

For my parents: Richard and Melody Dalman. You stood unwavering by my side through this and I couldn’t have asked for more. To my beautiful, smart daughters:

Isabella and Mackensie. I know you’ve only just started first grade, but I want you to know that I am humbled by the daughters you have been and excited to see the daughters you will become. I am so very proud of you two and I am positive you will change the world.

v ACKNOWLEDGEMENTS

I would like to whole-heartedly thank my members of my PhD committee, Dr.

Richard Londraville, Dr. Qin Liu, Dr. Zhong-Hui Duan, Dr. Ahmed Mustafa, and Dr.

Brian Bagatto. Thank you for your words of encouragement, your expertise, and your time. I am truly indebted to you for providing the research opportunities and scholarly experiences I cherish to this day.

I would also like to thank many undergraduate and graduate students who have helped me throughout my PhD. Graduate students: Donald Copeland, Hope Ball, Justin

Brantner, and Anthony Deeter. Undergraduate students: Mason King, Michael Graves,

Jessica Bucher, and Richard Ngo. Thank you for your support and advice. To my best friends John and Dee Warnock, I couldn’t have asked for better friends. To Dr. Amy

Hollingsworth- thank you for listening and keeping me sane.

vi TABLE OF CONTENTS Page

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

CHAPTER

I.GENERAL INTRODUCTION ...... 1

Mammalian Leptin Signaling ...... 2

Leptin’s Mode of Activation and Signaling Pathways ...... 5

The Adipostat Model of Leptin Signaling ...... 7

Development of Leptin Resistance ...... 9

Leptin Signaling and Secondary Effects ...... 10

Development ...... 11

Reproduction ...... 12

Immune Function ...... 12

Leptin in Fishes ...... 14

Identification of Fish Leptin and Questionable Role as an Adipostat ...... 15

Fish Leptin’s Role in Metabolism, Immune Function, and the Transcriptome .... 18

Overview of projects ...... 21

vii II. LEPTIN EXPRESSION AFFECTS METABOLIC RATE IN ZEBRAFISH EMBRYOS (D. RERIO) ...... 23

Introduction ...... 23

Materials and Methods ...... 26

Results ...... 29

Discussion ...... 34

Acknowledgements ...... 38

III.LEPTIN-A KNOCKDOWN SIGNIFCANTLY ALTERS INNATE IMMUNE RESPONSE AND IMMUNOCOMPETENCE OF DEVELOPING ZEBRAFISH EMBRYO ...... 39

Introduction ...... 39

Methods...... 43

Results ...... 46

Discussion ...... 51

IV. MICROARRAY ANALYSIS OF LEPTIN-A KNOCKOUT IN EARLY ZEBRAFISH DEVELOPMENT ...... 55

Introduction ...... 55

Methods...... 58

Results ...... 63

Discussion ...... 92

viii V. FOLD CHANGE AND P-VALUE CUTOFFS SIGNIFICANTLY ALTER MICROARRAY INTERPRETATIONS ...... 103

Introduction ...... 103

Methods...... 104

Results and discussion ...... 105

Conclusions ...... 112

Acknowledgements ...... 112

VI. CONCLUSIONS AND FUTURE DIRECTIONS ...... 113

The Power of the Comparative Approach ...... 113

Limitations and future directions of the study ...... 118

REFERENCES ...... 121

APPENDIX ...... 158

ix LIST OF TABLES Table Page

1. Sources of variation in metabolic rate ...... 29

2. Sources of variation in innate immune function ...... 47 3. Significantly regulated genes per gene selection criterion for zebrafish leptin-A knockdown at 72 hours post fertilization (hpf)...... 80

4. Top 80 significantly up regulated genes (morphant relative to control) involved in leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf)...... 81

5. Top 80 significantly down regulated genes (morphant relative to control) involved in leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf)...... 84

6. Collective enriched Gene Ontology (GO) groups for leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf) ... 87

7. Collective enriched KEGG Pathway Analysis for leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf) ...... 90

8. Genes categorized by both fold change and p-value in response to chronic constant hypoxia. The data presented is before any post normalization filtering ...... 108

LIST OF FIGURES Figure Page

1. Schematic of leptin binding and downstream signaling activation ...... 4

2. Generalized adipostat model in vertebrates ...... 8

3. Oxygen consumption and cumulative acid production assays through developmental time ...... 30

4. Stroke volume, embryonic heart rate, and cardiac output of developing zebrafish embryos ...... 33

5. Respiratory burst activity for zebrafish challenged by bacteria inoculation at 72 hpf ...... 48

6. Survival through developmental time for zebrafish challenged by bacterial injection ...... 49

7. Bacterial load clearance through zebrafish development ...... 50

8. Effects of leptin knockdown at 72 hpf on zebrafish lipid size ...... 69

9. Microarray analysis of leptin knockdown is reproducible and selectively impacts a large number of transcripts ...... 70

10. The number of genes (X) vs. Gene Ontology (GO) category (Y) for the effect of leptin A knockdown in 72 hpf zebrafish ...... 71

11. Directed Acyclic Graph (DAG) depicting significantly enriched down regulated Gene Ontology (GO) category, Biological Process ...... 72

12. Directed Acyclic Graph (DAG) depicting significantly enriched up regulated Gene Ontology (GO) category, Biological Process ...... 73

13. Directed Acyclic Graph (DAG) depicting significantly enriched down regulated Gene Ontology (GO) category, Cellular Component ...... 74

14. Directed Acyclic Graph (DAG) depicting significantly enriched up regulated Gene Ontology (GO) category, Cellular Component ...... 75

15. Directed Acyclic Graph (DAG) depicting significantly enriched down regulated Gene Ontology (GO) category, Molecular Function ...... 76

16. Directed Acyclic Graph (DAG) depicting significantly enriched up regulated Gene Ontology (GO) category, Molecular Function ...... 77

17. GENEMAPP KEGG Pathway of Adipocytokine Signaling pathway for leptin-A knockdown in zebrafish embryos at 72 hours post fertilization (hpf)...... 78

18. Validation of microarray data by selected functional enzyme and gene expression ...... 79

19. Differentially regulated genes for GO Annotation categories...... 106

20. Comparative fishers exact test and fold enrichment for significant GO groups within the zebrafish total gene array ...... 110

xii

CHAPTER I

GENERAL INTRODUCTION

Vertebrates maintain energy homeostasis by a network of neuroregulatory, endocrine, and metabolic pathways. Disruptions in the complex signaling cascade of energy balance leads to an inability to effectively coordinate energy stores with metabolic demands. Although many hormones are involved in signaling metabolic requirements, the discovery of leptin in 1994 was a significant step forward, for it provided connections among behavior (appetite), physiology (metabolism), and neurobiology (brain- adipose connection; Zhang et al., 1994). Leptin was named for the Greek word leptós, meaning thin; it is a 167 amino-acid peptide encoded by the ob gene that is primarily produced by adipocytes in mammals (Zhang et al., 1994). A mutation in the ob gene results in increased food intake and reduced lipolysis in mice. Furthermore, its tertiary structure is highly conserved across vertebrates even though its primary sequence is not (Zhang et al.,

1997; Copeland et al., 2011; Prokop et al., 2012; Londraville et al., 2014). In the two decades since its discovery in mice, over 90% (>25,000 articles) of leptin studies have focused on mammalian leptin signaling and its role as an anorexigen (appetite inhibiting molecule). The first evidence of a nonmammalian leptin came over a decade after mammals, and consequently little is known of leptin’s function across vertebrates

(Johnson et al., 2000; Kurokawa et al., 2005). In its original description in fish, fed sunfish (Lepomis cyanellus) had higher blood leptin than food-restricted animals, despite no difference in body fat between groups (Johnson et al., 2000). Leptin was later cloned

1 in fish, where its expression was primarily in liver and gonads (Kurokawa et al., 2005) vs. adipose in mammals. Leptin function in fish is still poorly characterized. Therefore, I tested the hypothesis that mammalian leptin function is conserved in fishes.

Mammalian Leptin Signaling

Leptin is encoded by the ob gene and synthesized primarily by both brown and white adipose tissue in mammals (Zhang et al., 1994; MacDougald et al., 1995). It is also weakly expressed in brain (Morash et al., 1999), stomach (Bado et al., 1998), skeletal muscle (Wang et al., 1998), placenta (Hoggard et al., 1997), bone (Laharrague et al.,

1998), heart (Hoggard et al., 2000), liver (Soukas et al., 2000), and pituitary gland

(Morash et al., 1999). It is secreted into the blood where it circulates and binds to leptin receptors centrally in the hypothalamus and peripherally in many other tissues (Ahima and Flier, 2000b). The ob gene product is highly conserved among mammals as mouse and human homologues share >80% primary sequence identity (McGarry, 1995). Leptin is a member of the type I helical cytokine family and its four anti-parallel α-helix structure is highly similar to other long-chain helical cytokines such as interleukin-4 (IL-

4), IL-6, IL-11, granulocyte-macrophage colony stimulating factor (GM-CSF), prolactin, and growth hormone (GH; Madej et al., 1995; Huising et al., 2006). Furthermore, leptin’s three binding sites to its receptor are orthologous to IL-6 and G-CSF binding sites

(Peelman et al., 2004).

Six leptin-receptor isoforms have been identified in mammals (OB-R a-f; Lee et al., 1996; Bates and Myers, 2003), and all bind a single leptin isoform. Leptin receptors are expressed throughout the central nervous system (CNS; De Matteis and Cinti, 1998;

Elmquist et al., 1998; Fei et al., 1997) and periphery (Tartaglia et al., 1995; Cao et al.,

1997; Goiot et al., 2001; Martin-Romero et al., 2000). All OB-R isoforms are membrane bound, except for OB-Re, which is soluble in blood (Ahima and Osei, 2004). OB-Re binds circulating leptin, thus reducing ‘free’ leptin capable of activating OB-Rb

(Lammert et al., 2001; Elefteriou et al., 2004; Robertson et al., 2008). The long-form leptin receptor (with a full-length intracellular domain; OB-Rb) is the best-studied receptor isoform, and it is thought to mediate all of leptin’s known physiological effects

(Bjorbaek et al., 1997; Tartaglia, 1997; Fong et al., 1998).

The OB-R gene has been identified in a wide range of metazoan taxa (Prokop et al., 2012). It contains 17 exons (in most species), which are alternately spliced to produce all isoform variants (Robertson et al., 2008). All six receptor isoforms share similar extracellular binding domains but differ in their length at the 3’-carboxyl end (Chua et al.,

1996; Leggio et al., 2012; Mancour et al., 2012). The long form receptor (OB-Rb) contains four extracellular domains: cytokine receptor homology module (CHR1), immunoglobulin (Ig)-like fold, CRH2, and fibronectin type III (FNIII); all participate in binding to and activation by leptin. CRH2 is the main receptor-binding site that makes contact with binding site II on leptin (Niv-Spector et al., 2005). Furthermore, the Ig-like fold is permissive for leptin activation as variants without it showed unimpaired binding of leptin site III but lack receptor activation (Zabeau et al., 2004). Additionally, FNIII binding domains (containing WSXWS motifs) are critical for activation of OB-R (Dagil et al., 2012). OB-Rb binding stoichiometry with leptin is thought to be through dimerization (2 leptin: 2 receptor; Mancour et al., 2012), though others debate this

(Peelman et al., 2004; Robertson et al., 2008; Leggio et al., 2012).

Figure 1. Schematic of leptin binding and downstream signaling activation. Note. Leptin binding causes homodimerization of leptin receptors and the formation of the leptin receptor/ JAK2 complex. The conformational change causes cross- phosphorylation of tyrosine residues 985, 1077, and 1138 by JAK2. Tyrosine phosphorylation causes activation of STAT3, which causes SOCS3 expression to negatively inhibit leptin signaling. SHP-2 binds to phosphyrolated tyrosine 985 and activates MAPK pathway. Additionally, JAK2 phosphorylation activates PI-3K pathway which then leads transcriptional regulation of target genes. Leptin receptor long isoform: Ob-Rb; POMC: pro-opiomelanocortin; AgRP: Agouti related protein; NPY: neuropeptide Y; JAK2: Janus kinase 2; STAT: signal transducers and activators of transcription; SOCS: suppressor of cytokine signaling; AP-1: activator protein 1; SHP2: SH2-containing protein tyrosine phosphatase 2; PI3K: phosphatidylinositol 3- kinase; MAPK: Mitogen-activated protein kinase; AKT: protein kinase B; P: phosphate; Y: tyrosine. Once leptin is bound, cascade of activation occurs with positive feedback (shown by green line) causing increased SOCS3 expression. Subsequently, through negative feedback (shown in red line), SOCS3 inhibits activation of JAK2 and STATs 1, 3, and 5.

Leptin receptor expression at the cell surface is regulated by interaction with another protein as it is processed through the Golgi apparatus. The OB-R gene related product (OB-RGRP) is encoded via an alternate start site on the mammalian OB-R gene, resulting in a protein with no sequence similarity to OB-R (Bailleul et al., 1997; Mercer et al., 2000; Seron et al., 2011). OB-RGRP contains four exons and has highest expression in the heart, placenta, and pancreas of humans (Bailleul et al., 1997). OB-

RGRP is membrane bound and associated with the Golgi and endosomes, it is highly conserved across vertebrates, and is inversely related to receptor activation and cell surface recruitment of leptin receptor. Silencing of OB-RGRP results in increased recruitment of leptin receptors and prevents development of diet-induced obesity (DIO;

Couturier et al., 2007). OB-RGRP is related to vesicle trafficking proteins in yeast and thus may be a key regulator in leptin sensitivity (Belgareh-Touze et al., 2002).

Leptin’s Mode of Activation and Signaling Pathways

Leptin’s central mode of activation is through the arcuate nucleus and ventromedial hypothalamus (Ghilardi et al., 1996). Here, two distinct (and opposing) classes of neurons are present; one expresses anorexigenic proopiomelanocortin (POMC) and cocaine-amphetamine related transcript (CART), whereas the other expresses orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AgRP; Coll et al.,

2007; 2008). These two pathways are widely implicated in regulating food intake, energy expenditure, and metabolism (Broberger et al., 1998). Intracerebroventricular (ICV) injections of leptin centrally into the third ventricle result in a dose-related reduction in body weight in mice (Halaas et al., 1997). Given that delivery of leptin behind the blood/ brain barrier (BBB) can produce systemic effects (in body weight and other systems),

5 many researchers hold the opinion that all, or most, of leptin’s effects are mediated from the CNS. Conversely, the idea that leptin acts directly on the peripheral systems without involvement of the CNS is gaining support, largely because several leptin receptor isoforms (including OB-Rb) are expressed throughout the body, including in heart, pancreatic beta islet cells, and immune cells (Margetic et al., 2002; Bjorbaek et al., 2004).

Administration of recombinant leptin results in lower serum free fatty acids (FFA), increased phagocytosis by macrophages, and increased STAT-3 and adenosine monophosphate-activated protein kinase (AMPK) expression, results in reduced apoptosis and increased glucose uptake (Fruhbeck et al., 1998; Shimabukuro et al., 1997;

Siegrist-Kaiser et al., 1997; Papathanassoglou et al., 2006; Mcgaffin et al., 2011).

How and where leptin is administreted alters the phenotype observed. ICV infusion into the third ventricle reduces food intake and body weight by 15% whereas peripheral infusion by subcutaneous pump at the same dose has no effect (on either) in mice (Halaas et al., 1997). Peripheral injections require larger doses to elicit the same effets in both mice and rats (Halaas et al., 1997; Niimi et al., 1999). Collectively, there are data to support actions for leptin from the CNS and directly in the periphery.

Leptin signals via the long form of OB-Rb, a Janus kinase (JAK) receptor that interacts with a signal transducer and activator of transcription (STAT) pathway (Figure

1). Upon ligand binding, the leptin receptor undergoes homodimerization and once phosphorylated, receptor-associated Janus kinases (JAK; Jak2) are activated (White et al.,

1997; St-Pierre and Tremblay, 2012). The phospho-tyrosine residues of Jak2 form binding targets for STATs 3 and 5 (Vaisse et al., 1996; Bjorbaek et al., 1997). Once bound, STATs become phosphorylated, dimerize and then translocate to the nucleus

6 where they modulate transcription of target genes, such as peroxisome proliferator- activated receptor gamma coactivator 1-alpha (PGC-1alpha) and silencer of cytokine signaling 3 (SOCS3; White et al., 1997; Carpenter et al., 1998; Sainz et al., 2009). The

JAK2/ STAT3 intracellular pathway is critical for all of leptin’s effects on energy balance and body weight (Bjorbaek et al., 1999). Leptin’s actions are opposed by induction of

SOCS3 (Bjorbaek et al., 1998). Phosphorylated STAT3 acts on two opposing neuronal circuits in the hypothalamus, both activating expression of anorexigenic pro- opiomelanocortin (POMC)/ cocaine and amphetamine related transcript (CART) via alpha-melanocyte stimulating hormone (alpha-MSH) and inhibiting orexigenic neuropeptide Y (NPY)/ agouti-related peptide (AgRP; Wauman and Tavernier, 2010; St-

Pierre and Tremblay, 2012).

The Adipostat Model of Leptin Signaling

The diversity of leptin signaling possibilities allows for a myriad of physiological effects. Administration of recombinant leptin to wildtype mice leads to reduced food intake and increased metabolic rate (Halaas et al., 1995; Friedman and Halaas, 1998).

Circulating leptin titers are positively correlated with adipose mass (Maffei et al., 1995;

Havel et al., 1996) and leptin titer decreases with fasting and its response precedes fat mass reduction (Keim et al., 1998). Leptin titers do not immediately respond to fasting or feeding, implying leptin’s functions are biased towards long-term maintenance rather than short-term response (Weigle, 1994; Mistry et al., 1997a; Doring et al., 1998;

Berthoud, 2004). Collectively, these observations led researchers to the idea that leptin acts as a sensor of adipose stores. This “adipostat” model of leptin signaling (Figure 2) is supported by the observations that administered leptin increases sympathetic nervous

7 tone (metabolic rate), and activates brown adipose tissue (BAT) thermogenesis (lipolysis) in mice along with activation of reward centers involved in perception of satiety (reduced food intake; Collins et al., 1996; Haynes et al., 1997; Scarpace et al., 1997; Robertson et al., 2008). Simply put, lower adipose stores result in low leptin titers and increases in appetite, higher adipose stores result in the opposite (although not immediately).

Together, these reports suggest that leptin acts as both a neuroendocrine gauge of total lipid available and as a signaling molecule to increase energy expenditure and feeding behavior.

Figure 2. Generalized adipostat model in vertebrates

The adipostat model of leptin action, although simple, is somewhat of a straw man. Early studies found a positive correlation between adiposity and leptin titer (Maffei et al., 1995; Ahima and Flier, 2000a; Al Maskari and Alnaqdy, 2006) however correlation between leptin titer and fat stores is highly variable among individuals

(Considine and Caro 1997; Cnop et al., 2002). As predicted by the adipostat model, lean humans have lower circulating titers compared to obese patients (Masuzaki et al., 1997;

Havel, et al., 1996; Hube, et al., 1996; Montague, et al., 1997; and Dussere, et al., 2000), which is similar to most mammals (Concannon et al., 2001; Florant et al., 2004). Leptin

8 administered to leptin deficient (ob/ob) mice reduces obesity, however leptin infusion does not significantly reduce fat mass in obese humans (Heymsfield et al., 1999; Myers et al., 2008; Van Heek et al., 1997). Additionally, variation in leptin titer among humans at a given body mass index (BMI) can vary as much as 10X (Cnop et al., 2002).

Interestingly, most hibernating mammals (e.g. little brown bats, Myotis lucifugus) show dissociation between circulating leptin and body mass that varies with season (Kronfeld-

Schor et al., 2000; Concannon et al., 2001).

Development of Leptin Resistance

Leptin resistance is the decreased response to increased circulating leptin. Mice that do not express leptin are obese, however obese humans are hyperleptinemic

(Hamilton et al., 1995; Caro et al., 1996). Obese humans have a high concentration of circulating leptin, but are resistant to its anorexigenic effects. Additionally, peripherally administered leptin in rodents fed a high fat diet elicited reduced food intake after four days, however they became resistant to leptin by day 16 (Van Heek et al., 1997). Leptin resistance may be caused by impaired transport of leptin across the BBB and/or by dysfunctional leptin receptor function and signaling (Myers et al., 2008; Scarpace and

Zhang, 2009). Evidence for impaired transport across the BBB is based on the fact that circulating leptin concentrations in the cerebrospinal fluid of obese humans is similar to non-obese humans (Schwartz et al., 1996). Conversely, at the receptor, negative feedback regulators such as suppressor of cytokine signaling 3 (SOCS3) are over-activated in energy surplus states (Figure 1; Dardeno et al., 2010). Increased SOCS3 activation by leptin binding causes decreased activation of leptin receptors by a negative feedback loop.

The db/db mouse model plays a prominent role in leptin biology. This model was co-identified along with the ob/ob model and does not express the long-form leptin receptor (Tartaglia, 1997). The mutant leptin receptor model expresses hyperphagia, decreased lean body mass, insulin resistance, hypothermia, and infertility with an obese phenotype observable by 5 weeks (post natal) and is completely unresponsive to peripheral or centrally administered leptin (Bray and York, 1997; Tartaglia, 1997). Thus the db/db mouse model shares similar traits to leptin resistance observed in mammals

(though at one extreme) as increased leptin has no effect on food intake or lipolysis in this model. Of note, leptin resistance in the db/db model may be restricted to metabolic effects, as effects on hypertension remain (Correia et al., 2002). In conclusion, leptin resistance has been observed across several mammalian model systems, including humans, however the exact mechanism, trigger, and spatiotemperal distribution of leptin resistance is still poorly resolved.

Leptin Signaling and Secondary Effects

Friedman’s group demonstrated leptin’s potent anorexigenic effects early after its identification (Zhang et al., 1994; Friedman and Halaas, 1998). Furthermore, body mass mirrored circulating leptin and long-term fasting results in reduced body fat (Bjorbaek et al., 1999). Perhaps unexpectedly, secondary effects were also observed in response to changes in leptin, such as decreased locomotor activity, sympathetic tone, thermogenesis, and gastric clearance of food (Van Heek et al., 1997). Additionally, neonatal developmental changes, puberty and reproductive status, and health status are significantly affected by adipose mass and thus circulating leptin (Moran and Phillip,

2003). Clearly, we now understand leptin’s effects as pleiotropic.

Development

Most of our knowledge of leptin’s biology comes from studies on adults, however leptin is also critical in development. Leptin concentrations in the human fetus increase throughout embryonic development (Jaquet et al., 1998). Some mammals show a postnatal leptin surge around postnatal day (PND) 5- 9 and human leptin titers slowly decrease after birth into adulthood (Marinoni et al., 2010). Although the leptin receptor is expressed and functional during neonatal development, leptin is unable to modulate feeding or energy expenditure until the second postnatal week in mice (PND10; Stehling et al., 1996, 1997; Matsuda et al., 1999; Mistry et al., 1999; Proulx et al., 2002). In rodents, the anorexigenic and orexigenic hypothalamic circuits remain functionally and structurally plastic until PND 21, and leptin deficency compromises neuronal development (Bouret et al., 2004; Bouret and Simerly, 2007). Leptin deficient (ob-/ob-) mice show marked decreases in brain mass, myelination, and cell density (Bereiter and

Jeanrenaud, 1979; Sena et al., 1985; Ahima et al., 1999b), which can be rescued in early development but not in adulthood, implying a critical developmental period (Steppan and

Swick, 1999). Thus leptin may be playing a neurotrophic role rather than an adipostat role during very early development. During fetal development, maternal diet, whether energetically restrictive or permissive, also affects postnatal circulating leptin (Ong et al.,

1999). For example, rats born from hypoleptinemic mothers (via maternal under nutrition) develop obesity in adulthood (Ferezou-Viala et al., 2007). Leptin deficiency in humans results in normal linear growth and adrenal function, unlike in mice, which have excessive circulating glucocorticoids (Farooqi et al., 1999; Chan et al., 2008). This may explain why leptin-deficent mice have reduced growth and insulin insensitivity as

11 glucocorticoids have significant effects on growth and insulin secretion. Collectively, these studies demonstrate that leptin is not only critical for brain development and hypothalamic circuits, but also that the leptin environment during development is critical for mammals to reach adulthood.

Reproduction

Leptin’s role in promoting reproductive maturity has direct fitness consequences

(Hausman et al., 2012). Ob-/ob- mice are infertile, but can be rescued by leptin injections

(Schneider et al., 2000). High leptin titers, from increases in fat mass and total weight, result in early puberty in mammals (Chehab et al., 1996; 1997). In humans, both male and female leptin titers increase until the onset of puberty, with males declining and females continuing to rise thereafter (Ahmed et al., 1999; Blum et al., 1997). Women with abnormally low adipose stores and hypoleptinemia are amenorrhic, and leptin injections rescue estrus and fertility (Chehab, 2000; Pallares et al., 2010; Chou et al.,

2011). Leptin may optimize pregnancy outcome through maintenance of maternal fuel homeostasis (Holness et al., 1999; Hausmen et al., 2012). Together, these data point to leptin signaling the nutritional state is optimal for reproductive function (Tataranni et al.,

1997).

Immune Function

Leptin deficient mice are hypothermic, hyperphagic, and immunocompromised

(Mandel and Mahmoud, 1978; Friedman and Halaas, 1998; Lord et al., 1998). Humans with leptin deficiency have increased infection-related deaths, and leptin injections restore lymphocyte function (Farooqi et al., 2002). Epidemiological studies indicate a

12 correlation between malnutrition and infection susceptibility, implying energy stores are critical for proper immune response (Blackburn, 2001). Malnourished mammals have a weakened immune response to bacterial infections Mycobacterium tuberculosis and

Streptococcus pneumonia via reduced clearance and defective phagocytosis (Chan et al.,

1996; Mancuso et al., 2002a; Hsu et al., 2007). Mice lacking leptin show dysfunction in both the innate and adaptive arms of the immune response, and leptin injections can rescue both innate and adaptive immune function (Fraser et al., 1999; Zhang et al., 2002;

Procaccini et al., 2012).

Leptin receptors are expressed on macrophages, dendritic cells, natural killer

(NK) cells, B cells, T cells, and regulatory T cells (Procaccini et al., 2012). With leptin administration, macrophages have increased phagocytic activity and increased proinflammatory cytokine secretion (such as TNF-alpha, IL-6, IL-12; Mancuso et al.,

2002; 2004; Zarkesh-Esfahani et al., 2001). In culture, leptin acts as a potent chemoattractant as it up-regulates migratory performance of dendritic cells and induces the release of oxygen radicals (superoxide radical and hydrogen peroxide) from neutrophils (Caldefie-Chezet et al., 2001; 2003; Gruen et al., 2007). Neutrophils’ actions are thought to be indirect and possibly mediated by TNF-alpha secreted by monocytes

(Zarkesh-Esfahani et al., 2004). Leptin injections increase recruitment of neutrophils in wildtype mice unlike leptin-deficient mice (ob-/ob-; Rummel et al., 2010). Mice lacking leptin show impaired T-cell stimulation, perhaps mediated through secretion of immunosuppressive cytokines, such as TGF-beta (Macia et al., 2006). Leptin also activates human B cells to produce IL-6, IL-10, and TNF-alpha by JAK-STAT and

MAPK signaling pathways; conversely leptin deficiency results in 70% fewer B cells

(Agrawal et al., 2011; Claycombe et al., 2008). Leptin promotes the proliferation of memory and naïve T cells, and survival of thymic T cells through decreased apoptosis

(Howard et al., 1999). Ob-/ob- mice show no signs of autoimmune damage from experimentally induced hepatitis, colitis, encephalomyelitis, or glomerulonephritis, suggesting leptin plays a proinflammatory role in immune response (Matarese et al.,

2001, Faggioni et al., 2000, Siegmund et al., 2002). Hypoleptinemia increases T- regulatory cells, thus increasing susceptibility to bacterial infectious diseases such as pneumonia and tuberculosis (Procaccini et al., 2010). Hyperleptinemia results in a decoupling of T- regulatory cells from induction of proinflammatory cytokines, resulting in increased autoimmune susceptibility such as rheumatoid arthritis (RA) and type-1 diabetes (De Rosa et al., 2007; Procaccini et al., 2010). Together, these results indicate that leptin acts as a pro-inflammatory cytokine in immune function and is critical for the proper balance between protection from infection and protection from autoimmunity.

Leptin in Fishes

Leptin biology has been extensively studied in mammals, undoubtedly due to the human obesity epidemic (Keen-Rhinehart et al., 2013; Schneeberger et al., 2014). To date, it is established that leptin in mammals modulates appetite, adipose stores, metabolic rate, reproductive maturity, and immunology. Are these leptin functions unique to mammals? To address this question, I used the zebrafish model. Zebrafish belong to the earliest evolved class of vertebrates. Zebrafish also have a large percentage

(~70%) of its genes that are orthologous to human genes (Howe et al., 2013;

Langheinrich, 2003). Furthermore, almost 50% of human proteins have a zebrafish orthologue (Howe et al., 2013). Zebrafish also share physiological and anatomical

14 characteristics to mammals during early development through adulthood (Kimmel et al.,

1995; Kalinka et al., 2010). For example, the zebrafish immune system shares mammalian characteristics, such as the presence of macrophages, neutrophils, and natural killer (NK) cells that are present by 36 hours post fertilization (HPF). However, unique to fish, the adaptive immune response is not functional until 4-6 weeks post fertilization

(Trede et al., 2004; Lam and Lu, 2007; Meeker and Trede, 2008).

The power of the zebrafish model lies in a fully mapped genome, the high diversity of molecular tools available, visually recordable embryogenesis, ease of husbandry, low cost, high fecundity, and rapid development (Westerfield, 2000; Spence et al., 2008). Currently, there are approximately 32,000 extant species of fish, and the zebrafish is in the most abundant class of vertebrates (Actinopterygii) as well as a member of the largest fish family (Cyprinidae; Eschmeyer et al., 2003). Teleost fish are the most diverse of all vertebrate groups, with genomes that reflect a whole genome duplication (WGD) event (Meyer and Schartl, 1999; Taylor et al., 2001; Taylor and Raes,

2004). Some duplicated genes resulting from the WGD were lost while others acquired new function as taxa evolved (Wagner et al., 1998; McClintock et al., 2001). Zebrafish are not representative of all fish species, however they share many physiological, developmental, genetic, and behavioral characteristics of fish species (Streisinger et al.,

1981; Grunwald and Eisen, 2002).

Identification of Fish Leptin and Questionable Role as an Adipostat

The first evidence of leptin in fishes used an anti-mouse leptin antibody (Johnson et al. 2000). Five years later (2005), Kurokawa and colleagues identified a bonafide leptin gene in fish, using gene synteny between mouse and pufferfish genomes. They found the

15 major expressing tissue was liver with undetectable expression in adipose (unlike in mammals; Kurokowa et al., 2005). This study was the breakthrough that allowed fish leptins and their receptors to be described in many fish species, including goldfish (de

Pedro et al., 2006), green sunfish (Londraville and Duvall, 2002), rainbow trout

(Murashita et al., 2008), zebrafish (Gorissen et al., 2009), common carp (Huising et al.,

2006), catfish (Kobayashi et al., 2011), pufferfish (Kurokawa et al., 2005), Atlantic salmon (Kling et al., 2009), fine flounder (Fuentes et al., 2013), arctic charr (Froiland et al., 2010), medaka (Chisada et al., 2014), striped bass (Won et al., 2012), and orange- spotted grouper (Zhang et al., 2013a). Leptin in fishes is mainly expressed in liver and gonads with minimal expression in brain and viscera (Gorissen et al., 2009; Liu et al.,

2010; 2012a; Ronnestad et al., 2010; Won et al., 2012). All mammals, amphibians, and birds express one isoform of leptin whereas fish may express one or two paralogs

(Gorissen et al., 2009; Copeland et al., 2011; Denver et al., 2011; Prokop et al., 2012,

Londraville et al., 2014). To date, there are insufficient data to resolve which of the fish isoforms is/are ancestral to mammalian leptins (Copeland et al., 2011; Won et al., 2012;

Londraville et al., 2014). Most functional studies of fish leptin are restricted to leptin A

(Murashita et al., 2011; Dalman et al., 2013; Liu et al., 2012) with leptin-B studies simply reporting tissue expression (Huising et al., 2006; Gorissen et al., 2009). Leptin-A is expressed mainly in the liver and leptin-B is more ubiquitously expressed among tissues

(Gorissen et al., 2009; Huising et al., 2006; Liu et al., 2012, Londraville et al., 2014). In silico simulations predict that leptin A binds with 10X more binding energy to its receptor than does leptin B (Prokop et al., 2012). These data, together with the fact that leptin B is expressed at 10X lower copy number than leptin A, suggest that leptin A is the

16 predominant functional isoform in fish (Londraville et al. 2014). Due to differences in number of isoforms, a lack of conservation of primary sequence and a lack of expression in adipose tissue in teleosts, there is support for the hypothesis that leptin signaling is functionally different between teleosts and mammals.

The mammalian paradigm of leptin acting as an adipostat is not supported across teleosts. Unlike mammals, leptin does not positively correlate with fat mass across several species of fishes (Londraville and Duvall, 2002; Huising et al., 2006; Cao et al.,

2011; Ronnestad et al., 2010; Liu et al., 2010). Earlier studies in fish found leptin-A expression increased with food intake (Tinoco et al., 2012) however mRNA expression was unresponsive to long-term fasting as in mammals (Huising et al., 2006). Zebrafish leptin-A transcript in liver did not respond to a one week fast whereas leptin-B was significantly down regulated (Gorissen et al., 2009). Interestingly, starved Atlantic

Salmon, Rainbow trout, and flounder show higher leptin levels than fully fed controls

(Murashita et al., 2008; Kling et al., 2009; Trombley et al., 2012; Fuentes et al., 2013) which is counter to the mammalian paradigm.

Leptin does appear to decrease appetite in fishes, as it does in mammals.

Intraperitoneal (IP) and intracerebroventricular (ICV) injections of leptin in rainbow trout and goldfish decrease food intake (de Pedro et al., 2006; Volkoff et al., 2003; Murashita et al., 2008). Though more studies are needed to validate responses to both leptin isoforms and in response to food intake, current data suggest that leptin does not consistently reflect energy reserves in fishes. Collectively, these studies do not support a role for leptin as an indicator of fat stores, but do support its appetite reducing effects.

Fish Leptin’s Role in Metabolism, Immune Function, and the Transcriptome

Initially, the most pressing question in comparative leptin biology was its effects on food intake. However, use of mammalian leptins in fish studies has made interpretation of appetite data challenging (Denver et al., 2011) though others suggest these early studies still have value (Londraville et al., 2014; Prokop et al., 2014).

Cumulatively, there are several fish studies on leptin that report decreases in growth, food intake, fatty acid binding protein (FABP), glucose sensing and body mass with leptin administration (Londraville and Duvall, 2002; Aguilar et al., 2010; de Pedro et al., 2006;

Murashita et al., 2011). For example, in mammals injections of leptin increase metabolic rate, thus reducing adipose stores and food intake (Makimura et al., 2001). Its metabolic affects in mammals are thought to be mediated by changes in heart rate, mean arterial pressure, and sympathetic tone (Winnicki et al., 2001; Carlyle et al., 2002; Chu et al.,

2010). Of note, leptin’s effects in early mammalian development may be purely sympathoexcitatory (Correia et al., 2002). In nonmammals, metabolic rate has been poorly characterized. Fasting surprisingly increases leptin expression in common carp, rainbow trout, and flounder (Huising et al., 2006; Kling et al., 2009; Fuentes et al., 2013).

In adult zebrafish, fasting has no effect on leptin-A expression, however leptin-B decreases (Gorissen et al., 2009). In conclusion, leptin does seem to affect metabolism in fishes, however all studies to date have either manipulated food or increased leptin via injection. I aimed to understand the impacts on metabolic rate in the developing zebrafish using a leptin knockdown model.

Fish and mammals share immune cell-type but differ in the demarcation between acquired and innate immune systems (Trede et al., 2004; Lam et al., 2004; Meeker and

Trede, 2008). The immune system in fish has been widely tested throughout early development, finding similar phagocytic responses to prebiotic oligosaccharides by increasing bacterial clearance as in mammals (Zhao et al., 2011). Phagocytic macrophages in zebrafish are present within the first 48 hours of development and are functional before 72 hours post fertilization (hpf). Leptin receptors (OB-Rb) have been identified on immune cells in mammals (Carbone et al., 2012) but are uninvestigated in fish. However, recombinant rainbow trout leptin activates leucocytes’ STAT3 and MAPK cascades, and decreases superoxide anion production in adult immune cells (Mariano et al., 2013). Additionally, fluorescently labeled bacteria and mutant zebrafish strains with fluorescently labeled macrophages have been used to visualize interaction and clearance of bacterial infections (Torraca et al., 2014). Leptin’s impact on immune function in fish is poorly studied with the exception of a few studies (Aoki et al., 2008; Sieger et al.,

2009; Kobayashi et al., 2011).

Modeling leptin signaling in fish has progressed slowly since its discovery in

2005 by Kurokawa’s group. Collectively, this is due to a lack of leptin clones, leptin mutants, resolved genomes, and multiple isoforms to investigate in fishes. Non-mammal leptin clones are relatively recent tools (Prokop et al., 2014; Londraville et al., 2014).

Furthermore, despite identification of several fish leptin genes, the production of leptin and/or leptin receptor knockdown/knockout models in fishes has eluded researchers until recently (Liu et al., 2012; Chisada et al., 2014). For mammals, the availability of leptin and leptin receptor null mutants has enhanced our understanding of the complex signaling of leptin. Interestingly, large-scale transcriptomic studies using microarrays have augmented and enhanced phenotypic observations of dysfunctional lipolysis by pointing

19 to pathways involved in mitochondrial, lipid metabolic and catabolic pathways, carboxylic acid, iron ion binding, and glutathione S-transferases (Sharma et al., 2010).

Transcriptomic data also corroborated secondary effects on immune function, finding shifts in pathways involved in inflammatory response and lysosomal activity (Sharma et al., 2010). Many of these pathways were previously identifed, albeit through low- throughput studies (Gracey et al., 2011; Mariano et al., 2013). High-throughput microarray technology has not been applied to non-mammalian leptin biology as microarray technology lagged behind the discovery of leptin in zebrafish (Affymetrix, personal communication). A recently developed (2012) zebrafish oligonucleotide microarray replaced the outdated 2003 array technology by incorporating more probes per gene along with including theoretically all expressed genes (among others, leptin and its receptor). These recent technical advancements along with our development of the leptin knockdown model, sets the stage to test leptin knockdown in the developing zebrafish using a transcriptomic approach.

To test whether leptin signaling is conserved from mammals to fish, we developed and characterized a leptin knockdown model in the zebrafish (which is analogous to the ob/ob mouse model). As described above, earlier fish studies either modified food intake or injected (native or heterologous) leptin, and our group was the first to reduce leptin signaling molecularly (Liu et al., 2012). There are two common strategies to reduce expression of genes in zebrafish. The first is using a morpholino oligonucleotide (MO) that is antisense to the mRNA and blocks the translation or the splicing of the mRNA (Nasevicus and Ekker, 2000). Since not all copies of the transcript will necessarily bind to the morpholino, it’s considered a knockdown and not a knockout

20 model. The second is using a vivo-morpholino (vivo-MO), which uses an octaguanidine dendrimer, which ionizes to guanidinum at physiological pH to help delivery into the embryo or adult tissue (Ferguson et al., 2014). Both modes of knockdown are transient, with MO’s effective for approximately the first four days of development and vivo-MOs less than that (Nasevicus and Ekker, 2000). The former has been empirically tested on leptin and significantly down regulates leptin-A protein expression (Liu et al., 2012;

Chapter 2). Recently in Medaka, Chisada’s group created a true leptin receptor knockout using TALEN technology (Chisada et al., 2014). This method resulted in no leptin receptor expression, increased growth and increased adipose deposition (Chisada et al.,

2014). There are no known leptin or leptin receptor knockout models available in zebrafish.

Overview of projects

I tested functional aspects of leptin biology using a recently developed zebrafish leptin- knockdown model. The primary focus of the research was to characterize the metabolic consequences of leptin knockdown in early developing zebrafish (chapter 2) and expand our understanding of secondary (immune- chapter 3) and global impacts

(transcriptome- chapter 4) in a representative nonmammal. Taken together, I identified both conservation of leptin function with mammals, and apparently unique leptin function in zebrafish.

Chapter II analyzes the metabolic consequences of leptin signaling knockdown in the developing zebrafish embryo from 24- 96 hpf. This developmental window was chosen due to the effective period of the morpholino and because of embryonic transparency for in situ observation of cardiac output (stroke volume and heart rate). I

21 examined both the metabolic consequences (oxygen consumption and carbon dioxide production) and the cardiovascular (cardiac output) impacts of leptin knockdown. Data produced from this study was previously published and was the first to report the effects of leptin on metabolic rate (Dalman et al., 2013; See Chapter 2 for details).

Chapter III analyzes one aspect of leptin biology that is considered secondary to its major function. The immune response is critical for the survival of an organism and responds to circulating leptin titers in mammals. We used a zebrafish to address host effects of hypoleptinemia on immunocompetence when challenged by a systemic bacterial infection. The zebrafish model system is distinct from mammals in that there is a clear developmental shift from innate to both innate and adaptive arms of immune function (both are present throughout development in mammals). This difference allows us to test leptin’s effect specifically on the innate arm of immune function.

Chapter IV analyzes leptin’s impact globally on the organism through analysis of transcriptomics at 72 hpf. Leptin’s developmental effects are poorly studied in all organisms, and in fishes are completely unknown. I utilized a recently developed oligonucleotide microarray that covers the entire genome and analyzed differences in transcription in the leptin morphant.

Chapter V was a general analysis of microarray studies similar to Chapter IV. I examined the consequences of data filtration on normalization and data interpretation in microarray analyses. I used a previously published data set on zebrafish response to hypoxia and reexamined data output through a multitude of gene selection criteria.

CHAPTER II

LEPTIN EXPRESSION AFFECTS METABOLIC RATE IN ZEBRAFISH EMBRYOS

(D. RERIO)

Dalman, M.R., Liu, Q., King, M.D., Bagatto, B., and Londraville, R.L. (2013). Leptin

expression affects metabolic rate in zebrafish embryos (D. rerio). Frontiers in

Physiology 4, 160.

Introduction

Leptin is a 16 kD cytokine hormone that was discovered nearly two decades ago; mice with a homozygous (ob−/ob−) mutation have signiﬁcant hyperphagia and increased adipose storage (Zhang et al., 1994). Leptin has been cloned across several vertebrate species, including mammals, amphibians, reptiles, and ﬁsh (Kurokawa et al., 2005;

Crespi and Denver, 2006; Boorse and Libbon, 2010). In mammals, leptin expression is highest in adipose tissue whereas among ﬁshes, liver is the major expressing tissue

(Gorissen et al., 2009; Liu et al., 2010). Mammalian leptin binds to JAK-STAT receptors in the hypothalamus, which activate anorexigenic pathways; leptin signaling is also present in ﬁshes (Londraville and Duvall, 2002; Murashita et al., 2008). Non-mammal leptins do not share conserved primary structure with mammalian leptins (10–30%), yet tertiary structure and some aspects of leptin function appear to be well conserved across vertebrates (Johnson et al., 2000; Londraville and Niewiarowski, 2010). In mammals,

23 leptin injections result in a dose-dependent reduction in food intake and increased lipolysis, metabolic rate, and thermogenesis (Makimura et al., 2001). In goldfish, intracerebroventricular (ICV) injection of murine leptin reduces food intake (Volkoff et al., 2003; de Pedro et al., 2006). Intraperitoneal (IP) injections of fish leptin reduce appetite (short term) in rainbow trout (Murashita et al., 2008), though IP delivery does not affect appetite in Coho salmon, catfish, or green sunfish (Silverstein and Plisetskaya,

2000; Londraville and Duvall, 2002). These contradictory results are likely confounded by leptin source (native or heterologous), method of delivery (IP or ICV), dosage, and sampling interval. Therefore, it is difﬁcult to assess which aspects of leptin function are conserved from ﬁsh to mammals.

The availability of mutants without leptin signaling has been invaluable to advancing leptin biology. Ob−/ob− mice are leptin null mutants and signal energy stores to the brain as if nutrient starved, and elicit similar dysfunction to fasted animals such as decreased thermogenesis, hypometabolism, infertility, and hyperphagia. All of these abnormalities can be rescued via leptin injections, however injections in fed wildtype mice do not signiﬁcantly increase metabolic rate or decrease food intake (Mistry et al.,

1997a; Doring et al., 1998). Moreover, diet induced obesity (DIO) and ob−/ob− mice show similar but not identical gains in body weight (de Pedro et al., 2006). These studies suggest that manipulating leptin in an animal that does not express the hormone is fundamentally different than manipulating leptin in one that does. To date, leptin investigations in non-mammals have been hampered by the lack of leptin-null mutants.

We now know that leptin’s effects go beyond appetite, with inﬂuence on immune function, reproduction, bone, and metabolism (see Chapter 1 for review). To date, the

24 effects of acute leptin injection are unresolved with variable responses depending on development age and time of the injection (Fruhbeck, 1999; Mistry et al., 1999). The current paradigm is that leptin increases oxygen consumption in mammals and reptiles while also increasing heart rate, mean arterial pressure, and sympathetic tone (Winnicki et al., 2001; Carlyle et al., 2002; Chu et al., 2010). Zebraﬁsh (D. rerio) embryos provide a robust model to visualize cardiac output noninvasively due to their transparency.

Additionally, the availability of molecular tools (Liu et al., 2010, 2012a) and visualization techniques (Barrionuevo and Burggren, 2006) makes the zebrafish a useful model to test leptin function in early vertebrates

We have developed and characterized both zebrafish leptin and leptin receptor- knockdown models (Liu et al., 2010, 2012a). We used antisense morpholino oligonucleotides against zebrafish leptin-A, resulting in embryos with dramatically reduced leptin expression and effects on the development of heart, eye, inner ear, and notochord (Liu et al., 2012). We established that the leptin A-knockdown is specific through a series of controls, including (1) leptin mRNA knockdown reduces zebrafish leptin expression (2) the morphant phenotype is produced with either several morpholinos against leptin or the leptin receptor, and control morpholinos produce no apparent phenotype (3) the morphant phenotype is rescued with recombinant leptin (Liu et al., 2012). Here we present effects of that knockdown on metabolic rate and cardiac function. Leptin’s effects on metabolic rate were measured directly by oxygen microprobe and indirectly via a pH sensitive dye for acid production. Cardiac output was analyzed using a high-speed camera and image software. In general, leptin A knockdown

25 reduced metabolic rate and was rescued by recombinant leptin, although the effect was dependent on developmental age and assay method.

Materials and Methods

Animals

Wild-type adult zebraﬁsh (Aquatic Tropicals, Bonita Springs, FL), Danio rerio, were maintained and bred at 28.5ºC with a light cycle of 14L:10D, according to The

Zebrafish Book (Westerfield, 1994). Immediately after fertilization, zebrafish embryos were transferred to fish tank water with fungicide (0.05% methylene blue) and allowed to develop. Ages of the embryos or larvae are given as hours post-fertilization (hpf). All animal-related procedures were approved by the University of Akron Institutional Animal

Care and Use Committee (IACUC).

Morpholino design and rescue

Morpholino antisense oligonucleotides were designed and manufactured by Gene

Tools (Philomath, OR) and reconstituted in Daneau buffer [58 mM NaCl, 0.7 mM KCl,

0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6]. A leptin-A translation blocking morpholino (lepMO: 5’-TTG AGC GGA GAG CTG GAA AAC GCA T -3’), a zebraﬁsh leptin- receptor translation blocking morpholino (lepRMO: 5’- TCA AGA CAG

ACA TCA TTT CAC TTG C -3’), and a control MO with ﬁve-mismatched nucleotides to

LepMO (5-misMO: 5’ - TTG AcC GcA GAc CTG cAA AAg GCA T -3’) were used in this study (Liu et al., 2012). These morpholinos have been previously validated for both leptin and leptin receptor (Liu et al., 2010, 2012a). Embryos at the 1–8 cell stage were injected with 2 nl of morpholino at a concentration of 0.4 mM using a Narishige MI300

26 microinjector. Subsamples of embryos at each age were used for each experiment at each respective age. Zebraﬁsh leptin A protein (recombinant, GenScript) was co-injected with the leptin morpholino to rescue the morphant phenotype. A leptin protein stock solution

(30 µ M in 50 mM Tris, pH 8.0, > 90% pure) was co-injected with the leptin-A morpholino and the embryos allowed to develop as above (Liu et al., 2012).

Oxygen consumption

Dechorionated zebraﬁsh embryos at desired ages (10/ trial) were placed into 5 ml vials and allowed to acclimate for 10 min at 28.5ºC. Each measurement was repeated with previously untested ﬁsh 2– 9X, with 10 embryos/ measurement. Three initial measurements were conducted using an oxygen microelectrode connected to Power Lab

(ADInstruments, Colorado Springs, CO) with LabChart software (ADInstruments,

Colorado Springs, CO). After initial readings, vessels were sealed with paraﬁlm M

(American Screening, Shreveport, LA) and incubated for an hour and then three post oxygen consumption measurements were taken. Mean consumption rates were displayed as nmol of O2 per individual per hour. Parafilm is slightly permeable to gas exchange but the small surface area of the vial potentially exposed to atmospheric gases and the small duration of the measurement (1 h) had no significant influence on oxygen consumption measurements.

Colorimetric whole-animal acid production assay

This assay was adapted from Makky et al. (2008). One dechorionated embryo was placed in a series of three washes of phenol red (0.02% w/v) assay medium (consisting of

RO water supplemented with Instant Ocean to a conductivity of ∼ 350 µ S at 28.5 ºC)

27 adjusted to pH 8.0 with concentrated sodium bicarbonate solution. Once rinsed and acclimated for 10 minutes, 100 µl of assay medium (with embryo) was transferred to one well of a sterile polystyrene 96-well microplate (Evergreen Scientiﬁc, Los Angeles, CA).

Coverslip mineral oil (100 µl) was overlaid to diminish diffusion of environmental oxygen into each well. Absorbance was measured at 570 nm over 1 h at 28.5 ºC and 10 second intervals using a spectrophotometer (Spectramax 384 Molecular Devices,

Sunnyvale, CA). Cumulative acid production was then derived from absorbance using a previously described equation (Makky et al., 2008).

Heart rate, stroke volume, and cardiac output

Video of the beating heart was captured for each individual embryo using a high- speed digital camera (Red Lake MASD, San Diego, CA) on an inverted microscope with a temperature-controlled stage. Early larva rested on the chamber bottom and typically did not move, however after swim bladder inﬂation, larval movement required anesthesia with 0.002% tricaine (MS-222). This low concentration of MS-222 does not affect heart rate compared to free swimming larvae (Moore et al., 2006). Heart rate was measured by recording time elapsed over 15 heartbeats and extrapolating to beats per minute (3x/ larvae). Stroke volume was estimated from end systole and diastole area recorded in a single frame for each during the cardiac cycle (Bagatto and Burggren, 2006).

Statistics

Oxygen consumption, microplate assay and cardiovascular data were analyzed using a two-way analysis of variance (ANOVA) at a p = 0.05 using standard linear models analysis (SAS Institute, Cary, NC) with developmental age and treatment

(wildtype, leptinMO, and rescue) as factors. Data were distributed normally. A Tukey’s multiple comparison procedure was performed to assess for speciﬁc pair-wise comparisons post-hoc.

Results

Oxygen consumption

Oxygen consumption rate of leptin morphants was signiﬁcantly decreased compared to both control and rescue embryos at all time points (p < 0.01) except for 60 hpf (Figure 3A, Table 1A).

Table 1. Sources of variation in metabolic rate Source dF MS F Pr > F (A) TWO- WAY ANOVA FOR OXYGEN USE Age 4 25.1 113.8 <0.001 Treatment 2 6.6 29.8 <0.001 Age x 8 1.2 5.3 <0.001 Treatment Error 63 0.2

(B) TWO- WAY ANOVA FOR ACID PRODUCTION Age 2 208.7 149.5 <0.001 Treatment 4 102.1 73.0 <0.001 Age x 8 30.15 21.6 <0.001 Treatment Error 240 1.4

An ANOVA was run using Age, Treatment, and Age by Treatment interaction as the modeled sources of variation. For oxygen consumption, each datum represented the average of 10 embryos of a standardized mass for that developmental age as previously described by Barrionuevo and Burggren (1999) and Bang et al. (2004). For acid production, each data point is an individual embryo.

Figure 3. Oxygen consumption and cumulative acid production assays through developmental time. (A). Oxygen consumption per individual for wildtype (control), morphants (LepMO), and rescue (morpholino + recombinant zebrafish leptin) embryos. All time points are significantly different (p < 0.05) between morphants and control and morphants and rescue, with the exception of 60 hpf (N = measurements / age, 10 individuals/measurement). (B) Cumulative acid production measured per embryo/hour by change in absorbance @570 nm. (N = individual fish). All data are mean ± SE. Control,wildtype embryo; LepMO, leptin morpholino injected zebrafish embryo; Rescue, Co-injected LepMO morpholino with recombinant zebrafish leptin; 5-mismo, control morpholino with mismatch basepairing at 5 sites; LepRMO, leptin receptor morpholino. All fish are morphologically aged matched. No significant differences among treatments at 24 hpf; Control and 5-mismo are significantly higher than other treatments at 48–50 hpf, and LepMO and LepRMO are significantly lower than other treatments at 72–76 hpf. p < 0.05.

Colorimetric aggregate acid production assay

All assay plate experiments were run in parallel such that a control well (assay medium only), a wildtype embryo, and a leptin-MO injected embryo (age matched) were placed in adjacent wells and each embryo only assayed once. The rate of total acid production was interpreted as the linear regression of changes in optical density at 570 nm over time. Blank wells showed no significant drift throughout the 1-h measuring window. Data were adjusted for morphological age based on developmental cues but not for size or mass as previously described (Liu et al., 2010, 2012a). Although morphant embryos are slightly smaller than wildtype (7.5% difference in total length), we compared morphants to wildtype and rescues using the same standardized mass (Bang et al., 2004), because any decrease in length is compensated by an increase in yolk (Liu et al., 2012). Control (wildtype) and morphant embryos absorbance values were blanked at time zero. In aggregate, there were significant effects of treatment, age, and an interaction between treatment and age (Figure 3B, Table 1B, p < 0.001), although these effects were driven by differences at 48 and 72 hpf, not 24 hpf (Figure 3B). The morpholino’s efficacy is reduced beginning ∼ 4 dpf (Liu et al., 2010, 2012a), therefore morphants were not assayed at 96 hpf. Leptin morphants rescued with recombinant leptin had metabolic rates equivalent to wildtype at 24 and 72 hpf, but not 48 hpf (Figure 3B).

Cardiac output

There is inherently high variability among individuals in embryonic development at 24 hpf (Westerﬁeld, 1994; Bang et al., 2004), therefore ventricle area was measured at

48 and 72 hpf only. Heart rate was lower in morphants with a pronounced pause between

31 each fluid heartbeat. Heart rate increased between 48 and 72 hpf, except for morphants

(Figure 4). Stroke volume and cardiac output increase dramatically between 48 and 72 hpf for wildtype embryos, but not morphants. Coinjection of recombinant leptin A returns all cardiac variables to that of wildtype (Figure 4).

Figure 4. Stroke volume, embryonic heart rate, and cardiac output of developing zebrafish embryos. (A) Average stroke volume for wildtype (control), leptin morphants (morphant), and rescue (leptin morphant + recombinant zebrafish leptin) at two developmental timepoints. (B) Embryonic heart rate for wildtype (control), leptin morphants (morphant), and rescue (morphant + recombinant zebrafish leptin) at two developmental timepoints. (C) Average cardiac output for wildtype (control), leptin morphants (morphant), and rescue (leptin morphant + recombinant zebrafish leptin) embryos. All data are mean ± SE. (N = 5 for A, B, N = 7 for C). Bars that do not share letters are significantly different (p < 0.05).

Discussion

Leptin’s effect on metabolic rate was one of the first functions described for the hormone, but its effects on fish metabolic rate were undescribed. Here we tested the effects of leptin A knockdown on metabolic rate for the first time in a non-mammal. In general, the results indicate that leptin knockdown reduces metabolic rate and cardiac output, and that these effects can be rescued by recombinant leptin A. These effects differ depending on developmental age and assay.

The end metabolite of both aerobic and anaerobic metabolism is acid. CO2 is produced during aerobic metabolism and when hydrated produces carbonic acid, whereas lactic acid is the end metabolite during anaerobic metabolism. These products of cumulative metabolism are commonly used as indicators of metabolic rate (Hu et al.,

2000; O’Connor et al., 2000; Stackley et al., 2011), however the validity of oxygen consumption methodologies are debated for aquatic organisms, especially those observed under a microscope (Makky et al., 2008). Unstirred boundary layers, diffusion, tissue density, probe precision, and clutch effects all can influence oxygen consumption measurements (Feder and Pinder, 1998; Bang et al., 2004; Moore et al., 2006; Makky et al., 2008). However, previous studies have found that absolute metabolic rate does increase over developmental time, mass-specific metabolic rate decreases, and both neuroendocrine and autonomic nervous systems can influence metabolic rate (Zhang and

Wang, 2006; Fraisl et al., 2009).

We calculated oxygen consumption at 3.5– 8.0 nmol O2/ h (Figure 3A) during development, which corroborates previously documented ﬁsh embryo data Bang et al.,

2004 (4.54– 8.29 nmol O2/ h). Lower published values (2 nmol O2) may be explained by

34 differences in methodology (Barrionuevo and Burggren, 1999). Our data are reported per embryo rather than per gram as approximately 60% of an embryo’s surface area is yolk sac that is facilitating only 33% of the overall oxygen uptake through diffusion (Wells and Pinder, 1996). Therefore, metabolically inactive yolk may unduly influence metabolic rate calculations on a per gram basis (Breslow et al., 1999). Finally, as further verification of this assay, we document a slowing of metabolic rate > 48 hpf. This is consistent with Kimmel et al. (1995), who demonstrated that the rate of change for the head-trunk angle dramatically slows during this time period, as does cardiac output in control zebrafish (Bagatto et al., 2006).

We used an aggregate acid production colorimetric assay to expand our data set due to its higher precision (individual measurements vs. groups of 10) and greater throughput (96 embryos at one time). Oxygen consumption and acid production have been widely used and validated for multiple dyes and microplate assays (Rowell, 1995;

Campbell et al., 2003; Nieman et al., 2003; O’Mahony et al., 2005; Makky et al., 2008;

Stackley et al., 2011). Total aggregate acid for developing zebraﬁsh embryos increases over developmental time (Makky et al., 2008; Stackley et al., 2011). Our data fall within

2.10– 6.95 nanomoles of H+ produced per hour per embryo, and are consistent with

Makky et al.’s original study (Makky et al., 2008). Leptin morphants had signiﬁcantly reduced rates of aggregate acid production (Figure 3B, p < 0.01). To minimize effects of decreasing pH as the assay proceeds, all data were collected over 1 h. Further, pH change

(aggregate acid production) ﬁt to a linear regression with R2 > 0.95 (Figure A1) suggesting assay conditions did not change signiﬁcantly during the assay, or if they did, they did not affect metabolic rate.

We used two measures of metabolism to test the effects of leptin knockdown; both are indirect measures of organismal metabolic activity. Although we can verify that our data are consistent with previously published control zebrafish data for each technique, and we can demonstrate an effect of knockdown and rescue with each assay, the response of each developmental age is not the same between assays. For both assays, there is a significant interaction between developmental age and treatment. For oxygen consumption, the effect of leptin knockdown decreases with time (Figure 3A), and for acid production, the effect increases with time (Figure 3B). Clearly, even though both of these assays are routinely used to measure metabolic rate (Nieman et al., 2003; Makky et al., 2008; Stackley et al., 2011), leptin expression affects each in distinct ways. Leptin knockdown decreasing its effect on oxygen consumption over time is consistent with the activity profile of morpholinos (losing their efficacy over the course of 4 days). We speculate that leptin knockdown canalizes (Waddington, 1942) some event early in development that accounts for more and more of acid production as development proceeds (perhaps proton leak; Stackley et al., 2011). Going forward, each assay has advantages. Oxygen consumption certainly has the weight of decades of research supporting its use, but the sensitivity and “ease of use” of oxygen electrodes make it impractical for individual embryos. The colorimetric assay has the advantage of precision, throughput, and ease of use. We recognize that the colorimetric assay does not distinguish between anaerobic or aerobic metabolism, however the relative contribution of lactic acid to hydrated CO2 for developing zebrafish embryos is minimal, suggesting most of the aggregate acid measured is from the hydration of CO2 (Stackley et al., 2011).

We assert that the colorimetric assay is precise and robust in determining relative, if not absolute differences in metabolic rate among treatments.

Cardiac output is tightly coupled with metabolism and the cardiovascular development of the zebrafish heart has been widely documented along with the ontogeny of its control (Hu et al., 2000; Bagatto, 2001). Our data demonstrate that leptin knockdown in developing embryos significantly decreases heart rate and stroke volume, resulting in significant decreases in cardiac output (Figure 4). Ob−/ob− mice have reduced arterial pressure and heart rate compared to wildtype (Mark et al., 1999). The mechanism by which leptin exerts its action on cardiac output is proposed to be by increased sympathetic tone, causing changes in heart rate, vascular tone, and the contractile properties of ventricular myocytes in mammals (Ozata et al., 1999; Nickola et al., 2000). Until this study, leptin’s effect on cardiac output has not been measured in fish. Morphant zebrafish rescued with recombinant leptin returns heart rate to that of wildtype embryos, suggesting that leptin may exert its effects on metabolic rate partially through modulating heart rate (Figure 4). Thyroid hormone (TH), in particular, has been shown to modulate cardiac physiology in zebrafish through increased ATP availability and increased metabolic gene expression (Little et al., 2013). In particular, thyroid hormone mediates the proportion of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) available thereby regulating the vascular and contractile properties of the heart tissue

(Carr and Kranias, 2002). An increase in metabolic rate by leptin may be facilitated by increased ATP availability (Portner and Knust, 2007; Little et al., 2013), and/ or through modulation of calcium cycling by SERCA (Aho and Vornanen, 1999; Carr and Kranias,

2002).

We sought to determine if leptin A influences metabolic rate in zebrafish, similar to how leptin affects metabolic rate in mammals. Although the assays we used are not in agreement as to the magnitude or timing of the effect, we assert that the data supporting a role of zebrafish leptin A in modulating metabolic rate are robust. In addition, we demonstrate that zebrafish cardiac performance is affected by leptin expression. By comparing how fish and mammal leptins are similar and divergent in structure and function, we hope to gain insight into the evolution of this hormone.

Acknowledgements

Funded by NIH 1R15DK079282-01A1 to Richard L. Londraville, Qin Liu, and

Brian Bagatto and 2 R15 DK079282-02 to Richard L. Londraville and Qin Liu.

CHAPTER III

LEPTIN-A KNOCKDOWN SIGNIFICANTLY ALTERS INNATE IMMUNE

RESPONSE AND IMMUNOCOMPETENCE OF DEVELOPING ZEBRAFISH

EMBRYO

Introduction

Leptin is a 16 kD non-glycosolated cytokine hormone expressed primarily by and in proportion to adipose tissue in mammals (Zhang et al., 1994). Leptin is encoded by the ob gene and its tertiary structure is highly conserved across many vertebrate species including mammals (Zhang et al., 1994), amphibians (Crespi and Denver, 2006), birds

(Prokop et al., 2014), and fish (Kurokowa et al., 2005). It is known to regulate energy expenditure and food intake (Zhang et al., 1994; Ahima and Flier, 2000a; Morton et al.,

2006), angiogenesis (Sierra-Honigmann et al., 1998; Bouloumie et al., 1998), bone density and linear growth (Ducy et al., 2000), and fertility (Chehab et al., 1996) in mammals. Serum leptin circulates throughout the body and binds to receptors present both centrally in the central nervous system (CNS; De Matteis and Cinti, 1998; Elmquist et al., 1998) and in peripheral tissues and circulating cells (Tartaglia et al., 1995; Cao et al., 1997; Goiot et al., 2001; Procaccini et al., 2012). All of leptin’s known effects are attributed to its long form receptor (OB-Rb), which is one of six leptin receptor isoforms

(OBR a-f). Ob-Rb is the only one capable of inducing downstream JAK-STAT activation

(Mancour et al., 2012). Leptin signaling also modulates immune function (Lord et al.,

1998; Howard et al., 1999). Leptin receptors are expressed on diverse immune cells including macrophages, natural killer (NK) cells, B cells, T cells, and regulatory T cells

(Procaccini et al., 2012). Leptin signaling in immune cells reaffirms links between nutrition and immune function that were documented prior to leptin’s discovery

(Sauberlich, 1984). It is now established that leptin plays a clear role in immune function in mammals, however its role in non-mammalian immunity is largely untested.

Much of what we know in mammals stems from the use of knockout mice for leptin (ob-/ob-). Leptin knockout results in hyperphagia, decreased thermogenesis, and many hormonal and immune abnormalities (Ahima et al., 1996; Howard et al., 1999;

Faggioni et al., 2000). Ob-/ ob- and db-/ db- (leptin receptor deficient) mice share similar defects in cell and humoral immunity and these effects are observed throughout development (Mandel and Mahmoud, 1978; Ozata et al., 1999). Interestingly, leptin belongs to the type I cytokine family, which includes many pro- and anti-inflammatory cytokines such as prolactin, growth hormone, erythropoietin, and interleukins (2, 3, 4, 6, and 12; Kau et al., 2011). Obesity is a chronic “low grade” inflammatory state and leptin acts both as an anti-inflammatory (acutely) and proinflammatory (chronically) signal

(Hotamisligil et al., 1993; Mancuso et al., 2012). Pro-inflammation is marked by increased T-lymphocyte stimulation, increased IL-1β, TNF-α, IL-6, reactive oxygen species production, phagocytosis, and decreased bacterial burden (Loffreda et al., 1998;

Fantuzzi and Faggioni, 2000; Martin-Romero et al., 2000; Faggioni et al., 2001; Hermann et al., 2004; Mito et al., 2004; Ferreira-Dias et al., 2005). Leptin also has stimulatory effects on adiponectin, an anti-inflammatory that is decreased in obesity (Li et al., 2006).

This reduction is most likely associated with a state of leptin resistance in obesity pathologies.

Leptin acts on and responds to immune function in mammals. Hypoleptinemia

(due to starvation, malnutrition, or low adipose concentration) reduces cell-mediated

(Th1) immunity, and increases wound healing time and mortality. High leptin titers result in a dysregulation between cell and humoral (Th2) mediated immunity, leading to the progression of autoimmune diseases, such as type 1 diabetes in mammals (Howard et al., 1999). In addition, several studies have reported that infection, sepsis, and LPS increase leptin levels in vivo, implying that leptin participates in acute inflammatory response and also is regulative in the host response to infection (Loffreda et al., 1998;

Faggioni et al., 1999; Faggioni et al., 2000; Grunfield et al., 1996; Bornstein et al., 1998).

Leptin receptors have been identified on phagocytic immune cells like macrophages and neutrophils in mammals and inflammation was linked to the overexpression of leptin

(Bruno et al., 2005). Both diet-induced obesity (DIO; high leptin) and ob-/ ob- mice (low leptin) have increased mortality from influenza as a result of increased wound healing time and altered antiviral immune response (Smith et al., 2007; O’Brien et al., 2007;

Karlsson and Beck, 2010). A unique challenge in mammals is identifying the relative effectiveness of acquired vs. innate immune response. Zebrafish are ideally suited for these questions.

The adaptive immune response arose uniquely in vertebrates ~450 million years ago, whereas innate immune function can be traced back almost a billion years

(Magnadottir, 2006). Adaptive immune function allowed for increased memory for pathogen recognition and, in some cases, innate immune function utility decreased over

41 evolutionary time (Fearon and Locksley, 1996; Lo et al., 1999). Zebrafish are unique in that there is a clear shift from innate to a combination of adaptive and innate immunity 4-

6 weeks post fertilization (Novoa and Figueras, 2012). Immune function in fish is also characterized by reduced reliance on antibody detection and production (Trede et al.,

2004). This reliance on innate immunity may be related to ectothermy, slow lymphocyte proliferation and the role of innate immune response during early development (Du

Pasquier, 1982; Trede et al., 2004). To date, there have been several studies on the genes and mechanisms involved in fish immune function (Levraud et al., 2007; Rauta et al.,

2012), such as how it responds to food intake (Cerezuela et al., 2012), and how these responses may change through developmental time (Galindo-Villegas et al., 2012).

However, none have tested immune function in a leptin-deficient fish. I argue that the zebrafish model system is uniquely positioned to test leptin’s integration in innate immune function due to available molecular tools, embryo transparency, and a clear demarcation between the innate and adaptive immune function, which is not a feature of mammalian development (Herbomel et al., 1999; Van der Sar et al., 2003; Stein et al.,

2007; Clatworthy et al., 2009).

In this study, I quantified the immuncompetence of leptin A- knockdown zebrafish embryos challenged by fluorescently labeled Pseudomonas aeruginosa bacteria. I tested the hypothesis that leptin morphant zebrafish embryos are immunocompromised. We found that nonmammalian leptin morphants share similar immune function response to mammals through measures of survival, immune response, and bacterial load clearance.

Methods

Zebrafish husbandry

All animal-related procedures were approved by the University of Akron

Institutional Animal Care and Use Committee (IACUC Approval ID#08-6B). Wild-type adult zebrafish (Aquatic Tropicals, Bonita Springs, FL), Danio rerio, were maintained and bred at 28.5ºC with a light cycle of 14L:10D, according to The Zebrafish Book

(Westerfield, 1994). Immediately after fertilization, zebrafish embryos were transferred to fish tank water with fungicide (0.05% methylene blue) and allowed to develop

(Kimmel et al., 1995). Embryos were staged according to morphological criteria

(Westerfield, 1994).

Morpholino injection

A start-site antisense morpholino oligonucleotide (MO) was designed and manufactured by Gene Tools (Philomath, OR) and reconstituted in Daneau buffer (58mM

NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6). It has been characterized and validated for both efficacy and specificity (Liu et al., 2010; Liu et al., 2012; Dalman et al., 2013). Notably, we previously determined that this morpholino is specific in its effects on leptin and does not simply slow embryonic development.

Embryos at the 1-8 cell stage were injected with 2 nl of 0.4 mM morpholino using a

Narishige MI300 microinjector.

Infection conditions and survivability

Zebrafish embryos were dechorionated by 36 hpf. Liquid bacterial cultures of

Pseudomonas aeruginosa PAO1 (MH539-PAO1 (MRP169), Tc80 Gm60, lasB::gfpASV fusion + rfp-tag, Gfp++ rfp+) were grown overnight from a single isolated colony.

Bacterial cells were centrifuged at 5000*g for 5 mins, supernatant removed, and the pellet was then reconstituted in autoclaved water. Prior to injection, 48 hpf embryos were anesthetized with buffered ethyl 3-aminobenzoate methanesulfonate (MS-222; 140

µg/ml), placed in an agarose injection plate, and excess water removed. Bacterial cells (in a volume of 1 nl) were microinjected into the laterodorsal portion of the yolk sac circulation valley. To reduce clumping of cells, bacteria solutions of 3x103 CFU were passed through a microinjection needle 10 times. Once injection was completed, embryos were gently washed in fresh E3 medium (Nusslein-Volhard and Dahm, 2002) and monitored at regular intervals for survival. Embryo water was replaced daily with E3 medium water supplemented with PTU. Embryo survival over time was documented.

Respiratory Burst

Immune cells phagocytose pathogens and produce reactive oxygen species to kill these cells in a respiratory burst, which was first assayed by Hermann et al. (2004). I modified the Hermann et al. assay by measuring the amount of fluorescent 2’,7’- dichlorofluorescein (DCF). DCF is the oxidized form of the acetyl ester of 6-carboxy-

2’,7’-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA; Molecular Probes Inc.,

Eugene, OR), which is a cell-permeant indicator. On the day of analysis, embryos (~20) were placed into a 50ml conical tube containing fish water and 25 µM carboxy-

H2DCFDA, and incubated at 28.5 ºC for 3 hours in the dark. Embryos were then immediately transferred to a 24-well plate containing only fish water at 28.5ºC. Embryos were then individually placed in a 96-well microplate and fluorescence was measured on a SpectraMAX Gemini Plus fluorescent microplate reader (Molecular Devices,

Sunnyvale, CA). Data were collected over three hours at ten-minute intervals.

Bacterial load

At 72 hpf, embryos were euthanized with an overdose of MS-222 and then homogenized using a 27.5 gauge needle in autoclaved water. The homogenate was then plated onto TSB medium supplemented with gentamicin (60µg/ml) using a Spiral

Biotech Autoplate 4000 (Advanced Instruments, Norwood, MA). Colonies were allowed to grow 24 hours at 37 ºC and then counted.

Statistical Analysis

Survival and respiratory burst activity were graphed using JMP (v. 11, SAS,

Richfield, OH). Respiratory burst was analyzed using an ANCOVA within JMP.

Treatments were compared with time as a covariate. Bacterial load clearance was analyzed using ANOVA within SIGMAPLOT 11 software (Systat, San Jose, CA).

Survival at 96 hpf was analyzed by a two-way ANOVA with treatment and injected/ uninjected as first and second factors, respectively.

Results

Respiratory burst activity

Microinjection of Pseudomonas aeruginosa at a concentration of 3000 CFU/nl caused a significant increase in respiratory burst in control embryos at 72hpf (Figure 5 and Table 2; p <0.0001). Leptin A morphants show an initial lower starting respiratory burst compared to controls. Both morphants and controls increased respiratory burst activity upon infection (p <0.0001). There is also a significant interaction between treatment and incubation time (p <0.0001; Figure 5).

Bacterial Load Clearance

The inoculum concentration of bacteria was based on previous studies using both

Pseudomonas and zebrafish (Clatworthy et al., 2009; Brannon et al., 2009). Injection of

3000 CFU into the laterodorsal portion of the yolk circulation valley resulted in an increase in bacterial load at 72 hours post fertilization, 26 hours post injection for both control and leptin morphants (Figure 7 and Table 2b; p=0.022 and p<0.001, respectively).

However, by 48 hpf, control embryos and morphant embryos CFU count was equivalent.

At 72 hpf, bacterial load increased in leptin morphants to 5.354 x104 and control embryos decreased to 1.774 x104. A significant change in bacterial load was observed from 48 to

72 hpf in both control and morphant embryos (p=0.013 and p=0.041,Table 2b).

Survival

Embryos were injected at 36 hpf and routinely checked every 12 hours for the presence of a heartbeat. After microinjection of leptin MO, less than 10% of embryos

46 were misinjected and thus discarded (Liu et al., 2012). Survival declines 12 hours post injection (Figure 6) with morphant survival at 20% by 96 hpf. Uninjected control embryos have 100% survival rate and microinjection of bacterial inoculum causing 50% survival (Figure 6). Morphants have continued mortality beyond 96 hpf but efficacy of the leptin morpholino (its ability to reduce leptin expression) declines after five days (Liu et al., 2012; Dalman et al., 2013). A two-way ANOVA of survival data at 96 hpf indicates a significant effect of treatment on survival (Table 2c; p <0.0001) in addition to a significant reduction in survival based on bacterial challenge (Table 2c; p <0.0.001).

There was also a significant interaction between treatment and bacterial challenge

(p<0.05; Table 2).

Table 2. Sources of variation in innate immune function

Source dF SS F ratio Pr > F (A) ANCOVA FOR RESPIRATORY BURST ACTIVITY Assay Time 1 269893656 7303.623 <0.0001 Treatment 4 561827265 3800.918 <0.0001 Assay Time x 4 154004867 1041.886 <0.0001 Treatment

(B) TWO- WAY ANOVA FOR BACTERIAL LOAD Age 1 4615148.148 0.0559 0.819 Treatment 2 1548642564.815 9.372 <0.010 Residual 8 660944785.185 21.6 Error 11

Bacterial Challenge 1 7956.75 328.113402 <0.0001 Treatment X 1 168.75 6.958763 <0.050 Bacterial challenge

12000 control +INJ

10000 morphant +INJ

control embryo 8000

6000

4000 Relative Fluorescent Units Fluorescent Relative 2000

0 0 50 100 150 Time (minutes)

Figure 5. Respiratory burst activity for zebrafish challenged by bacteria inoculation at 72 hpf. Each data point is the average of 8 measurements. The data were collected continuously over 3 hours in the dark. Control (diamond)= wildtype embryo, morphant (triangle)= leptin morphant, inj= microinjected with P. aeruginosa (control injected= square, morphant injected= X, no embryo= asterisk). Respiratory burst activity is proportional to line slope; (injected control> injected morphant, etc.). Slopes (treatments) are significantly different (p<0.0001) and the interaction between assay time and treatment is significant (p<0.0001; Table 2).

100

Survival

% 40 control embryo

control +INJ 20 morphant embryo 0 0 24 48 72 96 Time (hours post fertilization)

Figure 6. Survival through developmental time for zebrafish challenged by bacterial injection. Embryos were injected with 3000 CFU and monitored every 24 hours. Control = wildtype embryo, morphant= leptin morphant, inj= microinjected with P. aeruginosa. The data represent three replicates, n=3 at each measurement. Two way ANOVA at 96 hpf indicates significant effects for treatment (p< 0.0001), bacterial challenge (injected/ uninjected; p< 0.0001), and a significant interaction between bacterial challenge and treatment (p< 0.05).

Figure 7. Bacterial load clearance through zebrafish development. All data are mean ± SE. (N=3 at each time point). Control = wildtype embryo, morphant= leptin morphant. Bars that do not share letters are significant at p <0.05.

Discussion

Immune function is significantly altered in both leptin deficient and hyperleptinemia states in mammals, however how leptin affects immune function in nonmammals is poorly characterized. Here I demonstrate that 1) leptin-A knockdown significantly reduces respiratory burst activity in developing zebrafish embryos 2) leptin-

A knockdown alters the ability of zebrafish to survive a systemic bacterial infection 3) leptin knockdown results in a significant increase in bacterial burden. These data support a conserved immune response with that of leptin-deficient mammals and suggest that leptin-A plays a critical role in fish immunocompetence during early development.

As in all vertebrates, the presence of both the cell and humoral immune response work collectively to defend the organism from invading pathogens. The first line of defense for fish during early development is the innate response (Novoa et al., 1996). As in mammals, teleosts’ repertoire of cellular defense consists of several types of phagocytic cells including macrophages, neutrophils, and natural killer cells (Jimeno,

2008). Phagocytic-capable macrophages are present as early as 1 day post-fertilization

(Herbomel et al., 1999) with neutrophil granulocytes present by 48 hpf (Lieschke et al.,

2001). The primitive macrophages are capable of inducing a respiratory burst in response to invading pathogens (Robinson, 2009). Respiratory burst activity is generated continuously by oxidative metabolism, however the major source in response to infection

- is from immune cells which produce O2 and H2O2 (Robinson, 2009). Leptin receptors are expressed on macrophages in mammals (Lord et al., 1998) and leptin deficiency results in phenotypic alterations to macrophages as well as generally reduced immunocompetence

(Lee et al., 1999). I found that injection of P. aeruginosa into the developing zebrafish

51 embryo induced a respiratory burst response, and that leptin knockdown results in a significant reduction in this activity (Figure 5 and Table 2). In mammals, leptin increases survival of macrophages by modulating apoptosis and, moreover, leptin stimulates oxidative burst activity in monocytes/ macrophages (Sanchez-Pozo et al., 2003; Caldeﬁe-

Chezet et al., 2001). Conversely in fish, cytokines such as macrophage-deactivating factor (MDF) and nitric oxide (NO) are found in the blood circulation and they modulate macrophage function and thus respiratory burst activity (Stafford et al., 1999). Unlike mammals, isolated leukocytes from adult rainbow trout given recombinant leptin reduced superoxide anion production (Mariano et al., 2013). I report here that leptin deficiency results in lowered respiratory burst activity at 72 hpf and assert that the differences observed between Mariano and Colleagues (2013) and my study may be explained by mature vs. developing macrophages or in vivo versus in vitro analysis of immune function.

Lethality of infection hinges greatly on the inoculation concentration as well as the pathogen. In this study, injection volume of 1nl of P. aeruginosa resulted in a significant inability to fight invading pathogens in the zebrafish as evidenced by an increased bacterial load (Figure 6). Leptin morphants show, 70% survival 12 hours after injection versus control-injected at 75% and uninjected with 100% survival. By 96 hpf, less than 20% of morphants survive (p<0.0001; Table 2). Conversely, 50% of wildtype embryos survive to 96hpf. Thus leptin deficiency results in a reduced ability to survive bacterial challenge. In a previous study, zebrafish embryos submerged in a suspension of

P. aeruginosa (vs. injection in this study) required a high concentration of bacteria (109

CFU/ml) to induce mortality, and lethality was independent of bacterial viability

(Clatworthy et al., 2009). Interestingly, microinjection of 1700 CFU at 28 hpf result in embryo death by 48 hpi. Furthermore, at 50hpf, a much larger inoculum concentration

(>4000 CFU) was required to induce 100% lethality (Clatworthy et al., 2009). The increased inoculum concentration is attributed to higher immunocompetence through the presence of both macrophage and neutrophil presence. Thus mode of pathogen delivery can impact immunosusceptibility.

Injections of bacteria such as Streptococcus iniae, a gram-positive bacterium, at

103 CFU into the muscle of adult zebrafish results in 50% lethality and death by 2-3 days post injection (dpi; Neely et al., 2002). In leptin-deficient mice, microinjection of

Klebsiella pneumonia and Streptococcus pneumoniae results in 85% lethality by day 10 after injection (Mancuso et al., 2002; Hsu et al., 2007). If these same mice are followed by leptin injection after the infection, survival increases >2.5-fold 10 days post injection.

Of note, Mancuso and colleagues (2002b) found that long-term leptin deficiency had a more potent immunosuppressive effect on survival compared to short-term immunosuppressive effects of starvation (Mancuso et al., 2006). In other words, leptin knockdown and knockout models have a more severe impact on survival compared to starved animals with intact leptin signaling.

Injection of P. aeruginosa into the developing zebrafish resulted in an overall increase in bacterial load for both control and leptin morphants (Figure 7). Furthermore, bacterial load increased in morphants post infection, whereas control embryos reduced their bacterial load 36 hours post-infection. Bacteria or LPS injections into control mammals results in increased leptin expression, which causes a host of proinflammatory cytokines to be expressed such as TNF-alpha and IL-6 (Fernandez-Riejos et al., 2010).

These cytokines play an active role in clearing invading pathogens and thus reducing bacterial load. Leptin deficiency in mammals results in a significantly attenuated immune response. Leptin injection in fish results in a series of mammalian-like responses (Procaccini et al., 2009) that induce NF-kB and MAP kinase pathways, which activate and promote inflammatory responses and anti-apoptotic effects, as well (Mariano et al., 2013). Leptin induces chemotaxis toward mouse immune cells (Gruen et al., 2007).

I speculate that observation of macrophages in leptin morphants would show decreased sensing and motility resulting in increased bacterial burden.

In summary, our data demonstrate that leptin knockdown in zebrafish not only decreases respiratory burst activity but also alters bacterial clearance and thus embryo survival. This supports the mammalian paradigm of immune related function of leptin in a nonmammal. These data are the first report of leptin knockdown effects on immune function in the developing zebrafish.

CHAPTER IV

MICROARRAY ANALYSIS OF LEPTIN-A KNOCKOUT IN EARLY ZEBRAFISH

DEVELOPMENT

Introduction

Leptin is a 16kD, circulating adipocytokine that is critical to the maintenance of energy homeostasis and lipolysis (Zhang et al., 1994). It was first identified in mammals and subsequently identified in all major vertebrate classes, including amphibians (Crespi and Denver, 2006), reptiles (Denver et al., 2011), fish (Kurokowa et al., 2005), and recently birds (Prokop et al., 2014). Leptin is a member of the class 1 cytokine family and with its receptor is expressed both centrally in the brain and peripherally in many tissues including the stomach (Bado et al., 1998), placenta (Hoggard et al., 1997), muscle (Wang et al., 1998), brain (Morash et al., 1999), and bone (Laharrague et al., 1998). All of leptin’s downstream signaling is mediated through a transmembrane leptin receptor (OB-

Rb), which is the only known leptin receptor that engages in JAK-STAT signaling pathway, although other leptin receptors are expressed (OB-R a-f; Bjorbaek et al., 1997;

Tartaglia, 1997; Fong et al., 1998). Leptin and its receptor were identified as the gene products responsible for two obese mouse models (ob-/ob- and db-/db-, respectively) which have wide range of hormonal and metabolic dysfunctions including hyperphagia, hyperinsulinemia, hyperglycemia, and hypercorticosteronemia (Paz-Filho et al., 2010).

Wild type and mutant models differ in response to calorie manipulation, illustrating that

55 signaling in intact animals differs from ob-/ob- and db-/db- mutants (Mistry et al., 1997a;

Doring et al., 1998). The ob-/ob- mouse model signals as if energy starved, and can be rescued via leptin. The db-/db- model shares many of the ob model dysfunctions except that it cannot be rescued by leptin injections (de Luca et al., 2005). The use of models with either intact or disrupted leptin signaling has filled many of the gaps in our understanding leptin’s physiology and neuroendocrine signaling in mammals. However, for non-mammals, models with disrupted leptin signaling have not been available until recently (Liu et al., 2012; Chisada et al., 2014).

The first nonmammalian leptin was identified in 2005 in fish (Kurokawa et al.,

2005) and a year later in amphibians (Crespi and Denver, 2006). Leptin’s identification in non-mammals was hindered by low conservation of primary sequence among vertebrate leptins. Fish leptin is only 10-30% conserved with human leptin primary sequence, whereas mouse and human share >80% sequence identity. Despite this low primary sequence identity, threading algorithms predict (and functional reporter assays support) that the tertiary structures of all vertebrate leptins are highly conserved (Londraville et al., 2014). All mammals and amphibians express one isoform of leptin (Clarke et al.,

2001; Comuzzie et al., 1997; Crespi and Denver, 2006; Zhang et al., 1994) whereas most fish express two leptins (A & B; attributed to a whole genome duplication (WGD);

Gorissen et al., 2009; Copeland et al., 2011; Londraville et al., 2014). Leptin-A is expressed in higher abundance than leptin B and the two isoforms have distinct tissue expression with leptin-A in liver and gonads and leptin B in other peripheral tissues

(Huising et al., 2006; Kurokawa and Murashita, 2009; Gorissen et al., 2009; Liu et al.,

2010; 2012a).

Zebrafish are ideal fish models due to a sequenced and annotated genome, low cost, small size, high embryo generation, and a wide variety of genetic toolsets available

(Westerfield, 2000). We recently developed a leptin-deficient model using morpholino oligonucleotides to knockdown expression of leptin A (Liu et al. 2012a). The leptin- deficient embryos have a lower metabolic rate than controls (Chapter 2), and in that respect are similar to ob-/ob- mice (Zhang et al., 1994). Additionally, they also have dramatically compromised sensory systems (Liu et al., 2012) and the knockdown in zebrafish is lethal which is not the case in mammals (Liu et al., 2012; Chapter 2). In mammals, fasting decreases leptin as fat stores deplete whereas in fish, fasting usually increases leptin expression (Kling et al., 2009). Together, these data suggest that some aspects of fish leptin signaling are conserved with mammals and some aspects of the hormone differ dramatically.

The field of transcriptomics originated with large-scale DNA array technology, with tens of thousands of genes analyzed in one hybridization step, providing a powerful toolset to identify and compare complex gene-expression profiles. Mammalian obesity models have been analyzed using many different array and next-generation sequencing technologies to uncover the underlying complexity of many physiological processes. For example, liver in the obese mouse has reduced protein synthesis (Fu et al., 2011) and obesity causes changes in the gut microbiota in humans (Paliy and Agans, 2011). Fish array technology has lagged behind mammalian transcriptomics, with complete transcriptomes only available in recent builds (Affymetrix, 2012).

Many ‘omic profiling studies have examined leptin signaling in mammals, including in db-/db- mice (Guo et al., 2005), high fat diet (HFD) mice (Kim et al., 2004),

Zucker rats (Serkova et al., 2006) and ob-/ob- mice (Liang and Tall, 2001; Sharma et al.,

2010; Sainz et al., 2009; Won et al., 2012). In fish, no microarray studies directly address leptin signaling. The majority of fish microarray studies focus on changes through development (Mathavan et al., 2005), assays for drug screening (Rubinstein, 2006), response to infection (van Soest et al., 2011; Papaioannou et al., 2013), response to gut microbiota (Rawls et al., 2004), and nutrient availability (Calduch-Giner et al., 2014).

Here I applied transcriptomics to compare gene expression profiles between control and leptin-A knockdown zebrafish embryos to test the hypothesis that energy- producing biochemical pathways are compromised in the knockdown fish. The data support the hypothesis that leptin-A is critical for both anaerobic and aerobic metabolic processes and that leptin plays a significant role in sensory development.

Methods

Zebrafish husbandry

All animal-related procedures were approved by the University of Akron

(Westerfield, 1994). Immediately after fertilization, zebrafish embryos were transferred to fish tank water with fungicide (0.05% methylene blue) and allowed to develop.

Embryos were staged according to morphological criteria (Westerfield, 1994). A total of

8 control (wildtype embryo) and 4 leptin morphant batch samples were take for further microarray analysis at 72 hours post fertilization. Each batch contained 50 embryos.

Morpholino injection

A morpholino antisense oligonucleotide (MO) was designed and manufactured by

Gene Tools (Philomath, OR) and reconstituted in Daneau buffer (58mM NaCl, 0.7 mM

KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6). A previously characterized translation blocking leptin-A morpholino (5’-TTG AGC GGA GAG CTG

GAA AAC GCA T -3’) was used. It has been validated for efficacy and specificity, including determination that the morpholino does not simply slow development (Liu et al., 2012; Dalman et al., 2013). Embryos at the 1-8 cell stage were injected with 2 nl of

0.4 mM morpholino using a Narishige MI300 microinjector. Embryos were pooled for microarray and subsequent molecular and biochemical analyses.

RNA isolation

Total RNA was extracted by TRIzol, according to the manufacturer’s protocol

(Invitrogen, Carlsbad, CA). Any potential genomic contamination was removed by

Ambion Turbo DNA-free (Ambion, Austin, TX) and RNA was subsequently reconstituted and concentrated using Qiagen RNeasy MinElute Cleanup Kit (Qiagen,

Inc., Valencia, CA). Total RNA integrity (RIN) was measured using an Agilent 2100

Bioanalyzer (Agilent Technologies, Palo Alto, CA) and only RIN values ≥9.0 were used for microarray and qPCR. RNA samples were submitted to the University of Michigan

Microarray Core facility (Ann Arbor, MI) for microarray analysis. RNA samples used for qPCR were converted to cDNA using Quanta Biosciences qScript cDNA SuperMix

(Quanta Biosciences, Gaithersburg, MD) per manufacturer’s protocol. RNA was quantified using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and/or Agilent

2100 Bioanalyzer.

Microarray hybridization and analysis

A zebrafish 1.1 ST whole-transcriptome Gene Array Strip (Affymetrix, Santa

Clara, CA) was used to measure the expression of 59,302 gene-level probe sets using

1,255,682 probes with ~22 probes per gene. This array has high sensitivity (≥1.5pM) and dynamic range (~3 logs) with four internal Poly-A and Hybridization controls

(Affymetrix). A total of twelve arrays were hybridized (8 control and 4 morphant) with

RNA pooled at 72 hpf (hours post fertilization) across five clutches for each of 12 independent arrays. Biotinylated cDNA was prepared with the Ambion WT kit (Ambion,

Carlsbad, CA) from 250 ng total RNA. Following fragmentation, 5.5 µg of labeled cDNA was hybridized for 20 h at 48ºC and processed using the Affymetrix Gene Atlas System

(software version: 1.0.4.267; Affymetrix , Santa Clara, CA). Raw data from CEL files were uploaded to PARTEK Genomics Suite 6.6 (Partek, St. Louis, MO), and pre- processed using the Robust Multi-Array Average (RMA) algorithm method while accounting for the GC content of the array probes (Irizarry et al., 2003). This technique uses quantile normalization to make the distribution of probe intensities the same for each array under the assumption that all data come from similar distributions. Relative log2 expression of each array was analyzed by internal QA/QC methods in Partek software and found not to be significantly different among arrays, indicating high reproducibility.

Principle Component Analysis (PCA) was performed on all samples (Partek software, St.

Louis, MO).

As previously documented (Chapter 5), we analyzed our microarray data using several selection criteria for significance cutoff (≥ 1.5 Fold Change (FC), ≥ 2.0 FC, p- value with False detection rate (FDR) ≤.05 , FDR of ≤.02, 1.5 FC and 0.05 FDR, and 2.0

FC and 0.02 FDR). Each gene set selection criterion was then clustered based on

Hierarchical Clustering (Johnson, 1967). Gene sets were then analyzed using Partek’s

GO enrichment to identify significantly affected GO categories. Using Partek Pathway analysis (which is linked to the KEGG database), each gene selection criteria list was then analyzed for significantly enriched annotated pathways. Gene lists for GO and pathway analyses was also conducted using EnRichR (Chen et al., 2013) and

WEBGestalt software (Zhang et al., 2005; Wang et al., 2013).

Biochemical Assays

Embryos were homogenized by sonic disruption (5x 15 second bursts) in 3 volumes ice-cold homogenization buffer (0.1 M Phosphate buffer (pH 7.4) and frozen (-

80 oC). All enzyme activities were determined at 28.5 °C. Total protein concentration was measured using the Bradford method (Bradford, 1976) with bovine serum albumin (BSA) as the standard. Samples were assayed in triplicate. Enzyme assays were monitored at 5- second intervals over 4 minutes using 96-well microplates with the SpectraMax 384 Plus

UV-Vis spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

The lactate dehydrogenase (LDH) assay was adapted from Hansen and Sidell

(1983). LDH activity was measured by the oxidation NADH at 340 nm in UV-transparent microtiter plates. Tissue was homogenized in 50 mM HEPES, pH 7.4 @ 28.5 °C and the assay was initiated with 0.1 M sodium pyruvate. Citrate Synthase activity was measured by reduction of 5,5’-Dithio-bis-(2-nitrobenzoic acid) (DTNB) at 412 nm as adapted from

Hansen and Sidell (1983). Activity was measured in embryo homogenates in 50 mM

HEPES, pH 7.4 @ 28.5 °C and the assay was initiated with 0.5 mM Oxaloacetic acid.

The catalase assay was adapted from Grim et al. (2010). Catalase activity was measured

61 at 240 nm using UV-transparent microtiter plates (Thermo Scientific, Thermo Scientific,

Ottawa Ontario). Activity was measured in embryo homogenates in 50 mM HEPES, pH

7.4 @ 28.5 °C. Hydrogen peroxide (30%) was diluted in 0.05 M phosphate buffer (pH

7.0) to final concentration of 1.76% (v/v) to initiate the assay. 3-Hydroxyacyl-CoA dehydrogenase (HOAD) enzyme activity was monitored by following the oxidation of

NADH at 340nm as adapted by Hansen and Sidell (1983). Activity was measured in embryo homogenates in 50 mM HEPES, pH 7.4 @ 28.5 °C and assays were initiated with 0.1mM Acetoacetyl CoA. The carnitine palmitoyltransferase (CPT) assay was adapted for microplate analysis from Bieber et al. (1972). CPT activity was estimated from the reduction of 5,5’-Dithio-bis-(2-nitrobenzoic acid) (DTNB) at 412 nm, initiating the assay with 1.25 mM Carnitine HCL in 0.04 M HEPES. Absorbance was recorded at

10-second intervals over 15 minutes.

Nile red staining

Embryos were raised in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM

CaCl2, 0.33 mM MgSO4) and supplemented with 1-phenyl-2-thiourea (PTU) at 0.003%.

Starting at 24 hours post fertilization (hpf), 20-50 embryos were placed into cups containing 12.5 ng/ml nile red in E3, diluted from a Nile red 40,000X stock solution at

500µg/ml in acetone. Embryos were transferred to new E3 medium with Nile red each day until imaging. At 3 days post-fertilization (dpf), larvae were examined by fluorescent microscopy. Fluorescence and bright-field images were obtained using a SPOT digital camera (SPOT imaging Solutions, Sterling Heights, MI) mounted on an Olympus BX51 microscope (BX51, Olympus America Inc., Center Valley, PA). Area and perimeter of yolk sac was measured using SPOT imaging software.

Data Analysis

The comparative analysis including probe normalization, filtering, Principal

Component Analysis, and gene set production was generated using Partek Genomics

Suite 6.6 (Partek, St. Louis, MO). Web-based Gene Set Analysis Toolkit was used to produce GO and directed acylic graphs (DAG) and GENEMAPP software (Salomonis et al., 2007) was used to simultaneously display multiple selection criteria on one KEGG database pathway (adipocytokine signaling). Nile red staining yolk sac area and perimeter was analyzed using a paired students t-test.

Results

Effects of leptin knockdown on sensory and lipid area at 72 hpf

Leptin morphants showed the typical leptin A knockout phenotype, including significant reductions in growth and development with reduced melanin formation, smaller eyes, curved notochord, reduced otoloith formation, decreased linear growth, and reduced yolk absorption at 72 hpf as previously described (Figure 8; Liu et al. 2012a).

Morphant embryos express significantly larger yolk area (Figure 8d; p<0.01).

Identification of genes affected by leptin knockdown

Using the Zebrafish Gene 1.1 ST Array platform referenced to the danRer6 and

Zv9 genomic builds, 1.255,682 probes were used (59,302 gene-level probes; median of

22 probes/ gene) to assess the gene expression profile of leptin knockdown in the developing zebrafish embryo. This build includes 26,000 genes from 51,169 transcripts

(Collins et al., 2012).

Multiple gene-selection cutoff criteria have been used in transcriptomic data and their effects on interpretation have been discussed (Chapter 5). Using the least stringent feature selection criteria of a ≥1.5 fold change (FC), 10,129 genes were differentially affected with 4,373 (43%) of them up regulated and 5,756 (57%) down regulated (Table

3). A selection criterion of ≥2 FC decreased the number of significantly altered genes to

3,103 genes. Of those 3,103 genes, 1,236 (40%) were up regulated and 1,867(60%) were down regulated (morphant relative to control). In addition to fold change, false detection rate has been widely used in large data sets (Pawitan et al., 2005; Pounds, 2006). Using a false detection rate of ≤ 0.05 resulted in 1,511 genes significantly altered with 780 (52%) up regulated and 731 (48%) down regulated. Increasing selection criteria to ≤ 0.02 reduced the amount of differentially regulated genes to 779 with 441 (57%) up regulated and 338 (43%) down regulated. Combining the least or most stringent selection criterion

(p-value and fold change; ≥1.5FC and ≤ 0.05, ≥2.0FC and ≤ 0.02) the most stringent selection criteria had 515 genes differentially regulated with 265 (51%) up and 250

(49%) down regulated genes. The least-stringent combined criteria resulted in 1,361 genes with 679 (50%) up up regulatedregulated genes and 682 (50%) down regulated genes. Based on 26,152 genes (Collins et al., 2012), 1.97%, 2.98%, 5.20%, 5.78%,

11.87%, and 38.73% of annotated genes were differentially regulated at selection criteria cutoffs: ≥2.0FC and ≤ 0.02FDR, ≤ 0.02FDR, ≥1.5FC and ≤ 0.05FDR, ≤ 0.05FDR,

≥2.0FC, and ≥1.5FC, respectively (Table 3).

Using combined selection criteria, I performed a multivariate, principal components analysis (PCA) to determine whether leptin knockdown and control embryos could be seprated by their gene expression signals. Analysis of the principal component

64 model performance cumulatively explained 70% of the total variability of the data

(Figure 9a). PCA 1 accounts for 46.3% of the variance with PCA 2 and 3, accounting for

13% and 10.7% of the total variance, respectively. The two groups are well defined by separate elipses (Figure 9a). Volcano plot analysis qualitatively indicates more than half of the genes assayed do not change more than ±1.1 FC nor are significantly affected beyond p= 0.10 (Figure 9b). The top 80 (greatest FC) up and down regulated genes are tabulated Tables 3 and 4. Transcripts are ranked according to the magnitude of their fold change. Table A1 lists all significantly affected genes.

Genes positively regulated by leptin knockdown

As expected, the expression of leptin receptor (lepr) was up regulated (2.10 FC, morphant relative to control) in response to leptin A knockdown. Mean expression of

Leptin A and B transcripts were also up regulated (1.6 and 3.0 fold, respectively) and the change was not significant under the adopted criteria. Notch homologs-1a, -1b, and -3

(implicated in notch signaling and Dorso-ventral axis formation), deltaA through D, and jagged 1a were significantly up regulated. RNA transport genes such as survival motor neuron 1 and aminoacyl-tRNA biosynthesis genes (alanyl-, isoleucyl-, arginyl-, valyl-, and glutamyl-prolyl- tRNA synthetases) were also significantly up regulated with leptin knockdown. Splicesome related genes such as catenin- beta-like 1 and heat shock cognate

70-kD protein were up regulated in response to leptin knockdown. Leptin knockdown also up regulated transcription factor Dp-2, cyclin-dependant kinase 6, and cyclin D1 all of which are implicated in cell cycle and apoptosis (Ptak et al., 2013). The top three up regulated genes were matrix metalloproteinase 9 (mmp9), FOS-like antigen 1a (fosl1a),

65 and heat shock cognate 70kD (hsp70l) with fold change increases of 9.85, 8.45, and 7.67, respectively (Table 4 and Table 2A).

Genes negatively regulated by leptin knockdown

Genes involved in phototransduction such as rhodopsin and calmodulin 1a were down regulated (morphant relative to control) along with glutamate receptors (grin1b, grm7, grm1a, gria3a, and grin2b), corticotropin releasing hormone receptor 1, oxytocin receptor, thyroid hormone receptor alpha b, and opiod receptor mu 1 (Table 5, Tables 1A and 2A). Genes implicated in calcium signaling pathways such as calcium/calmodulin- dependent protein kinase (CaM kinase) II delta 2, protein kinase C- beta b, and ATPase

Ca2+ transporting plasma membrane 3b were also significantly down regulated. Cell adhesion related molecules such as neural cell adhesion molecule 2, neuronal growth regulator 1, and neuroligin 2a and 4a were also affected, Adiponectin receptor 2, Signal transducer and activator of transcription 2 (stat2), T-cell immune regulator 1- ATPase

(tcirg1), and synaptotagmins (Va, Vb, and Ia) were also negatively regulated in response to leptin A knockdown. The top three down-regulated genes were opsin 1 (cone pigments)- short wave sensitive 1 (opn1sw1), rhodopsin (rho), and peripherin 2b (retinal degeneration-slow; prph2b) with negative fold changes of -56.45, -31.72, and -27.83, respectively (Table A1 and A2).

Gene Ontology Pathway Analysis

The most enriched Gene Ontology (GO) term was binding (Molecular Function), with the “top level” organization of GO indicating the most significantly affected

Molecular Function, Cellular Component, and Biological Processes are binding, cell

66 parts, and cellular processes, respectively (Figure 10-16). The most enriched GO terms for molecular and cellular functions were nucleus, photoreceptor outer segment, GABA receptor activity, photoreceptor activity, and helicase activity (Figure 10-16; Table 6).

GO Biological processes involved in sensory and visual perception to stimulus and RNA metabolic process were the most enriched (Figures 10-16).

KEGG Pathway Analysis

Enrichment analysis of gene expression profiles against known D. rerio pathways identified 7 significantly regulated pathways at p<0.05 level (Table 7). Partek Pathway analysis identified Phototransduction as the most significantly affected pathway (p =

9.76e-07), followed by Notch signaling (p = 2.5e-05), Ribosome Biogenesis (p = 1.3e-

04), RNA Transport (p = 3.2e-03), Neuroactive Ligand-receptor interaction (p = 0.10),

Aminoacyl-tRNA Biosynthesis (p=0.012), and Calcium signaling (p = 0.035; Table 7).

Furthermore, Adipocytokine signaling pathway did not meet gene selection criteria chosen within this study, however, because it is related to the hypothesis, I examined it separately (Figure 17). Based on listed gene cutoff criteria, only adiponectin and leptin receptors were found significantly affected based on combined selection criteria within

Adipocytokine signaling pathway. Reducing the gene cutoff threshold, collectively leptin isoforms A (+1.69FC) and B (+3.00FC) and leptin receptor (+1.73FC) increased while adiponectin receptor (-1.87FC) mRNAs decreased (Figure 17).

Confirmation Analyses of Microarray Expression Data

To independently verify the results of the array, I used absolute quantification and functional enzyme assays. Enzymes of intermediary metabolism were selected as

67 independent indictors of the microarray signal, because of leptin’s well-known effects on metabolism in mammals. 3-hydroxyacyl-Coenzyme A dehydrogenase (HOAD), carnitine palmitoyltransferase (CPT), citrate synthase, and catalase all validated the microarray data, in that change in enzyme activity was in the same direction as change in message expression (Figure 18). LDH enzyme activity, with contributions from all active isozymes, increased with leptin A knockdown, whereas LDH message response depended on the LDH orthologue. Multiple isoforms of lactate dehydrogenase were measured on the microarray, however only isoforms LDH-D and LDH-A4 were significantly affected

(-1.673 and -3.048, respectively; Table A1). Other LDH isozymes were not present under the least gene selection criteria containing >10,000 genes (≥1.5 FC). We postulate that differences in activity between mRNA and protein may be due to timeshifts in transcript, lifetime of transcript, and/or processivity of LDH isozymes.

B D

Figure 8. Effects of leptin knockdown at 72 hpf on zebrafish lipid size. Panels A and B fluorescent imaging using Texas red filter. Nile red stain fluoresces in presence of lipid. Panel A control and panel B morphant embryo at 72hpf. Scale bar represents 200 µm. Panels C and D are 72 hpf zebrafish embryo data of lipid circumference and area. Morphant embryos have significantly decreased yolk circumference and increased yolk area as compared to control embryos. * = p<0.05. N=5 for each measurement.

•Leptin MO A A •Control

2 2 13% - PC

PC-1 46.3% PC-3 10.7%

B B

value

Adjusted p Adjusted value Corrected p Corrected

Fold Change Figure 9. Microarray analysis of leptin knockdown is reproducible and selectively impacts a large number of transcripts. A. Multivariate, Principal Components Analysis (PCA) of zebrafish embryo gene expression profiles at 72 hours post fertilization (hpf). Red and blue dots represent morphants and control embryo samples, respectively. B. Volcano plot of gene expression significance as a function of fold change. Fold change expression data is log2 transformed. Red is p value of 1.0 and blue is a p value of p<1.0e- 6.

Figure 10. The number of genes (X) vs. Gene Ontology (GO) category (Y) for the effect of leptin A knockdown in 72 hpf zebrafish. A.) Molecular Function B.) Cellular Component C.) Biological Process. GO categories are nested from top to bottom, with green up regulated and red down regulated (morphant relative to control).

Figure 11. Directed Acyclic Graph (DAG) depicting significantly enriched down regulated Gene Ontology (GO) category, Biological Process. Comparing controls with morphant zebrafish embryos at 72 hpf, GO categories in red are the top 10 categories that also have a p value <0.05. Black categories are nonenriched parent nodes. Each category indicates number of enriched genes in category and adjusted p value.

Figure 12. Directed Acyclic Graph (DAG) depicting significantly enriched up regulated Gene Ontology (GO) category, Biological Process. Comparing controls with morphant zebrafish embryos at 72 hpf, GO categories in red are the top 10 categories that also have a p value <0.05. Black categories are nonenriched parent nodes. Each category indicates number of enriched genes in category and adjusted p value.

Figure 13. Directed Acyclic Graph (DAG) depicting significantly enriched down regulated Gene Ontology (GO) category, Cellular Component. Comparing controls with morphant zebrafish embryos at 72 hpf, GO categories in red are the top 10 categories that also have a p value <0.05. Black categories are nonenriched parent nodes. Each category indicates number of enriched genes in category and adjusted p value.

Figure 14. Directed Acyclic Graph (DAG) depicting significantly enriched up regulated Gene Ontology (GO) category, Cellular Component. Comparing controls with morphant zebrafish embryos at 72 hpf, GO categories in red are the top 10 categories that also have a p value <0.05. Black categories are nonenriched parent nodes. Each category indicates number of enriched genes in category and adjusted p value.

Figure 15. Directed Acyclic Graph (DAG) depicting significantly enriched down regulated Gene Ontology (GO) category, Molecular Function. Comparing controls with morphant zebrafish embryos at 72 hpf, GO categories in red are the top 10 categories that also have a p value <0.05. Black categories are nonenriched parent nodes. Each category indicates number of enriched genes in category and adjusted p value

Figure 16. Directed Acyclic Graph (DAG) depicting significantly enriched up regulated Gene Ontology (GO) category, Molecular Function. Comparing controls with morphant zebrafish embryos at 72 hpf, GO categories in red are the top 10 categories that also have a p value <0.05. Black categories are nonenriched parent nodes. Each category indicates number of enriched genes in category and adjusted p value.

Figure 17. GENEMAPP KEGG Pathway of Adipocytokine Signaling pathway for leptin A knockdown in zebrafish embryos at 72 hours post fertilization (hpf). Red genes are down regulated and green genes are up regulated (morphant relative to control at 72 hpf). Within each gene block square, specific gene selection criteria are displayed.

Enzyme assayed Enzyme Fold Gene ID Gene Assignment Fold change Microarray assay change data 3-hydroxyacyl- hadh NM_001003515 -1.56 Coenzyme A hadhaa ENSDART00000079734 -1.74 dehydrogenase -1.27 (HOAD) hadhab ENSDART00000076009 -1.64

carnitine cpt1b ENSDART0000008113 -2.28 palmitoyltransfer -1.37 2 ase cpt2 ENSDART0000013593 -1.87 (CPT) 8 LOC100333227 ENSDART0000008816 -1.50 8

Catalase -1.29 cat ENSDART00000007781 -3.95

Citrate Synthase -2.01 idh1 ENSDART00000007789 -1.93

Lactate ldhd ENSDART00000056721 -1.67 Dehydrogenase +1.19 ldha ENSDART00000059886 -3.05 (LDH)

TNFSF13b ≈ tnfsf13b ENSDART00000017939 +1.66 qPCR

Figure 18. Validation of microarray data by selected functional enzyme and gene expression. Fold change expression is referenced to control embryos. Red and green arrows indicate decreased and increased fold change, respectively for morphants relative to controls. Gene assignment and ID is taen directly from PARTEK software.

Table 3 - Significantly regulated genes per gene selection criterion for zebrafish leptin-A knockdown at 72 hours post fertilization (hpf). Each column is independently tabulated. FDR represents false detection rate.

≥1.5 ≥2 Fold ≤0.02 ≥1.5Fold ≤0.05 ≥2 Fold Fold Change FDR Change FDR Change Change ≤0.02 FDR ≤0.05 FDR UP regulated genes 265 441 679 780 1236 4373 DOWN regulated genes 250 338 682 731 1867 5756 TOTAL differentially expressed genes 515 779 1361 1511 3103 10129 % UP regulated genes 51 57 50 52 40 43 % DOWN regulated genes 49 43 50 48 60 57

Table 4 - Top 80 significantly up regulated genes (morphant relative to control) involved in leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf). Gene assignment and symbol is taken directly from microarray data gene

Gene Assignment Gene Adj P Fold Symbol value Change XM_003200280 // LOC100537029 // immunoglobulin superfamily DCC LOC100537 6.7E-08 35.84 subclass member 3-like / 029 XM_693882 // LOC570404 // uncharacterized LOC570404 // --- // 570404 LOC570404 2.1E-06 20.23 /// ENSDART0000005 XM_001339896 // LOC799595 // immunoglobulin superfamily DCC LOC799595 4.5E-08 17.15 subclass member 3-like // - BC151913 // si:dkey-204l11.1 // si:dkey-204l11.1 // --- // 100006301 si:dkey- 2.8E-07 10.22 204l11.1 ENSDART00000062845 // mmp9 // matrix metalloproteinase 9 // --- // mmp9 2.5E-04 9.85 406397 /// NM_213123 NM_001200012 // LOC562935 // heat shock cognate 70 kDa protein // --- LOC562935 2.3E-05 9.05 // 562935 /// ENS ENSDART00000055623 // hbbe3 // hemoglobin beta embryonic-3 // --- // hbbe3 2.5E-06 8.60 30596 /// NM_00101 NM_001161552 // fosl1a // FOS-like antigen 1a // --- // 564241 /// fosl1a 4.8E-05 8.46 ENSDART00000008373 / NM_001113589 // hsp70l // heat shock cognate 70-kd protein, like // --- // hsp70l 6.5E-05 7.67 560210 /// N NM_131099 // foxn4 // forkhead box N4 // --- // 30315 /// foxn4 2.7E-08 7.28 ENSDART00000008994 // foxn4 / ENSDART00000003646 // optc // opticin // --- // 445189 /// optc 8.8E-05 7.02 NM_001003583 // optc // opti ENSDART00000033848 // brf1a // BRF1 homolog, subunit of RNA brf1a 5.6E-09 6.84 polymerase III transcription NM_001173501 // whsc2 // Wolf-Hirschhorn syndrome candidate 2 // --- whsc2 1.2E-06 6.69 // 559677 /// ENSD NM_199896 // iars // isoleucyl-tRNA synthetase // --- // 334393 /// iars 6.1E-07 6.33 ENSDART00000004423 ENSDART00000006180 // dla // deltaA // --- // 30131 /// NM_130954 // dla dla 1.3E-06 6.27 // deltaA // - NM_001045353 // hes2.2 // hairy and enhancer of split 2.2 // --- // 751634 hes2.2 3.7E-06 6.13 /// ENSDART0 ENSDART00000122681 // nes // nestin // --- // 100150939 /// nes 2.8E-08 6.02 XM_001919887 // nes // nest NM_131441 // notch1a // notch homolog 1a // --- // 30718 /// notch1a 7.0E-07 5.95 ENSDART00000129224 // notc XM_682888 // wu:fi04f09 // wu:fi04f09 // --- // 559540 wu:fi04f09 1.3E-07 5.79 ENSDART00000111598 // cxcl-c1c // chemokine (C-X-C motif) ligand cxcl-c1c 4.1E-06 5.56 C1c // --- // 795785 / NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 1.7E-06 5.55 XM_690545 // wu:fc14a10 // wu:fc14a10 // --- // 567253 /// wu:fc14a10 2.8E-04 5.31 ENSDART00000112838 // wu:fc1 XM_001340105 // LOC799825 // protein lin-28 homolog A-like // --- // LOC799825 1.3E-04 5.23 799825 /// ENSDART ENSDART00000055428 // cbx7a // chromobox homolog 7a // --- // 550551 cbx7a 1.8E-04 5.16 /// NM_001017853 / NM_001045073 // hsp90aa1.2 // heat shock protein 90, alpha (cytosolic), hsp90aa1.2 1.2E-04 5.13 class A member ENSDART00000078563 // neurog1 // neurogenin 1 // --- // 30239 /// neurog1 1.6E-06 5.03 NM_131041 // neurog1 BC124167 // azi1 // 5-azacytidine induced gene 1 // --- // 563066 azi1 7.1E-06 5.03 NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 9.1E-06 4.83

ENSDART00000019259 // dlb // deltaB // --- // 30141 /// NM_130958 // dlb dlb 1.0E-08 4.79 // deltaB // - ENSDART00000151494 // wu:fj64h06 // wu:fj64h06 // --- // 336342 /// wu:fj64h06 1.2E-05 4.68 XM_002665873 // wu: NM_153673 // unc45b // unc-45 homolog B (C. elegans) // --- // 266640 /// unc45b 5.2E-04 4.65 ENSDART000000 XM_001339301 // LOC798927 // uncharacterized LOC798927 // --- // LOC798927 9.9E-04 4.56 798927 DQ360116 // prtgb // protogenin homolog b (Gallus gallus) // --- // 572241 prtgb 7.3E-07 4.47 ENSDART00000025428 // epha2 // eph receptor A2 // --- // 30689 /// epha2 4.8E-07 4.33 NM_131415 // epha2 / XM_001919922 // LOC557824 // heat shock protein 105 kDa-like // --- // LOC557824 1.7E-07 4.16 557824 /// ENSDA ENSDART00000021299 // nmd3 // NMD3 homolog (S. cerevisiae) // --- // nmd3 6.2E-05 4.08 541444 /// NM_0010 ENSDART00000122353 // LOC553492 // uncharacterized LOC553492 // LOC553492 4.1E-06 4.02 --- // 553492 /// ENSDA ENSDART00000091158 // irg1l // immunoresponsive gene 1, like // --- // irg1l 6.3E-04 3.96 562007 /// NM_00 XM_682888 // wu:fi04f09 // wu:fi04f09 // --- // 559540 wu:fi04f09 1.6E-04 3.95 ENSDART00000099224 // dld // deltaD // --- // 30138 /// NM_130955 // dld dld 2.7E-07 3.91 // deltaD // - ENSDART00000110219 // zgc:171476 // zgc:171476 // --- // 334271 /// zgc:171476 2.9E-04 3.87 NM_001114886 // zgc NM_001044310 // aars // alanyl-tRNA synthetase // --- // 324940 /// aars 6.4E-06 3.86 NM_001040035 // aar BC117615 // arhgef1b // Rho guanine nucleotide exchange factor (GEF) arhgef1b 3.8E-05 3.80 1b // --- // 55798 ENSDART00000038727 // DDX47 (1 of 2) // DEAD (Asp-Glu-Ala-Asp) DDX47 5.3E-05 3.75 box polypeptide 47 // -- ENSDART00000014127 // si:dkey-3j24.1 // si:dkey-3j24.1 // --- // 557055 si:dkey- 4.8E-08 3.72 /// ENSDART0000 3j24.1 NM_131531 // hoxc1a // homeo box C1a // --- // 58046 /// hoxc1a 5.2E-04 3.70 ENSDART00000103131 // hoxc1a / NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 1.0E-06 3.68 NM_199820 // eif3s10 // eukaryotic translation initiation factor 3, subunit eif3s10 4.1E-04 3.67 10 (theta) ENSDART00000109892 // SAMD9 // sterile alpha motif domain SAMD9 3.4E-07 3.64 containing 9 // --- // --- NM_001172556 // bxdc2 // brix domain containing 2 // --- // 402823 /// bxdc2 2.8E-06 3.63 ENSDART000000330 NM_001039636 // smyd1b // SET and MYND domain containing 1b // --- smyd1b 6.0E-04 3.63 // 569027 /// BC1630 DQ851840 // prdm1b // PR domain containing 1b, with ZNF domain // --- prdm1b 2.5E-04 3.58 // 569677 NM_001258317 // im:7137886 // im:7137886 // --- // 449866 /// HM114349 im:7137886 2.5E-04 3.56 // im:7137886 // NM_001145786 // dnttip2 // deoxynucleotidyltransferase, terminal, dnttip2 2.1E-04 3.54 interacting protein 2 ENSDART00000056005 // ascl1a // achaete-scute complex-like 1a ascl1a 1.5E-05 3.48 (Drosophila) // --- // 30 XM_002665422 // LOC100334443 // zinc finger protein 36, C3H1 type- LOC100334 1.3E-04 3.47 like 1-like // --- // 443 XM_002666924 // LOC100333070 // uncharacterized LOC100333070 // -- LOC100333 4.2E-04 3.40 - // 100333070 /// EN 070 ENSDART00000073440 // DNAJA4 // DnaJ (Hsp40) homolog, subfamily DNAJA4 2.2E-04 3.35 A, member 4 // --- // - ENSDART00000074499 // olig4 // oligodendrocyte transcription factor 4 olig4 5.1E-05 3.34 // --- // 324857 NM_200631 // srfl // serum response factor like // --- // 393604 /// srfl 7.8E-07 3.33

BC057414 // srfl / XM_688618 // LOC565341 // uncharacterized LOC565341 // --- // 565341 LOC565341 4.8E-05 3.33 NM_199658 // insm1b // insulinoma-associated 1b // --- // 323882 /// insm1b 4.8E-06 3.31 ENSDART00000075331 ENSDART00000084411 // LOC568788 // novel protein similar to LOC568788 6.3E-07 3.30 vertebrate ADAM metallopept XM_691849 // si:ch73-21g5.7 // si:ch73-21g5.7 // --- // 568516 /// si:ch73- 3.1E-05 3.29 ENSDART00000142921 / 21g5.7 ENSDART00000109394 // her13 // hairy-related 13 // --- // 550600 /// her13 3.6E-05 3.28 NM_001017901 // he ENSDART00000014822 // coe2 // coe2 // --- // 30692 /// NM_131418 // coe2 coe2 1.4E-04 3.28 // coe2 // --- BC135096 // zgc:163061 // zgc:163061 // --- // 100037379 /// zgc:163061 6.3E-04 3.25 ENSDART00000109720 // zgc: BC049436 // mphosph10 // M-phase phosphoprotein 10 (U3 small mphosph10 3.2E-05 3.25 nucleolar ribonucleoprotei NM_200573 // onecut1 // one cut domain, family member 1 // --- // 393545 onecut1 3.3E-06 3.24 /// ENSDART000 ENSDART00000022060 // atf3 // activating transcription factor 3 // --- // atf3 2.7E-05 3.20 393939 /// NM NM_214716 // hspa4a // heat shock protein 4a // --- // 335865 /// hspa4a 2.3E-04 3.18 ENSDART00000021037 // NM_001034986 // lama1 // laminin, alpha 1 // --- // 569971 /// DQ131910 // lama1 3.2E-06 3.16 lama1 // lam BC068429 // zgc:85936 // zgc:85936 // --- // 406563 /// zgc:85936 1.6E-05 3.16 ENSDART00000123163 // zgc:85936 ENSDART00000129223 // PHF21B (1 of 2) // PHD finger protein 21B // -- PHF21B 2.8E-05 3.13 - // --- ENSDART00000126063 // TTF2 // transcription termination factor, RNA TTF2 1.3E-06 3.13 polymerase II // -- ENSDART00000146005 // gtpbp1 // GTP binding protein 1 // --- // 378721 gtpbp1 5.4E-06 3.08 /// NM_213475 // XM_678195 // LOC555628 // inositol 1,4,5-triphosphate receptor- LOC555628 1.0E-04 3.07 interacting protein-like ENSDART00000129888 // LOC100537256 // 5-azacytidine-induced LOC100537 2.0E-04 3.05 protein 1-like // --- // 10 256 BC058295 // tab1 // TGF-beta activated kinase 1/MAP3K7 binding tab1 3.2E-07 3.02 protein 1 // --- // 4030 XM_001343253 // LOC100003830 // uncharacterized LOC100003830 // -- LOC100003 1.3E-04 3.01 - // 100003830 /// EN 830

Table 5 - Top 80 significantly down regulated genes (morphant relative to control) involved in leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf). Gene assignment and symbol is taken directly from microarray data generated

Gene Assignment Gene Adj P Fold Symbol value Change ENSDART00000067160 // opn1sw1 // opsin 1 (cone pigments), short- opn1sw1 2.8E-05 -56.45 wave-sensitive 1 // --- NM_001204332 // gngt2b // guanine nucleotide binding protein (G gngt2b 9.5E-07 -48.83 protein), gamma transdu ENSDART00000027000 // rho // rhodopsin // --- // 30295 /// NM_131084 // rho 2.3E-05 -31.72 rho // rhodopsin ENSDART00000020671 // prph2b // peripherin 2b (retinal degeneration, prph2b 3.2E-06 -27.83 slow) // --- // 55 ENSDART00000065940 // opn1lw2 // opsin 1 (cone pigments), long-wave- opn1lw2 7.9E-07 -24.87 sensitive, 2 // --- ENSDART00000005547 // gnb3b // guanine nucleotide binding protein (G gnb3b 2.8E-06 -23.70 protein), beta pol BC047826 // zgc:56085 // zgc:56085 // --- // 327506 /// zgc:56085 2.5E-05 -21.89 ENSDART00000101449 // zgc:56085 NM_200785 // pde6h // phosphodiesterase 6H, cGMP-specific, cone, pde6h 2.4E-05 -19.94 gamma // --- // 393758 ENSDART00000075513 // aqp9b // aquaporin 9b // --- // 570191 /// aqp9b 2.0E-04 -16.78 NM_001177744 // aqp9b ENSDART00000064896 // gnat1 // guanine nucleotide binding protein (G gnat1 4.6E-04 -16.62 protein), alpha tr BC060940 // zgc:73359 // zgc:73359 // --- // 393810 zgc:73359 1.1E-05 -15.97 NM_131192 // opn1sw2 // opsin 1 (cone pigments), short-wave-sensitive 2 opn1sw2 1.1E-05 -15.82 // --- // 30435 ENSDART00000106501 // pde6c // phosphodiesterase 6C, cGMP-specific, pde6c 9.4E-06 -15.46 cone, alpha prime / ENSDART00000023543 // rcv1 // recoverin // --- // 335650 /// NM_199964 rcv1 1.3E-05 -14.75 // rcv1 // recov ENSDART00000078996 // arr3a // arrestin 3a, retinal (X-arrestin) // --- // arr3a 3.7E-06 -14.66 436678 /// N ENSDART00000114673 // LOC100535672 // trypsin-1-like // --- // LOC10053 2.0E-04 -14.48 100535672 5672 NM_200825 // zgc:73075 // zgc:73075 // --- // 572207 /// zgc:73075 2.5E-05 -13.42 ENSDART00000128721 // zgc:7307 XM_001339170 // myhb // myosin, heavy chain b // --- // 100002040 /// myhb 3.9E-04 -12.68 ENSDART0000002625 NM_131869 // gnat2 // guanine nucleotide binding protein (G protein), gnat2 1.4E-06 -11.48 alpha transducing ENSDART00000036050 // rs1 // retinoschisis (X-linked, juvenile) 1 // --- // rs1 3.4E-04 -11.11 445044 /// ENSDART00000055415 // prph2a // peripherin 2a (retinal degeneration, prph2a 8.1E-06 -10.71 slow) // --- // 58 ENSDART00000080106 // zgc:158677 // zgc:158677 // --- // 100009626 /// zgc:158677 1.1E-04 -10.07 NM_001082995 // BC091979 // aglb // amylo-1, 6-glucosidase, 4-alpha-glucanotransferase b aglb 7.4E-05 -9.89 // --- // 5533 ENSDART00000111499 // IMPG1 // interphotoreceptor matrix IMPG1 1.6E-06 -9.63 proteoglycan 1 // --- // --- NM_001030248 // zgc:114180 // zgc:114180 // --- // 570333 /// zgc:114180 1.8E-04 -9.55 ENSDART00000074036 // zgc NM_131451 // irbp // interphotoreceptor retinoid-binding protein // --- // irbp 1.1E-04 -9.32 30735 /// EN ENSDART00000130128 // rgs9a // regulator of G-protein signaling 9a // -- rgs9a 3.1E-05 -9.00 - // 767636 ///

BC076192 // faimb // Fas apoptotic inhibitory molecule b // --- // 436668 /// faimb 1.1E-04 -8.92 ENSDART00 ENSDART00000105741 // AGL (3 of 3) // amylo-alpha-1, 6-glucosidase, AGL 1.2E-04 -8.91 4-alpha-glucanotran NM_001017711 // grk1b // G protein-coupled receptor kinase 1 b // --- // grk1b 8.9E-05 -8.51 550406 /// ENS XM_003199054 // LOC799480 // uncharacterized LOC799480 // --- // LOC79948 2.0E-04 -8.28 799480 /// ENSDART0000 0 ENSDART00000012673 // gnb3a // guanine nucleotide binding protein (G gnb3a 3.1E-06 -8.05 protein), beta pol NM_200794 // rom1a // retinal outer segment membrane protein 1a // --- rom1a 6.7E-05 -7.71 // 393767 /// EN XM_001336435 // LOC100000094 // uncharacterized LOC100000094 // --- LOC10000 2.1E-05 -7.69 // 100000094 0094 XM_693199 // LOC569792 // uncharacterized LOC569792 // --- // 569792 LOC56979 2.9E-04 -7.68 2 BC151864 // wu:fb15e04 // wu:fb15e04 // --- // 566445 wu:fb15e04 3.9E-05 -7.59 XM_682552 // LOC559232 // regulator of G-protein signaling 9-binding LOC55923 3.2E-06 -7.44 protein-like // -- 2 ENSDART00000126830 // opn1mw1 // opsin 1 (cone pigments), medium- opn1mw1 1.8E-04 -7.22 wave-sensitive, 1 // - NM_200751 // rpe65a // retinal pigment epithelium-specific protein 65a // rpe65a 4.0E-04 -7.10 --- // 393724 ENSDART00000055995 // sagb // S-antigen; retina and pineal gland sagb 1.0E-06 -7.02 (arrestin) b // --- // XM_002662494 // LOC566922 // gamma-aminobutyric acid receptor LOC56692 3.2E-06 -6.97 subunit beta-3-like // -- 2 ENSDART00000084011 // cplx4a // complexin 4a // --- // 768157 /// cplx4a 6.1E-05 -6.87 NM_001077300 // cplx4 NM_001190305 // slc1a2a // solute carrier family 1 (glial high affinity slc1a2a 6.2E-05 -6.75 glutamate trans BC049482 // rom1b // retinal outer segment membrane protein 1b // --- // rom1b 4.1E-04 -6.71 393989 /// ENS NM_001110473 // igsf21b // immunoglobin superfamily, member 21b // --- igsf21b 2.8E-07 -6.59 // 567714 /// EN ENSDART00000058936 // LOC100004357 // secretory carrier-associated LOC10000 9.9E-05 -6.59 membrane protein 5A- 4357 NM_001089376 // stxbp1b // syntaxin binding protein 1b // --- // 557717 /// stxbp1b 2.0E-04 -6.54 BC171526 // NM_205729 // nr1d1 // nuclear receptor subfamily 1, group d, member 1 nr1d1 4.5E-05 -6.13 // --- // 494487 ENSDART00000134719 // prom1b // prominin 1 b // --- // 378834 /// prom1b 4.9E-06 -6.12 ENSDART00000102768 // ENSDART00000006897 // rlbp1a // retinaldehyde binding protein 1a // --- rlbp1a 2.2E-04 -6.09 // 393678 /// N ENSDART00000054735 // LOC558290 // synaptoporin-like // --- // 558290 LOC55829 8.3E-04 -5.88 /// XM_681491 // 0 ENSDART00000054322 // cnrip1b // cannabinoid receptor interacting cnrip1b 2.6E-05 -5.81 protein 1b // --- // ENSDART00000133035 // syt5a // synaptotagmin Va // --- // 436686 /// syt5a 3.5E-04 -5.46 ENSDART00000059197 XM_001334934 // LOC794903 // collagen alpha-4(VI) chain-like // --- // LOC79490 4.9E-05 -5.30 794903 3 XM_001335844 // LOC100000241 // uncharacterized LOC100000241 // --- LOC10000 3.3E-04 -5.28 // 100000241 0241 ENSDART00000145035 // saga // S-antigen; retina and pineal gland saga 1.0E-04 -5.26 (arrestin) a // --- // XM_003199754 // LOC100535278 // uncharacterized LOC100535278 // --- LOC10053 5.5E-04 -5.12 // 100535278 5278 ENSDART00000016753 // mag // myelin associated glycoprotein // --- // mag 6.4E-05 -5.10 474346 /// NM_001

ENSDART00000058773 // rgs16 // regulator of G-protein signaling 16 // -- rgs16 3.2E-05 -5.09 - // 569828 /// NM_001020546 // syt5b // synaptotagmin Vb // --- // 553567 /// syt5b 4.4E-04 -5.04 ENSDART00000013117 // sy ENSDART00000145835 // tmx3 // thioredoxin-related transmembrane tmx3 6.4E-06 -5.01 protein 3 // --- // 553 ENSDART00000105952 // aqp8a.2 // aquaporin 8a, tandem duplicate 2 // aqp8a.2 3.3E-04 -4.97 --- // 563130 /// ENSDART00000100287 // grk7a // G-protein-coupled receptor kinase 7a grk7a 7.1E-05 -4.93 // --- // 566120 // ENSDART00000086936 // IMPG2 (2 of 3) // interphotoreceptor matrix IMPG2 2.3E-05 -4.92 proteoglycan 2 // --- NM_001144131 // grin1b // glutamate receptor, ionotropic, N-methyl D- grin1b 2.2E-05 -4.90 aspartate 1b // -- ENSDART00000104353 // atp2b1b // ATPase, Ca++ transporting, plasma atp2b1b 1.1E-05 -4.85 membrane 1b // --- / NR_030507 // mir726 // microRNA 726 // --- // 100033737 mir726 2.6E-05 -4.84 ENSDART00000004619 // IMPG2 (3 of 3) // interphotoreceptor matrix IMPG2 4.1E-07 -4.82 proteoglycan 2 // --- ENSDART00000127706 // lrit1b // leucine-rich repeat, immunoglobulin- lrit1b 8.6E-06 -4.75 like and transmembr XM_001345079 // LOC100006333 // teneurin-2-like // --- // 100006333 LOC10000 3.0E-04 -4.75 6333 NM_200693 // arl3l1 // ADP-ribosylation factor-like 3, like 1 // --- // arl3l1 3.8E-04 -4.70 393666 /// BC07 ENSDART00000066501 // zgc:163073 // zgc:163073 // --- // 100037358 /// zgc:163073 8.7E-04 -4.70 NM_001089511 // ENSDART00000124154 // LOC100331665 // phenylserine dehydratase- LOC10033 7.2E-06 -4.68 like // --- // 100331665 1665 ENSDART00000125743 // slc25a3a // solute carrier family 25 slc25a3a 2.4E-04 -4.67 (mitochondrial carrier; phos BC127586 // zgc:158340 // zgc:158340 // --- // 780836 /// zgc:158340 8.7E-05 -4.60 ENSDART00000102312 // zgc:158 ENSDART00000145775 // LOC100535324 // MAGUK p55 subfamily LOC10053 6.6E-06 -4.41 member 4-like // --- // 10053 5324 ENSDART00000150894 // LOC100331226 // MAGUK p55 subfamily LOC10033 9.1E-07 -4.31 member 4-like // --- // 10033 1226 ENSDART00000014661 // glmnb // glomulin, FKBP associated protein b glmnb 1.9E-04 -4.29 // --- // 100170791 NM_001017822 // zgc:110204 // zgc:110204 // --- // 550520 /// zgc:110204 5.3E-04 -4.28 ENSDART00000014454 // zgc ENSDART00000076322 // zgc:171544 // zgc:171544 // --- // 561738 /// zgc:171544 3.1E-04 -4.23 NM_001114742 // zgc

Table 6 - Collective enriched Gene Ontology (GO) groups for leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf). Enrichment score and p- value is calculated by PARTEK Genomics Suite 6.6.

Function type Enrichment Enrichment p- Score value sensory perception of light stimulus biological process 32.2275 1.01E-14 visual perception biological process 32.2275 1.01E-14 RNA metabolic process biological process 18.1826 1.27E-08 ncRNA metabolic process biological process 17.4992 2.51E-08 ncRNA processing biological process 15.4201 2.01E-07 nucleobase-containing compound metabolic biological process 13.8171 9.98E-07 process nucleic acid metabolic process biological process 13.2709 1.72E-06 single-organism process biological process 13.0364 2.18E-06 rRNA processing biological process 12.7106 3.02E-06 regulation of cellular process biological process 12.5695 3.48E-06 cellular nitrogen compound metabolic process biological process 12.438 3.96E-06 phototransduction biological process 12.3485 4.34E-06 rRNA metabolic process biological process 12.2148 4.96E-06 heterocycle metabolic process biological process 12.1968 5.05E-06 cellular component organization or biogenesis biological process 11.9339 6.56E-06 protein-chromophore linkage biological process 11.9033 6.77E-06 cellular aromatic compound metabolic process biological process 11.8895 6.86E-06 cell differentiation biological process 11.7747 7.70E-06 detection of light stimulus biological process 11.5404 9.73E-06 regulation of transcription, DNA-dependent biological process 11.5312 9.82E-06 regulation of cellular macromolecule biosynthetic biological process 11.5139 9.99E-06 process regulation of RNA biosynthetic process biological process 11.4962 1.02E-05 regulation of macromolecule biosynthetic process biological process 11.4118 1.11E-05 regulation of cellular biosynthetic process biological process 11.3421 1.19E-05 regulation of biosynthetic process biological process 11.2751 1.27E-05 regulation of biological process biological process 11.2568 1.29E-05 ribosome biogenesis biological process 11.2201 1.34E-05 regulation of macromolecule metabolic process biological process 11.0502 1.59E-05 regulation of RNA metabolic process biological process 11.0486 1.59E-05 ribonucleoprotein complex biogenesis biological process 10.7552 2.13E-05 organic cyclic compound metabolic process biological process 10.6874 2.28E-05 regulation of gene expression biological process 10.6858 2.29E-05 chromatin modification biological process 10.5862 2.53E-05 neurological system process biological process 10.5468 2.63E-05 RNA processing biological process 10.4922 2.78E-05 cellular component biogenesis biological process 10.3191 3.30E-05

87 biological regulation biological process 10.2739 3.45E-05 sensory perception biological process 10.2335 3.59E-05 regulation of cellular metabolic process biological process 10.1454 3.93E-05 cellular macromolecule metabolic process biological process 10.1034 4.09E-05 transcription, DNA-dependent biological process 10.0437 4.35E-05 regulation of nucleobase-containing compound biological process 9.91475 4.94E-05 metabolic process regulation of nitrogen compound metabolic biological process 9.88464 5.10E-05 process axonogenesis biological process 9.77132 5.71E-05 nervous system development biological process 9.77066 5.71E-05 ribonucleoprotein complex assembly biological process 9.68911 6.20E-05 RNA biosynthetic process biological process 9.68866 6.20E-05 regulation of primary metabolic process biological process 9.61791 6.65E-05 cellular developmental process biological process 9.60116 6.76E-05 nitrogen compound metabolic process biological process 9.51782 7.35E-05 cell projection morphogenesis biological process 9.49118 7.55E-05 nucleobase-containing compound biosynthetic biological process 9.36462 8.57E-05 process Notch signaling pathway biological process 9.32715 8.90E-05 cell part morphogenesis biological process 9.22176 9.89E-05 neuron fate determination biological process 9.12505 0.000108903 neuron projection morphogenesis biological process 9.11147 0.000110392 multicellular organismal process biological process 9.09607 0.000112105 single-multicellular organism process biological process 9.09607 0.000112105 multicellular organismal development biological process 8.94748 0.000130065 cellular component morphogenesis biological process 8.78754 0.000152623 chromatin organization biological process 8.56773 0.000190143 cellular nitrogen compound biosynthetic process biological process 8.53327 0.000196811 cellular component organization biological process 8.46585 0.000210537 detection of stimulus biological process 8.36501 0.000232875 regulation of metabolic process biological process 8.33845 0.000239142 heterocycle biosynthetic process biological process 8.24561 0.000262409 aromatic compound biosynthetic process biological process 7.95212 0.000351917 ion transport biological process 7.82186 0.000400875 response to external stimulus biological process 7.81654 0.000403013 ribonucleoprotein complex subunit organization biological process 7.70327 0.000451351 nucleus cellular component 15.5335 1.79E-07 photoreceptor outer segment cellular component 15.2163 2.46E-07 primary cilium cellular component 14.1194 7.38E-07 nonmotile primary cilium cellular component 13.9578 8.67E-07 synapse part cellular component 13.1831 1.88E-06 postsynaptic membrane cellular component 11.8793 6.93E-06 synaptic membrane cellular component 11.3419 1.19E-05

88 synapse cellular component 9.62962 6.58E-05 nucleolar part cellular component 9.53931 7.20E-05 intracellular part cellular component 9.48514 7.60E-05 nuclear part cellular component 8.86269 0.000141574 cell part cellular component 8.83911 0.000144952 membrane-bounded organelle cellular component 8.61856 0.000180721 intracellular membrane-bounded organelle cellular component 8.61856 0.000180721 extrinsic to internal side of plasma membrane cellular component 8.45725 0.000212355 heterotrimeric G-protein complex cellular component 8.45725 0.000212355

GABA receptor activity molecular function 14.1194 7.38E-07 photoreceptor activity molecular function 13.9829 8.46E-07

GABA-A receptor activity molecular function 13.0828 2.08E-06 helicase activity molecular function 11.372 1.15E-05 extracellular ligand-gated ion channel activity molecular function 10.2187 3.65E-05 ATP binding molecular function 9.57253 6.96E-05 adenyl ribonucleotide binding molecular function 9.47442 7.68E-05 adenyl nucleotide binding molecular function 9.44188 7.93E-05 sodium:dicarboxylate symporter activity molecular function 8.37773 0.000229932 anion:cation symporter activity molecular function 7.98081 0.000341962 signal transducer activity molecular function 7.97972 0.000342336 molecular transducer activity molecular function 7.97972 0.000342336 protein binding molecular function 7.90203 0.000369992

Table 7 - Collective enriched KEGG Pathway Analysis for leptin –A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf). Enrichment score and p value is calculated by Genomics Suite 6.6.

KEGG ID Pathway Name Enrichment enrichment score p value kegg_pathway_33 Phototransduction 13.8398 9.76E-07 kegg_pathway_42 Notch signaling pathway 10.5644 2.58E-05 kegg_pathway_18 Ribosome biogenesis in eukaryotes 8.9015 0.000136184 kegg_pathway_91 RNA transport 5.74185 0.00320883 kegg_pathway_25 Neuroactive ligand-receptor interaction 4.58091 0.0102456 kegg_pathway_82 Aminoacyl-tRNA biosynthesis 4.34505 0.0129708 kegg_pathway_78 Calcium signaling pathway 3.33475 0.0356236 kegg_pathway_49 Vascular smooth muscle contraction 2.73359 0.0649854 kegg_pathway_34 Dorso-ventral axis formation 2.50516 0.0816627 kegg_pathway_4 Purine metabolism 2.2678 0.10354 kegg_pathway_26 Melanogenesis 2.01323 0.133557 kegg_pathway_64 One carbon pool by folate 1.93906 0.143839 kegg_pathway_43 Cell adhesion molecules (CAMs) 1.86583 0.154768 kegg_pathway_119 Cytosolic DNA-sensing pathway 1.82853 0.16065 kegg_pathway_107 Taurine and hypotaurine metabolism 1.7837 0.168015 kegg_pathway_27 Fatty acid biosynthesis 1.7837 0.168015 kegg_pathway_70 Butirosin and neomycin biosynthesis 1.7837 0.168015 kegg_pathway_17 Mucin type O-Glycan biosynthesis 1.48766 0.225901 kegg_pathway_80 Sulfur relay system 1.42223 0.241175 kegg_pathway_90 p53 signaling pathway 1.41668 0.242519 kegg_pathway_133 Wnt signaling pathway 1.37013 0.254075 kegg_pathway_40 RNA polymerase 1.2254 0.293639 kegg_pathway_117 Phosphatidylinositol signaling system 1.19339 0.303192 kegg_pathway_35 mTOR signaling pathway 1.12752 0.323834 kegg_pathway_38 Pyrimidine metabolism 1.12265 0.325415 kegg_pathway_66 ABC transporters 1.1168 0.327326 kegg_pathway_56 Endocytosis 1.10784 0.330271 kegg_pathway_112 Primary bile acid biosynthesis 1.08148 0.339092 kegg_pathway_88 Hepatitis B 1.02335 0.359388 kegg_pathway_85 Gap junction 1.01716 0.361619 kegg_pathway_149 Selenocompound metabolism 0.997357 0.368853 kegg_pathway_93 alpha-Linolenic acid metabolism 0.997357 0.368853 kegg_pathway_132 Starch and sucrose metabolism 0.97555 0.376985 kegg_pathway_95 Lysine degradation 0.906744 0.403837 kegg_pathway_76 Alanine, aspartate and glutamate metabolism 0.893417 0.409255 kegg_pathway_131 Spliceosome 0.824065 0.438645 kegg_pathway_98 Hedgehog signaling pathway 0.767489 0.464177

90 kegg_pathway_134 Steroid biosynthesis 0.744123 0.475151 kegg_pathway_94 Glycosaminoglycan degradation 0.695518 0.498816 kegg_pathway_36 ECM-receptor interaction 0.650574 0.521746 kegg_pathway_39 Fanconi anemia pathway 0.636504 0.529139 kegg_pathway_127 Progesterone-mediated oocyte maturation 0.623666 0.535976 kegg_pathway_99 Biosynthesis of unsaturated fatty acids 0.610628 0.54301 kegg_pathway_7 NOD-like receptor signaling pathway 0.586162 0.556459 kegg_pathway_23 Galactose metabolism 0.573355 0.563631 kegg_pathway_47 Cell cycle 0.5733 0.563662 kegg_pathway_147 Regulation of autophagy 0.507267 0.602139 kegg_pathway_118 Intestinal immune network for IgA production 0.507267 0.602139 kegg_pathway_62 Protein processing in endoplasmic reticulum 0.497977 0.607759 kegg_pathway_1 Oocyte meiosis 0.492261 0.611243 kegg_pathway_72 mRNA surveillance pathway 0.397622 0.671916 kegg_pathway_106 Butanoate metabolism 0.379419 0.684259 kegg_pathway_75 Arginine and proline metabolism 0.376392 0.686333 kegg_pathway_73 beta-Alanine metabolism 0.358769 0.698536 kegg_pathway_16 MAPK signaling pathway 0.340182 0.711641 kegg_pathway_155 Drug metabolism - other enzymes 0.339437 0.712171 kegg_pathway_60 ErbB signaling pathway 0.337392 0.713629 kegg_pathway_124 Porphyrin and chlorophyll metabolism 0.321317 0.725193 kegg_pathway_65 Herpes simplex infection 0.30047 0.74047 kegg_pathway_115 RNA degradation 0.274484 0.759964 kegg_pathway_44 VEGF signaling pathway 0.253759 0.775879 kegg_pathway_19 Fatty acid metabolism 0.245874 0.782021 kegg_pathway_125 Propanoate metabolism 0.245874 0.782021 kegg_pathway_96 Cardiac muscle contraction 0.244001 0.783487 kegg_pathway_103 Peroxisome 0.244001 0.783487 kegg_pathway_54 Adipocytokine signaling pathway 0.234623 0.790869 kegg_pathway_111 Jak-STAT signaling pathway 0.234564 0.790916 kegg_pathway_130 Cysteine and methionine metabolism 0.233323 0.791898 kegg_pathway_22 Fructose and mannose metabolism 0.221482 0.80133 kegg_pathway_129 Tight junction 0.22077 0.801901 kegg_pathway_13 Basal transcription factors 0.210305 0.810337 kegg_pathway_83 Pyruvate metabolism 0.210305 0.810337 kegg_pathway_128 GnRH signaling pathway 0.205306 0.814398 kegg_pathway_116 N-Glycan biosynthesis 0.199747 0.818938 kegg_pathway_156 Circadian rhythm 0.189768 0.827151 kegg_pathway_92 Nucleotide excision repair 0.162951 0.849633 kegg_pathway_46 Adherens junction 0.158632 0.85331 kegg_pathway_30 Amino sugar and nucleotide sugar metabolism 0.154948 0.85646 kegg_pathway_136 Cytokine-cytokine receptor interaction 0.141814 0.867783 kegg_pathway_58 Natural killer cell mediated cytotoxicity 0.141051 0.868445

91 kegg_pathway_109 Focal adhesion 0.140695 0.868754 kegg_pathway_113 Insulin signaling pathway 0.140531 0.868897 kegg_pathway_37 RIG-I-like receptor signaling pathway 0.126899 0.880823 kegg_pathway_101 Apoptosis 0.125403 0.882141 kegg_pathway_159 Inositol phosphate metabolism 0.109419 0.896355 kegg_pathway_139 PPAR signaling pathway 0.08995 0.913977 kegg_pathway_24 Lysosome 0.066736 0.935442 kegg_pathway_102 Glycolysis / Gluconeogenesis 0.064064 0.937945 kegg_pathway_110 Glycerophospholipid metabolism 0.052848 0.948524 kegg_pathway_53 Phagosome 0.05051 0.950744 kegg_pathway_20 Oxidative phosphorylation 0.029955 0.970489 kegg_pathway_84 Toll-like receptor signaling pathway 0.024625 0.975676 kegg_pathway_55 TGF-beta signaling pathway 0.023483 0.976791 kegg_pathway_6 Energy Metabolism 0.009102 0.990939 kegg_pathway_12 Regulation of actin cytoskeleton 0.007275 0.992751 kegg_pathway_15 Ubiquitin mediated proteolysis 0.006838 0.993185 kegg_pathway_114 Metabolic pathways 0.001941 0.998061

Discussion

The primary goal of this study was to identify metabolic genes and pathways regulated at the transcriptional level by leptin-A in the zebrafish embryo. The leptin knockdown morphotype has previously been described (Liu et al., 2012) along with some of its physiological effects (Chapters 2 and 3). In this study, oligonucleotide microarrays were used to quantify the transcriptomic response to leptin knockdown (low leptin). This is the first application of oligonucleotide microarrays to leptin signaling in fish.

Leptin modulates neuronal and cellular processes

Because of leptin’s obvious effects on lipid metabolism, we expected the primary effect of leptin A knockdown to be metabolic. However, the most dramatically affected pathways were developmental and sensory (Figures 10-12, Tables 6-7). Leptin’s role in

92 neuronal development is documented in mammalian systems (Bouret et al., 2004, Harvey et al., 2005). Several genes identified in this study suggest a developmental role for leptin

A in fish. Synaptotagmins (Va, Vb, and Ia), synaptophysin, and syntaxin were down regulated, along with negr1 and glutamate receptors (grin1a and -2a). These genes are directly implicated in neuronal outgrowth and cell sensing (Hudson and Birnbaum, 1995;

Alladi et al., 2002; Schwartz et al., 2012). Negr1 is highly expressed in the hypothalamus and modulates neuron density (Lee et al., 2011; Walley et al., 2011). Negr1 is also implicated as a putative novel biomarker for obesity (O’Rahilly, 2009). Furthermore, syntaxins have reduced expression in ob-/ob- and db-/db- mice that can be rescued by leptin administration (Ahima et al., 1999a). Together, these data support the hypothesis that leptin plays a role in sensory development of both fish and mammals, however the relative contribution in fish may be higher as sensory and developmental genes and pathways were the most significantly impacted.

The leptin morphant phenotype is the result of a dramatically altered development

(Liu et al., 2012; Dalman et al., 2013; Londraville et al., 2014), and several developmental genes changed expression with leptin knockdown. Hoxc1a, hoxb8b, vsx2, dbx1a, dbx1b, 1bx1b, irx5b, hoxc4a, hoxb2a, pou3f1, hoxbba, and hoxb4a were all up regulated in response to leptin knockdown, and all influence body planning along the anterio-posterior axis (Kaji and Nonogaki, 2013; DeCarvalho et al., 2014). Irx5b is required for normal development of the heart (Kim et al., 2012; Mirzoyan and Pandur,

2013) and morphant zebrafish have compromised heart function (Chapter 2). Brip1 interacts with BRCA1 to facilitate DNA repair (Litman et al., 2008), and is up regulated in leptin A morphants, as is Smn1 which is involved in neuronal migration and

93 differentiation (Zanetta et al., 2014). Pescadillo (up regulated in morphants) is directly involved in embryonic development, has been previously identified in zebrafish, and is up regulated in malignant human astrocytomas (Allende et al., 1996; Li et al., 2009). It has also been implicated in ribosome biogenesis, and contains a BRCA1 C-terminal protein-protein interacting domain (Holzel et al., 2007). It is highly expressed during early development, and null mutations have small eyes, impaired brain development, and gastrointestinal malfunctions (Kinoshito et al., 2001). Leptin A morphants display decreased eye development, enlarged pericardial edema, and disrupted brain and ear development (Liu et al., 2012; Dalman et al., 2013; Londraville et al., 2014). The upregulation of many developmental genes such as pescadillo and irx5b suggests at least some developmental genes are sensitive to reduced leptin signaling, therefore stimulating changes in expression in the leptin morphants. For example, reduced development/angiogenesis may have in impact on nutrient supply and waste removal from tissues, hindering gene expression (Mathavan et al., 2005) and organ development

(Mendelsohn et al., 2008). Conversely, faimb expression was down regulated in leptin morphants and it counteracts Fas-mediated apoptosis when overexpressed (Schneider et al., 1999). Overexpression leads to enhanced neurite and sympathetic neuron growth

(Sole et al., 2004), and may play a role in obesity-related pathologies (Schmid et al.,

2012). I interpret the response of these developmental genes to leptin A manipulation as either the proximate cause of the developmental anomalies or an attempt by the embryo to compensate for them. It should also be noted that the specific developmental window chosen for this analysis is actively modulating developmental transcripts (Mathavan et al., 2005). Therefore, additional transcriptomic analyses during both early and late stages

94 of development should be pursued to observe whether changes observed are a byproduct of development.

Leptin knockdown significantly up regulated multiple aminoacyl tRNA synthetases (aars, iars, eprs, rars, vars). Aminoacyl tRNA synthetases recharge tRNAs with amino acids and thus are critical for protein expression (Woese et al., 2000).

Increased oxidative stress can impair translation via defective aminoacyl tRNA synthetases (Ling and Soll, 2010) thus reducing protein synthesis (Harding et al., 2000).

Leptin morphants show significantly reduced oxidative capacity (chapter 2) and reduced catalase function implying less oxidative metabolite formation. In combination with the small size of zebrafish and reduced oxidative function (enzymes and cardiac output; see

Chapter 2), these data can be interpreted as either a true lowered oxidative stress or conversely an increased oxidative load that cannot be matched. We would need to independently determine respective differences to fully interpret these changes. Once again, changes in nutrient and oxygen supply, waste removal, and stress signaled via the hypothalamic pituitary axis (HPA) may also be adding to the observed effect of leptin knockdown on metabolic activity (Spiegel et al., 2004a; Mendelsohn et al., 2008).

Additional assays as well as more time points can clear up whether these physiological responses are correlated with or a cause of the phenotype observed in leptin morphants.

Tcirg1 expression is negatively regulated by leptin A knockdown (Table A1;

Table 5). Tcirg1 is a Vacuolar H+-ATPase pump and is involved in the acidification of bone at the ruffled border by osteoclasts (Boyce et al., 2009; Boyce et al., 2012).

Inactivation results in reduced bone turnover. Autosomal mutations in this gene result in osteopetrosis (abnormally dense bones; Castro Domingoes et al., 2005; Bruder et al.,

2003; Ferron et al., 2010). Genome chip experiments in rats indicate that inhibition of leptin signaling causes decreased ossification and bone mineralization, while increasing reabsorption (Zhang et al., 2013b). Leptin A morphants share similar reduced ossification with no otolith development (Liu et al., 2012; Londraville et al., 2014). Conversely, when leptin is readministered, it binds to receptors in the hypothalamus, and through acting on the sympathetic nervous system (SNS), beta adrenergic receptors signal osteoblast activation of bone formation (Harada and Rodan, 2003; Elefteriou et al., 2004; Takeda et al., 2002). Thus based on this array and phenotypic observations (Londraville et al.,

2014), leptin’s impact on bone growth may share similar aspects to leptin deficient mammals (Hamrick et al., 2004; 2005).

Leptin modulates fatty acid oxidation

Ob-/ob- mice show decreased metabolic rate, lipolysis, and increased adipose storage along with increased food intake (Halaas et al., 1997; Ahima and Flier, 2000a).

Our hypothesis was that we would see similar effects in the zebrafish knockdown model.

Enzyme activities of β-oxidation, such as 3-hydroxyacyl-Coenzyme A dehydrogenase

(HOAD) and carnitine palmitoyltransferase (CPT) are reduced in the leptin morphants, despite their gene transcripts not meeting our selection criterion of 1.5 FC and p value <

0.05 (Figure. 14, Table A1).

A primary transcript involved in fat utilization, acyl-Coenzyme A oxidase 3

(acox3) was down regulated within the array and is involved in fatty acid beta-oxidation.

It catalyzes the first rate-limiting step in peroxisomal beta-oxidation of fatty acids. It is expressed ubiquitously throughout the embryo and found in the liver and intestines of adult fish (Morais et al., 2007). Furthermore, its expression increases in response to

96 feeding in both zebrafish and rainbow trout and the array confirms leptin morphants lack of lipid utilization (Morais et al., 2007). The biological roles of acyl-CoA oxidase

(acox3), HOAD, and thiolase have been identified in mice, and a homozygous mutation for acox3 results in viable but infertile mice with growth retardation (Fan et al., 1996).

This lack of growth has been observed in leptin morphant zebrafish (Liu et al., 2012) as well, and in newborn humans with low serum leptin (Jaquet et al., 1998). Interestingly, transcriptome analysis of ob-/ob- mouse liver found that leptin repletion increased beta- oxidation in liver compared to adipose (Liang and Tall, 2001). Thus fatty acid oxidation and lipid utilization in zebrafish embryos is significantly down regulated in fish, as it is in leptin deficient mammals.

Leptin’s impact on metabolism

Leptin is expressed by adipose tissue in mammals (Zhang et al., 1994), yet its highest expression is found in liver and gonads in fish (Liu et al., 2012). Leptin morphants have decreased capacity to modify lipid size (Figure 8; Liu et al., 2012).

Markers of aerobic oxidation (CPT, HOAD, CS) are significantly reduced with anaerobic capacity (LDH) increased (Figure 18). Catalase enzyme activity was also down regulated in leptin morphants and may be in response to lower production of hydrogen peroxide

(Figure 18). The enzyme data corroborate the microarray with the exception of lactate dehydrogenase (Figure 18). There are several isoforms of LDH in vertebrates (Markert and Faulhaber, 1965) and the only ones that were significantly down regulated in this study were ldha and ldhd (-1.67 and -3.05, respectively). Either LDH transcripts are out of phase with active protein concentrations, or certain transcripts contribute disproportionately to overall activity.

Other metabolic effects include the responses of agl and oprm1. Agl (amylo- alpha-1, 6-glucosidase, 4-alpha-glucanotransferase) was significantly down regulated in response to leptin knockdown. Agl catalyzes glycogen debranching/degradation

(Goldstein et al., 2010). Mutations in this gene cause glycogen storage disease (GSD) in humans with incomplete glycogenolysis and accumulation of small-chained glycogen resembling dextrins (Goldstein et al., 2010). Opiod receptor mu 1 (oprm1) was down regulated in leptin morphants. Mu opiod receptors (oprm1) are involved in orosensory reward to highly palatable food (Lynch and Libby, 1983) and leptin affects signaling in brain reward centers (Stice et al., 2013; Lim et al., 2014). Stimulation of mu opioid receptors increases intake of calorically dense food (Barnes et al., 2006). Mu opioid receptors are highly expressed in rats susceptible to diet-induced obesity (Zhang et al.,

1998). The reduced expression of mu opiod receptors and agl suggests that leptin knockdown not only decreases oxidative phosphorylation and increases lipid retention but that lipid is not properly identified by the reward centers in the central nervous system.

Leptins impact on phototransduction and sensory perception

The most significantly affected GO categories were related to visual and sensory perception, and the most significantly affected KEGG pathway was phototransduction (P

=9.76E-07; Tables 6 and 7). Most genes related to phototransduction were opsin related

(opsin 1 short-wave-sensitive 1 (opn1sw1), opsin 1short-wave-sensitive 2 (opn1sw2), opsin 1 medium-wave-sensitive 1 (opn1mw1), opsin 1long-wave-sensitive 2 (opn1lw2), opsin 3 (opn3), opsin 4.1(opn4.1), and opsin 4b (opn4b) as well as rhodopsin (rho), peripherin genes (prph-2a, -2b, -2l), and pde6h (phosphodiesterase 6H, cGMP-specific,

98 cone) which are all expressed in the retina and involved in the inhibitory transmission and amplification of visual signals (Jovanovic et al., 2011). These genes were all down regulated in response to leptin knockdown, and among the most significantly affected of all genes (Tables 4, 5, Table A1). Our initial description of the zebrafish leptin knockdown morphotype (Liu et al., 2012) showed significant alterations in eye development. Leptin’s role in sensory perception and phototransduction in nonmammals has received little attention and may be important for detecting photoperiod and thus metabolic cues.

There is an established link between light-dark cycles, color perception, and hormonal release of leptin and ghrelin (Spiegel et al., 2004a, 2004b; Schmid et al., 2008) in mammals. As leptin levels are low in the morning and generally peak during the day, sleep deprivation (5 hours of sleep compared to 8) reduces these circulating levels

(Figueiro et al., 2012). Furthermore, sleep deprivation in combination with awaking to specific light spectra (ie. red, green, or blue) could rescue effects of sleep deprivation and circulating leptin titer to control (Figueiro et al., 2012). Based on the data from this study and what is known in mammals, leptin may have long-term effects on circadian rhythm, color perception, and food intake in non-mammals.

Leptin knockdown and its similarities to diet induced obesity (DIO) and leptin deficency

Microarray studies have been used to uncover signaling pathways affected by food (Moraes et al., 2003; Lopez et al., 2003) and leptin manipulation (Soukas et al.,

2000) in mammals. Based on conserved sequence of leptin and its receptor across many vertebrates species, we hypothesized that leptin manipulation in the zebrafish would also

99 share a similar transcriptomic response to what is observed in mammals. Within my study

I found overall metabolism reduced, including lipolysis and other oxidative functional transcripts. At a combined selection criteria of 1.5FC and FDR <0.05, over 1300 genes were significantly affected. Leptin’s most well characterized pathways in mammals have been its impact on metabolism and lipolysis.

At one end of the spectrum in mice, increased leptin titer (hyperleptinemia) as a result of diet induced obesity (DIO), affected less than 500 genes and 70% of those genes were down regulated (Moraes et al., 2003). Additionally, body fat and mass almost doubles (Moraes et al., 2003). DIO mice most significantly impacted pathways were almost entirely involved in inflammation whereas the most negatively affected transcipts were involved in structural functions genes such as smooth muscle calponin gene, keratin complex 1, smoothelin, cytokeratin endo A or keratin 8, and myosin heavy chain 11. Of note, several genes involved in lipid metabolism were down regulated including fatty acid synthase (-1.3FC), GAPDH (-1.6FC), and glycerol kinase (-2.5FC) in DIO mice.

Similar observations of small genes sets have been observed in DIO rats (Lopez et al.,

2003). Interestingly in DIO rats, several markers of lipid use were up regulated including leptin (49.2 FC), uncoupling proteins (2.0 FC), and fatty acid binding protein (15.7 FC).

In DIO rats, some of the most up regulated pathways were involved in macronutrient metabolism, transcription factors, and cellular cytoskeleton (Lopez et al., 2003).

Additionally, redox and stress related genes were down regulated along with several hormone and signal transduction genes such as proenkephalin, inositol triphosphate receptor subtype 3 (IP3R-3), Phosphatidylinositol 3-kinase p85 alpha subunit, and MAP kinase kinase 1 (MEKK1). The discrepancy between DIO mice and rats may be a result

100 of spatiotemporal sampling or a species-specific response. Collectively, however, both arrays point to metabolic function and stress-related transcripts as the most enriched pathways.

Conversely, leptin deficient (~hypoleptinemia) mammals have a very small subset of genes that are affected (<500; Soukas et al., 2000) which shares similarity with DIO

(Lopez et al., 2003; Moraes et al., 2003). Leptin deficient mammals down regulate fatty acid and cholesterol biosynthesis in white adipose tissue. Leptin A morphants share similar reductions in metabolic function however they were not the most significantly enriched pathways (Soukas et al., 2000; Lopez et al., 2003; Moraes et al., 2003).

Additionally, leptin deficiency in mammals results in several inflammatory markers and acute phase proteins along with markers expressed on macrophages, including macrophage metalloelastase and macrophage specific cysteine-rich TM glycoprotein to increase and were surprisingly some of the top regulated transcripts. This increased inflammatory response is not observed in our leptin-A morphants. We postulate that a lack of enriched inflammatory markers may have to do with a lack of adaptive immune response presence during early zebrafish development and/or the sampling differences in adult versus embryonic zebrafish (i.e. kidney versus whole animal, respectively). To tease out the specific impact leptin has on these systems, ob-/ob- Tg mice (express 50% less leptin than wildtype) were used. These mutants are able to correct many of these immune related transcripts to wildtype levels but cannot rescue metabolic related transcripts (Soukas et al., 2000). Furthermore, ob-/ob- mice given daily leptin infusion are not able to rescue many of these inflammatory markers that are rescued by subnormal

101 leptin levels (ob-/ob- Tg mice). This suggests that developmentally, leptin signaling is complex and is not merely an on/off switch.

Leptin A morphants share similar affected pathways to mammals including metabolism and fatty acid utilization. These pathways were consistently some of the most enriched pathways in mammalian studies but were not in our study. In both DIO and ob-

/ob- models, inflammatory and immune related transcripts were significantly enriched and were also some of the most top affected transcripts but were interestingly not in our study. Within this study, the most affected pathways were involved in organismal development and sensory perception. Phototransduction was the most significantly enriched pathway, which is in stark contrast to both leptin deficient and DIO mammalian models. My study does corroborate leptin’s role in influencing metabolically intensive functions both at the transcript and enzymatic level. Nonmammalian leptin transcriptomic studies deviate here, showing significant alterations in sensory and developmental transcripts. Thus my data supports the leptin A morphant model as an excellent comparative system and point future studies to uncover the relative contributions of leptin isoforms as well as both upstream and downstream signaling molecules.

102

CHAPTER V

FOLD CHANGE AND P-VALUE CUTOFFS SIGNIFICANTLY ALTER

MICROARRAY INTERPRETATIONS

Dalman, M.R., Deeter, A., Nimishakavi, G., and Duan, Z.-H. (2012). Fold change and p-

value cutoffs significantly alter microarray interpretations. BMC Bioinformatics

13, S11.

Introduction

As more and more genomes are sequenced and annotated, the capacity to accurately and efficiently catalog the gene expression profiles of these organisms is becoming ever more apparent (Allison et al., 2006). With techniques such as in situ hybridization, QRT-PCR, and more recently absolute quantitation being used to assess gene expression, there are still lingering issues of minimal throughput and lack of massive parallel comparisons. Array technology has improved these conditions yet problems of standardizing statistical analyses are lacking, along with observed differences when comparing microarray platforms (Mah et al., 2003), though others have found significant reproducibility (MAQC Consortium, 2006).

Oligonucleotide arrays, for example, prove useful in not requiring cDNA library production (Enard et al., 2002), while cDNA microarray proves useful in cases of nonmodel organism and even been used to identify heterologous genes across multiple

103 species (Renn et al., 2004). Even so, the current microarray platforms are still several years behind the current state of knowledge of many organisms genome. Furthermore, classifying a differentially regulated gene is a problem of both array types with research even suggesting it should be dealt with in a tiered approach (Miller et al., 2001).

To assess the power of analysis, there are many ways when using expression data

(Jeffery et al., 2006; Witten and Tibshirani, 2007; Lin et al., 2010). The use of t-tests,

ANOVAs, Gene Ontology (GO) annotation, p-value cutoffs, Bonferroni corrections, array normalization, Fishers exact test, and fold change cut offs all lead towards a reduction in gene expression data which may inadvertently reduce or increase the power of analysis.

We obtained a data set from a recently published paper (Marques et al., 2008) and reanalyzed the raw data using multiple different approaches. From this data analysis, we hypothesized that by changing the significance level as well as the fold change cut off, more than one interpretation of the data can be obtained. Currently, there is only one microarray study on heart tissue response to hypoxia. Additionally, this study is analyzed using one of many selection criterion putatively perputating it is the sole means of data interpretation for that treatment. Essentially, the novelty of this study in light of Marques et al. (2008) is for future studies to unravel which significance criteria are relevant, biological and/or statistical.

Methods

In a previously published paper in which adult zebrafish heart tissue was assayed in response to chronic hypoxia (Marques et al., 2008), microarray files were downloaded from NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) (including

104 accession numbers GSM112796 and GSM112798 through 806). Raw files were imported into GeneSpring GX Version 11.0 (Silicon Genetics, Redwood City, CA) and intensities normalized using MicroArray Suite 5 method (Fujita et al., 2006). Due to small sample size, equal variance across data could not be assumed and data was analyzed using an unpaired T-test with unequal variance with no statistical corrections (Ruxton et al., 2006).

Results and discussion

Microarray technology has proven beneficial to directly identifying co-regulated genes, pathways, and systems allowing for a more informed snapshot of the transcriptome. As such, our results indicate that changes in the significance level of differential expressed gene products along with the fold change cut-offs can give very different results that imply different signaling pathways and functions involved (Figure

19). As T-tests have been widely used to identify deviation from the mean, large sampling sizes (~15,000 genes assayed) can influence the number of false positives and may infer little if anything about the biology (Lin et al., 2010; Nadon and Shoemaker,

2002). Fold change on the other hand lends itself to a more biologically meaningful assessment yet still encounters problems with identifying what is significant to the organism. Therefore, using both criteria may help but not fix the problem of microarray analysis.

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Figure 19. Differentially regulated genes for GO Annotation categories. A. Cellular component B. Molecular function C. Biological Function. Black shaded blocks are the intersection of genes with p-values ≤ 0.02 and fold change cutoff of ≥2.0. Gray shaded blocks are the intersection of genes with p-values ≤ 0.05 and fold change cutoff of ≥1.5. The genes found in the dark shaded blocks are also included in the number of genes in the gray shaded block. Categories are directly taken from the second level of GO annotation from GeneSpring.

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Within this study, the contributions of each set of differentially expressed gene criteria were evaluated. As indicated before, the criteria suggest different biological meaning. The number of significant genes are overwhelming at p ≤ 0.05 and even upon increasing to a p ≤ 0.02 level, the data are reduced almost in half yet still remains massive for understanding the biological response to hypoxia (Table 8). Fold change suggests more meaningful insight to the organism throughout development and into adulthood (McCarthy and Smyth, 2009) with 1.5 proving to be a better eliminator of background noise as there were fewer genes left after making a fold change cutoff of ≥1.5 as compared to using significance cutoffs (Table 8). As the fold change level increases to that of ≥2, the number of genes significantly decreases. This suggests that biologically, less genes change drastically and that the significant difference observed at p ≤ 0.05 and

0.02 are related to a possible whole animal response to treatment. To understand the change and ultimately the importance in interpretation of genes influenced by chronic constant hypoxia, an intersection of ≥1.5 fold change and p ≤ 0.05 was compared to ≥2 fold change and p ≤ 0.02. The data obtained from our analysis indicates that there were more up regulated genes as compared to down regulated, implying that even in a state of stress the organism is actively adjusting its transcriptome and, more specifically, the transcriptome of the heart (Figures 15).

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Table 8 - Genes categorized by both fold change and p-value in response to chronic constant hypoxia. The data presented is before any post normalization filtering.

Using the most stringent criteria (intersection of p ≤ 0.02 and ≥2 fold change), there were several genes that were up regulated and observed in both intersection groups

(Figures 15). Hypoxia inducible factor 1 was found to be up regulated and is known to be expressed in response to hypoxic conditions (Jacob et al., 2002). Interestingly, chemokine

(C-X-C motif) ligand 12a stromal cell derived factor 1 was only observed under ≥1.5 fold change and p ≤ 0.05 conditions. CXCL12 is involved in directing hematopoietic cells and angiogenesis (Askari et al., 2006). By omitting this chemokine from the biological interpretation, clinical researchers may overlook a corollary between tumor progression

(which requires oxygen) and ischemia, a state of desperate need for oxygen and nutrients.

Other genes such as bone morphogenetic protein 2a and insulin-like growth factor binding protein 1a were observed in each selection criteria indicating they are significant and change more than two fold in response to chronic constant hypoxia. IGFBP-1A has been known to be involved in regulating IGF and insulin pathways which can be linked to cell proliferation and protection against cell death along with BMP-2 involved with bone development (Kahn et al., 2004; Scarth, 2006).

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Those genes down regulated at the most stringent selection criteria show similar reductions to those observed by Marques et al. (2008). Metabolically related genes, such as acyl coenzyme A dehydrogenase, pyruvate dehydrogenase kinase, SOCS3, and creatine kinase indicate that a shift to oxygen independent metabolism is occurring.

These genes are involved in fatty acid oxidation, pyruvate oxidation, cytokine signaling disruption, and the rapid shuttling of ATP sources, respectively. Interestingly, changes both at a metabolic level as well as at a synaptic level were observed as synaptotagmin, uncoupling protein 2, and ATPase,

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Figure 20. Comparative fishers exact test and fold enrichment for significant GO groups within the zebrafish total gene array

Ca2+transporting, cardiac muscle, fast-twitch 1 like gene were found to be significantly down regulated indicating that mitochondrial membrane leak was reduced and calcium initiated cellular signaling may be attenuated in cardiac tissue (Figure 19). However, some genes not identified under the most stringent criteria and counter to ischemia/

110 reperfusion, are superoxide dismutase, heat shock protein 90, and lactate dehydrogenase

D. This suggests the response to chronic constant hypoxia may be tissue specific as these genes should be up regulated under hypoxic conditions (Iacobas et al., 2008). SOD has been found to be crucial in the breakdown of reactive oxygen species while HSP are used for stabilizing proteins under stressful conditions (Isaacs et al., 2003). Other genes such as programmed cell death 8 (apoptosis-inducing factor) and RAS association

(RalGDS/AF-6) domain family 8 were down regulated and differentially expressed but were not found under most stringent statistical criteria. Both of these genes are involved in cellular processes important to the survival of the cell or its interaction with other cells, respectively. As zebrafish have developed ways to respond and survive under hypoxic conditions, AIF-8 and RAS may bring insight into cellular mechanisms responsible for cell survivability and cell rigidity (van der Weyden and Adams, 2007). Thus understanding the integrative response to chronic constant hypoxia is beginning to look more and more like the response observed in tumorigenesis, which may be overlooked based on selection criteria of genes.

Using fold enrichment analysis and fishers exact tests of all sets in comparison to originally published, we found that though our most stringent criteria were similar to

Marques et al (2008), we had distinct changes in GO annotation groups involved in microtubule activity, hydrolase activity, nucleic acid binding and carbohydrate binding

(Figure 20). Interestingly, reducing the criteria down to 1.5 FC and p≤ 0.05, several different GO groups were found to be significant especially those involved in structural integrity, ribosome structure, and transcription regulation and factors, suggesting these may not vary widely in expression but that there are more significant genes thus pointing

111 towards the problems of microarray analysis (Figures 15 and 16). On one hand, transcriptomic studies revere novelty in research while on the other hand replication and accuracy to natural phenomena is very low, resulting in potentially skewed explanations.

We assert that reanalysis of microarray data is necessary along with follow up gene expression studies to accurately explain biological phenomena.

Conclusions

Our results point to the pitfalls of statistical selection criteria and shed light on genes crucial to understanding chronic constant hypoxia. As more genes are sequenced, microarray platform expanded, along with understanding the role of splice variants in zebrafish (D. rerio), a larger and more dynamic picture of the transcriptome can be gathered. We demonstrate how fold change and statistical cut-offs modulate the outcome of microarray data. Future studies should tie together previously unanalyzed genes such as leptin to microarray data in response to constant hypoxia in adult zebrafish. As the current microarray platform does not include them, problems still exist at the molecular level, influencing our understanding of the physiological and ultimately behavioural and ecological roles of these organisms.

Acknowledgements

This was supported in part by Choose Ohio First Bioinformatics Scholarship grant and was published in BMC Bioinformatics Volume 13 Supplement 2, 2012: Proceedings from the Great Lakes Bioinformatics Conference 2011. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/13/S2

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CHAPTER VI

CONCLUSIONS AND FUTURE DIRECTIONS

The Power of a Comparative Approach

Using a comparative approach, I posed questions as to whether a portion of physiological systems are conserved between mammals and non-mammals and in doing so, I hope to gain insight into how leptin function evolved. I assert this approach will inform how we understand leptin function in nonmammals and help to uncover previously unknown functions in mammals. For example, much of what we know about neurophysiology stems from studying the giant squid axon (Hodgkin and Huxley, 1939), our understanding of heritable genetic traits and development of the nervous system were advanced by the use of D. melanogaster (Morgan, 1910; Rubin and Lewis, 2000; Bellen et al., 2010), and our understanding of osmoregulation was advanced by the use of shark rectal gland (Burger and Hess, 1960). These comparative approaches accelerated our understanding of mammalian systems and have driven new discoveries in human biology.

My primary focus was to test leptin function using our leptin knockdown model and compare it to the well-characterized impacts on metabolism, immune function, and transcription in mammals. I characterized the leptin knockdown phenotype using respiration and cardiac output, immunocompetence indices (respiratory burst, survivability, and bacterial load clearance), and transcriptomic analyses (microarray on mRNA transcripts present). Together, these physiological and transcriptomic data reveal

113 how manipulation of leptin A expression identifies pathways that are functionally conserved with mammals while also highlighting distinct differences from mammals.

I measured zebrafish metabolic rate using traditional and high-throughput techniques (Chapter 2). I hypothesized that leptin knockdown in zebrafish mirrors a state of energy deficency as it does in leptin knockout mammals. Therefore, energetically costly functions are down regulated in animals with reduced leptin expression. Overall, leptin-knockdown embryos had reduced cardiac output, with significant reduction to both heart and stroke volume. Reduction in metabolism is usually associated with reduced lipolysis in mammals, resulting in increased body mass and lipid retention (Zhang et al.,

1994). During early development of mammals, metabolism is increased and leptin deficiency results in significantly attenuated metabolic rate (Carlyle et al., 2002). This effect can be rescued albeit most early effects in development are purely sympathoexcitatory (Correia et al., 2002; Spiegel et al., 2004a). A hypometabolic state in zebrafish embryos also results in increased yolk size (and presumed in reduced lipolysis as compared to controls (Chapter 2). The effect of leptin knockdown on metabolism and cardiac function were largely conserved between mammals and zebrafish.

Immune status of animals is widely regarded as a valid metric of the health of an organism (Blackburn, 2001). Leptin deficient mammals show an increased vulnerability to infection with in increased duration of infection and decreased survival.

Hyperleptinemia results in an overly active immune system, resulting in autoimmune diseases such as rheumatoid arthritis and type-1 diabetes (Howard et al., 1999). Leptin’s link to energy status and immune function in mammals was a logical next question to address in our leptin morphants. Developing externally offers a different immunological

114 environment for embryonic fish compared to embryonic mammals. Zebrafish also have a distinct window of development of innate vs. adaptive immunity (adaptive maturing 4-6 weeks after innate, Chapter 4) unlike mammals whose innate and adaptive immunity overlap in development. Leptin receptors have been identified on macrophages in mammals (Procaccini et al., 2012), yet leptin’s role in fish immunity is poorly characterized (Mariano et al., 2013). I tested whether leptin knockdown immunocompromised zebrafish embryos.

I hypothesized that leptin knockdown will significantly alter the developing zebrafish embryo to mount an immune response and to reduce the bacterial load and thus increase survivability. Leptin morphants show a significant reduction in a metric of immune response (respiratory burst activity) as compared to controls (Chapter 3). Though leptin morphants could still mount an immune response to bacterial infection, it was significantly attenuated compared to control embryos. Furthermore, morphants had reduced survivability and increased bacterial load. Interestingly, leptin infusion to isolated immune cells from rainbow trout caused similar mammalian-like MAPK cascades to be activated but with reduced superoxide production (Mariano et al., 2013).

Reduced superoxide production in rainbow trout cell culture and low (compared to mammals) respiratory burst activity in mature goldfish macrophages (Novoa et al., 1996), suggests that macrophage phagocytosis may be under complex, spatiotemporal regulation. Therefore, additionally studies are needed to address immune function in fish.

The field of transcriptomics has been applied to many model systems to address global changes in transcription. Moreover, transcriptomics has been applied to leptin- manipulated mammals (through genetics or diet), though much of the focus has been on

115 specific tissues rather than whole animal response (Chapters 1, 4). Transcriptomics typically applies one of two approaches: RNAseq and microarray. RNAseq based methods are useful for nonmodel systems and/ or systems in which the main focus is on understanding post-translation modifications. This method uses multiple primers to amplify sequences and then massive parallel library construction to identify transcripts significantly affected. Since the zebrafish genome is well annotated, microarray technology was chosen to take advantage of the sequenced zebrafish genome. Microarray technology, however, is only as good as its build date and subsequent annotation

(Dalman et al., 2012). Microarrays for mammals such as humans, mouse, and rat have been continually updated, and have included leptin’s sequence for almost two decades.

Array technology for zebrafish has only included leptin within the past five years. Thus, a transcriptional study such as mine (comparing response to leptin manipulation between mammals and fish) was only possible recently.

I used a recently developed microarray (2012) that includes over 1.1 million probes for approximately ~26000 zebrafish genes. We found 1361 genes were significantly altered by leptin knockdown (Chapter 4). The most significantly affected pathways were those in sensory development, such as phototransduction. To validate the observed transcriptional differences on the array, we assayed several intermediate metabolic enzymes. These enzyme transcripts were all down regulated on the microarray, and the enzyme assay provided an independent means of testing the microarray.

Enzymatic indicators of fatty acid oxidation such as CPT and HOAD corroborated microarray data. Furthermore, catalase and citrate synthase were also down regulated in leptin morphants, indicating that aerobic metabolism and hydrogen peroxide catabolism

116 are reduced. These data, together with reduced metabolic rate, reduced cardiac output, and compromised immune response, build a picture of an overall hypometabolic state in leptin morphants. Of note, LDH enzyme activity (anaerobic metabolism indicator) was up regulated in morphants yet its transcription was down regulated. We speculate this lack of agreement may be a result of sampling time (lag between standing pool of mRNA vs active enzyme) or that LDH isoforms switch between the two treatments (which would not be detected by the enzyme assay). Transcriptome analysis of leptin knockout in mammalian tissues results in significant reduction in transcription of genes involved in lipolysis and fatty acid oxidation, and enrichment of transcripts that participate in inflammation. This is in stark contrast to our observed findings and even within the top

80 significantly affected genes, PARTEK software failed to identify enriched immune function pathways in response to leptin-A knockdown. In fish, the fertilized embryo is self-sufficient and does not eat until approximately day 10 (@28.5ºC). Furthermore, their heart doesn’t begin to beat until 24 hpf. Mammals, on the other hand, from fertilization through development are continuously linked to the dynamic nutritional status of the mother. In other words, zebrafish energy stores are commited prior to development whereas mammalian lipid stores and energy use are continuously monitored and modulated. Fat acquisition and use during zebrafish early development may not be as sensitive to leptin and leptin may instead have a primary role in signaling the development of sensory structures. Furthermore, maternally present leptin in zebrafish embryos may prime leptin sensitivity in the adult (Liu et al., 2012). In our original description of the leptin morphant, compromised sensory development (reduced otic vesicle formation and reduced non-functional eyes) was observed (Liu et al., 2012).

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Sensory function is also influenced by leptin in mammals, as ob-/ob- mice are deaf (Liu et al., 2012). It is likely that there is overlap among pathways affected by leptin between fish and mammals.

In analyzing millions of present/absent calls of an array, multiple correction procedures arose to account for each transcript, which is inherently influencing the hybridization of its neighbor transcript. Invoking the assumption that all transcripts equally influence the next, false detection rate (FDR) and fold change (FC) metrics have been used to make calls on whether a transcript is significant. Different interpretations of significance include fold change as primary vs. others that suggest a multi-tiered approach (Dalman et al., 2012). As transcriptomic data collection increased over the past decade, surprising discontinuity has been observed surrounding the consistency of gene threshold criteria. I hypothesized that varying the selection criteria affects data interpretation. In comparing combined gene selection criteria datasetsI observed that increasing cutoff thresholds did not equally affect both up and down regulated gene sets.

Thus based on one’s selection criteria, a significant number of genes can be gained or lost from the analysis, and this may be critical for comparative analyses. In the case of the sample dataset I used (taken from zebrafish hearts in response to hypoxia), leptin was not present within the array (earlier microarray build).

Limitations and future directions of study

I set out to test the hypothesis that leptin A function was similar between mammals and zebrafish. That hypothesis is supported in the sense that decreased leptin expression reduced metabolic rate and immune function in both zebrafish and mammals.

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It was not supported in that the pathways most affected by reduced leptin expression are different between mammals (immune function and cytoskeleton) and zebrafish (sensory and development). In fact, leptin A knockdown is lethal in zebrafish (Liu et al., 2012) whereas leptin knockout is not lethal in mammals (Zhang et al., 1994). This aspect of leptin function may be key to understanding how its action is distinctly different in nonmammals.

Unlike mammals, fish leptin is not correlated with body mass, liver mass, or adipose mass (Chapter 1). All of my experiments used developing zebrafish and undoubtedly there are distinct features of fish versus mammalian development that modulate sensitivity and thus signaling. Furthermore, external fertilization and development by fish and nutrition through yolk versus placenta likely affects leptin’s action in early development. Since the bias of the leptin literature is on adult mammals, future studies on fish should look to adults to better understand conservation of leptin signaling among vertebrates.

An additional limitation to my thesis research was the focus on a single leptin isoform (A). Future experiments should aim to characterize both isoforms separately, and their interaction. Another shortcoming is the lack of a “true” knockout strain of leptin A

(or B). Antisense oligonucleotides are well characterized for their ability to knock down translation within the first ten days of development. The development of brain, muscles, and even immune function in many vertebrates does not reach maturity until much later.

The current leptin knockdown is only establshed for zebrafish and thus a comparative approach among other fish species is warranted to control for species-specific differences of leptin function. Although leptin is studied in many fish species (several salmonids,

119 striped bass, catfish, etc.), our knockdown model is the only model available to study reduced leptin function in any fish species. Therefore, future studies should aim to uncover isoform functionality and to expand our understanding of leptin physiology across other fish species.

In the case of immune function, the bacterium chosen for this study was based on its validated use in zebrafish. Future studies may wish to identify whether other bacterial and viral pathogens respond similarly. Studies on adult zebrafish and leptin/ leptin receptors may provide useful data in uncovering the degree to which adaptive and innate immune functions are affected by leptin knockdown. One weakness of the transcriptomic study was that embryo injections and thus array hybridization was done over the course of multiple years. Clutch effects and/or variation in morpholino delivery among trials likely affect the variation I observed. However my analysis indicated high reproducibility within both control and knockdown embryos as indicated by principal component analysis. Validation of the microarray analysis would benefit from gene expression assays of genes involved in phototransduction. Finally, the leptin knockdown model is not directly analgous to the ob-/ob- mouse. As such, a true leptin knockdown fish will be a superior model to address comparative function of leptin across taxa.

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REFERENCES

Agrawal, S., Gollapudi, S., Su, H., and Gupta, S. (2011). Leptin activates human B cells to secrete TNF-α, IL-6, and IL-10 via JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathway. Journal of Clinical Immunology 31, 472–478.

Aguilar, A.J., Conde-Sieira, M., Polakof, S., Míguez, J.M., and Soengas, J.L. (2010). Central leptin treatment modulates brain glucosensing function and peripheral energy metabolism of rainbow trout. Peptides 31, 1044–1054.

Ahima, R.S., and Flier, J.S. (2000a). Leptin. Annual Review of Physiology 62, 413–437.

Ahima, R.S., and Flier, J.S. (2000b). Adipose tissue as an endocrine organ. Trends in Endocrinology & Metabolism 11, 327–332.

Ahima, R.S., and Osei, S.Y. (2004). Leptin signaling. Physiology & Behavior 81, 223– 241.

Ahima, R.S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier, E., and Flier, J.S. (1996). Role of leptin in the neuroendocrine response to fasting. Nature 382(6588) 250-252.

Ahima, R.S., Bjorbaek, C., Osei, S., and Flier, J.S. (1999a). Regulation of neuronal and glial proteins by leptin: Implications for brain development. Endocrinology 140, 2755–2762.

Ahima, R.S., Kelly, J., Elmquist, J.K., and Flier, J.S. (1999b). Distinct physiologic and neuronal responses to decreased leptin and mild hyperleptinemia 1. Endocrinology 140, 4923–4931.

Ahmed, M.L., Ong, K.K., Morrell, D.J., Cox, L., Drayer, N., Perry, L., Preece, M.A., and Dunger, D.B. (1999). Longitudinal study of leptin concentrations during puberty: Sex differences and relationship to changes in body composition 1. The Journal of Clinical Endocrinology & Metabolism 84, 899–905.

Aho, E. and Varnanen, M. (1999). Contractile properties of atrial and ventricular myocardiumof the heart of rainbow trout Oncorhynchus mykiss: effects of thermal acclimation. Journal of Experimental Biology 202, 2663–2677.

121

Alladi, P.A., Wadhwa, S., and Singh, N. (2002). Effect of prenatal auditory enrichment on developmental expression of synaptophysin and syntaxin 1 in chick brainstem auditory nuclei. Neuroscience 114, 577–590.

Allende, M.L., Amsterdam, A., Becker, T., Kawakami, K., Gaiano, N., and Hopkins, N. (1996). Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development. Genes & Development 10, 3141–3155.

Allison, D.B., Cui, X., Page, G.P., and Sabripour, M. (2006). Microarray data analysis: from disarray to consolidation and consensus. Nature Reviews Genetics 7, 55–65.

Aoki, T., Takano, T., Santos, M.D., Kondo, H., and Hirono, I. (2008). Molecular innate immunity in teleost fish: Review and future perspectives. In: 5th World Fisheries Congress, pp. 263–276.

Askari, A.T., Unzek, S., Popovic, Z.B., Goldman, C.K., Forudi, F., Kiedrowski, M., Rovner, A., Ellis, S.G., Thomas, J.D., DiCorleto, P.E., Topol, E.J., Penn, M.S. (2003). Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. The Lancet 362, 697–703.

Bado, A., Levasseur, S., Attoub, S., Kermorgant, S., Laigneau, J.-P., Bortoluzzi, M.-N., Moizo, L., Lehy, T., Guerre-Millo, M., Le Marchand-Brustel, Y., Lewin, M.J. (1998). The stomach is a source of leptin. Nature 394, 790–793.

Bagatto, B. (2001). The developmental physiology of the zebrafish: influence of environment of metabolic and cardiovascular attributes. PhD dissertation, University of North Texas, Denton.

Bagatto, B., Francl, J., Liu, B., and Liu, Q. (2006). Cadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development. BMC Developmental Biology 6, 23.

Bagatto, B and W.W. Burggren. 2006. A three-dimensional functional assessment of heart and vessel development in the larvae of the zebrafish (Danio rerio). Physiological and Biochemical Zoology 79(1):194-201.

Bailleul, B., Akerblom, I., and Strosberg, A.D. (1997). The leptin receptor promoter controls expression of a second distinct protein. Nucleic Acids Research 25, 2752–2758.

Bang, A., Grønkjaer, P., and Malte, H. (2004). Individual variation in the rate of oxygen consumption by zebrafish embryos. Journal of Fish Biology 64, 1285–1296.

122

Barnes, M.J., Holmes, G., Primeaux, S.D., York, D.A., and Bray, G.A. (2006). Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity. Peptides 27, 3292–3298.

Barrionuevo, W.R., and Burggren, W.W. (1999). O2 consumption and heart rate in developing zebrafish (Danio rerio): influence of temperature and ambient O2. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 276, R505–R513.

Bates, S.H., and Myers Jr, M.G. (2003). The role of leptin receptor signaling in feeding and neuroendocrine function. Trends in Endocrinology & Metabolism 14, 447– 452.

Belgareh-Touzé, N., Avaro, S., Rouillé, Y., Hoflack, B., and Haguenauer-Tsapis, R. (2002). Yeast Vps55p, a functional homolog of human obesity receptor gene- related protein, is involved in late endosome to vacuole trafficking. Molecular Biology of the Cell 13, 1694–1708.

Bellen, H.J., Tong, C., and Tsuda, H. (2010). 100 years of Drosophila research and its impact on vertebrate neuroscience: A history lesson for the future. Nature Reviews Neuroscience 11, 514–522.

Bereiter, D.A., and Jeanrenaud, B. (1979). Altered neuroanatomical organization in the central nervous system of the genetically obese (ob/ob) mouse. Brain Research 165, 249–260.

Berthoud, H.-R. (2004). Neural control of appetite: Cross-talk between homeostatic and non-homeostatic systems. Appetite 43, 315–317.

Bieber, L.L., Abraham, T., and Helmrath, T. (1972). A rapid spectrophotometric assay for carnitine palmitoyltransferase. Analytical Biochemistry 50, 509–518.

Bjorbaek, C., Uotani, S., da Silva, B., and Flier, J.S. (1997). Divergent signaling capacities of the long and short isoforms of the leptin receptor. Journal of Biological Chemistry 272, 32686–32695.

Bjorbaek, C., Elmquist, J.K., Frantz, J.D., Shoelson, S.E., and Flier, J.S. (1998). Identification of SOCS-3 as a potential mediator of central leptin resistance. Molecular Cell 1, 619–625.

Bjorbaek, C., El-Haschimi, K., Frantz, J.D., and Flier, J.S. (1999). The role of SOCS-3 in leptin signaling and leptin resistance. Journal of Biological Chemistry 274, 30059–30065.

Bjorbaek, C., and Kahn, B.B. (2004). Leptin signaling in the central nervous system and the periphery. Recent Progress in Hormone Research 59, 305–332.

123

Blackburn, E.H. (2001). Switching and signaling at the telomere. Cell 106, 661–673.

Blum, W.F., Englaro, P., Hanitsch, S., Juul, A., Hertel, N.T., Müller, J., Skakkeb\a ek, N.E., Heiman, M.L., Birkett, M., Attanasio, A.M., Kiess, W., and Rascher, W. (1997). Plasma leptin levels in healthy children and adolescents: Dependence on body mass index, body fat mass, gender, pubertal stage, and testosterone 1. The Journal of Clinical Endocrinology & Metabolism 82, 2904–2910.

Boorse, G.C., and Libbon, J. (2010). Genomic characterization of two leptin genes and a leptin receptor gene in the Green Anole, Anolis carolinensis. In Integrative and Comparative Biology, (Oxford Univ Press Inc CARY, NC 2751,3 USA), pp. E207–E207.

Bornstein, S.R., Licinio, J., Tauchnitz, R., Engelmann, L., Negrao, A.B., Gold, P., and Chrousos, G.P. (1998). Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm in cortisol and leptin secretion. The Journal of Clinical Endocrinology & Metabolism 83, 280–283.

Bouloumié, A., Drexler, H.C., Lafontan, M., and Busse, R. (1998). Leptin, the product of Ob gene, promotes angiogenesis. Circulation Research 83, 1059–1066.

Bouret, S.G., Draper, S.J., and Simerly, R.B. (2004). Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 304, 108–110.

Bouret, S.G., and Simerly, R.B. (2007). Development of leptin-sensitive circuits. Journal of Neuroendocrinology 19, 575–582.

Boyce, B., Yao, Z., and Xing, L. (2009). Osteoclasts have multiple roles in bone in addition to bone resorption. Critical ReviewsTM in Eukaryotic Gene Expression 19.

Boyce, B.F., Rosenberg, E., de Papp, A.E., and Duong, L.T. (2012). The osteoclast, bone remodelling and treatment of metabolic bone disease. European Journal of Clinical Investigation 42, 1332–1341.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.

Brannon, M.K., Davis, J., Mathias, J.R., Hall, C.J., Emerson, J.C., Crosier, P.S., Huttenlocher, A., Ramakrishnan, L., and Moskowitz, S.M. (2009). Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cellular Microbiology 11, 755–768.

124

Bray, G.A., and York, D.A. (1997). Leptin and clinical medicine: a new piece in the puzzle of obesity. The Journal of Clinical Endocrinology & Metabolism 82, 2771–2776.

Breslow, M.J., Min-Lee, K., Brown, D.R., Chacko, V.P., Palmer, D., and Berkowitz, D.E. (1999). Effect of leptin deficiency on metabolic rate in ob/ob mice. American Journal of Physiology-Endocrinology and Metabolism 276, E443– E449.

Broberger, C., Johansen, J., Johansson, C., Schalling, M., and Hökfelt, T. (1998). The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proceedings of the National Academy of Sciences 95, 15043–15048.

Bruder, E., Stallmach, T., Peier, K., Superti-Furga, A., and Vezzoni, P. (2003). Osteoclast morphology in autosomal recessive malignant osteopetrosis due to a TCIRG1 gene mutation. Fetal & Pediatric Pathology 22, 3–9.

Bruno, A., Chanez, P., Chiappara, G., Siena, L., Giammanco, S., Gjomarkaj, M., Bonsignore, G., Bousquet, J., and Vignola, A.M. (2005). Does leptin play a cytokine-like role within the airways of COPD patients? European Respiratory Journal 26, 398–405.

Burger, J.W., and Hess, W.N. (1960). Function of the rectal gland in the spiny dogfish. Science 131, 670–671.

Caldefie-Chezet, F., Poulin, A., Tridon, A., Sion, B., and Vasson, M.P. (2001). Leptin: a potential regulator of polymorphonuclear neutrophil bactericidal action? Journal of Leukocyte Biology 69, 414–418.

Caldefie-Chezet, F., Poulin, A., and Vasson, M.-P. (2003). Leptin regulates functional capacities of polymorphonuclear neutrophils. Free Radical Research 37, 809–814.

Calduch-Giner, J.A., Echasseriau, Y., Crespo, D., Baron, D., Planas, J.V., Prunet, P., and Pérez-Sánchez, J. (2014). Transcriptional Assessment by Microarray Analysis and Large-Scale Meta-analysis of the Metabolic Capacity of Cardiac and Skeletal Muscle Tissues to Cope With Reduced Nutrient Availability in Gilthead Sea Bream (Sparus aurata L.). Marine Biotechnology 1–13.

Campbell, C.D., Chapman, S.J., Cameron, C.M., Davidson, M.S., and Potts, J.M. (2003). A rapid microtiter plate method to measure carbon dioxide evolved from carbon substrate amendments so as to determine the physiological profiles of soil microbial communities by using whole soil. Applied and Environmental Microbiology 69, 3593–3599.

125

Cao, G.Y., Considine, R.V., and Lynn, R.B. (1997). Leptin receptors in the adrenal medulla of the rat. American Journal of Physiology-Endocrinology and Metabolism 273, E448–E452.

Cao, L., Choi, E.Y., Liu, X., Martin, A., Wang, C., Xu, X., and During, M.J. (2011). White to brown fat phenotypic switch induced by genetic and environmental activation of a hypothalamic-adipocyte axis. Cell Metabolism 14, 324–338.

Carbone, F., La Rocca, C., and Matarese, G. (2012). Immunological functions of leptin and adiponectin. Biochimie 94, 2082–2088.

Carlyle, M., Jones, O.B., Kuo, J.J., and Hall, J.E. (2002). Chronic cardiovascular and renal actions of leptin role of adrenergic activity. Hypertension 39, 496–501.

Caro, J.F., Kolaczynski, J.W., Nyce, M.R., Ohannesian, J.P., Opentanova, I., Goldman, W.H., Lynn, R.B., Zhang, P.-L., Sinha, M.K., and Considine, R.V. (1996). Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. The Lancet 348, 159–161.

Carpenter, L.R., Farruggella, T.J., Symes, A., Karow, M.L., Yancopoulos, G.D., and Stahl, N. (1998). Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor. Proceedings of the National Academy of Sciences 95, 6061–6066.

Carr, A. N. and Kranias, E.G. (2002) Thyroid hormone regulation of calcium cycling proteins. Thyroid 12, 453–457.

De Carvalho Borges, B., Rorato, R.C., Uchoa, E.T., Marangon, P.B., Elias, C.F., Antunes-Rodrigues, J., and Elias, L.L. (2014). Protein tyrosine phosphatase 1b (PTP1B) contributes to lps-induced leptin resistance in male rats. American Journal of Physiology-Endocrinology and Metabolism 94.

Castro Domingos, A., Freitas, D.Q., Whaites, E.J., and Filho, A.M. (2005). Osteopetrosis–a review and report of two cases. Oral Diseases 11, 46–49.

Cerezuela, R., Guardiola, F.A., Meseguer, J., and Esteban, M. (2012). Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata) did not correlate with disease resistance to Photobacterium damselae. Fish & Shellfish Immunology 32, 1032–1040.

Chan, J.L., Wong, S.L., and Mantzoros, C.S. (2008). Pharmacokinetics of subcutaneous Rrcombinant methionyl human leptin administration in healthy subjects in the fed and fasting states. Clinical Pharmacokinetics 47, 753–764.

Chehab, F.F. (2000). Leptin as a regulator of adipose mass and reproduction. Trends in Pharmacological Sciences 21, 309–314.

126

Chehab, F.F., Lim, M.E., and Lu, R. (1996). Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nature Genetics 12, 318–320.

Chehab, F.F., Mounzih, K., Lu, R., and Lim, M.E. (1997). Early onset of reproductive function in normal female mice treated with leptin. Science 275, 88–90.

Chen, E.Y., Tan, C.M., Kou, Y., Duan, Q., Wang, Z., Meirelles, G.V., Clark, N.R., and Ma’ayan, A. (2013). Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128.

Chisada, S., Kurokawa, T., Murashita, K., Rønnestad, I., Taniguchi, Y., Toyoda, A., Sakaki, Y., Takeda, S., and Yoshiura, Y. (2014). Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation. General and Comparative Endocrinology 195, 9–20.

Chou, S.H., Chamberland, J.P., Liu, X., Matarese, G., Gao, C., Stefanakis, R., Brinkoetter, M.T., Gong, H., Arampatzi, K., and Mantzoros, C.S. (2011). Leptin is an effective treatment for hypothalamic amenorrhea. Proceedings of the National Academy of Sciences 108, 6585–6590.

Chu, D.L.H., Li, V.W.T., and Yu, R.M.K. (2010). Leptin: clue to poor appetite in oxygen-starved fish. Molecular and Cellular Endocrinology 319, 143–146.

Chua, S.C., Chung, W.K., Wu-Peng, X.S., Zhang, Y., Liu, S.-M., Tartaglia, L., and Leibel, R.L. (1996). Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271, 994–996.

Clarke, I.J., Henry, B., Iqbal, J., and Goding, J.W. (2000). Leptin and the regulation of food intake and the neuroendocrine axis in sheep. Clinical and Experimental Pharmacology & Physiology 28, 106–107.

Clatworthy, A.E., Lee, J.S.-W., Leibman, M., Kostun, Z., Davidson, A.J., and Hung, D.T. (2009). Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants. Infection and Immunity 77, 1293–1303.

Claycombe, K., King, L.E., and Fraker, P.J. (2008). A role for leptin in sustaining lymphopoiesis and myelopoiesis. Proceedings of the National Academy of Sciences 105, 2017–2021.

Cnop, M., Landchild, M.J., Vidal, J., Havel, P.J., Knowles, N.G., Carr, D.R., Wang, F., Hull, R.L., Boyko, E.J., Retzlaff, B.M., Walden, C.E., Knopp, R.H., and Kahn, S.E. (2002). The concurrent accumulation of intra-abdominal and subcutaneous fat explains the association between insulin resistance and plasma leptin

127

concentrations distinct metabolic effects of two fat compartments. Diabetes 51, 1005–1015.

Coll, A.P., Farooqi, I.S., and O’Rahilly, S. (2007). The hormonal control of food intake. Cell 129, 251–262.

Coll, A.P., Yeo, G.S., Farooqi, I.S., and O’Rahilly, S. (2008). SnapShot: the hormonal control of food intake. Cell 135, 572.

Collins, S., Kuhn, C.M., Petro, A.E., Swick, A.G., Chrunyk, B.A., and Surwit, R.S. (1996). Role of leptin in fat regulation. Nature 380, 677.

Collins, J.E., White, S., Searle, S.M., and Stemple, D.L. (2012). Incorporating RNA-seq data into the zebrafish Ensembl genebuild. Genome Research 22, 2067–2078.

Comuzzie, A.G., Hixson, J.E., Almasy, L., Mitchell, B.D., Mahaney, M.C., Dyer, T.D., Stern, M.P., MacCluer, J.W., and Blangero, J. (1997). A major quantitative trait locus determining serum leptin levels and fat mass is located on human chromosome 2. Nature Genetics 15, 273–276.

Concannon, P., Levac, K., Rawson, R., Tennant, B., and Bensadoun, A. (2001). Seasonal changes in serum leptin, food intake, and body weight in photoentrained woodchucks. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 281, R951–R959.

Considine, R.V., and Caro, J.F. (1997). Leptin and the regulation of body weight. The International Journal of Biochemistry & Cell Biology 29, 1255–1272.

Copeland, D.L., Duff, R.J., Liu, Q., Prokop, J., and Londraville, R.L. (2011). Leptin in teleost fishes: an argument for comparative study. Frontiers in Physiology 2: 26.

Correia, M.L., Haynes, W.G., Rahmouni, K., Morgan, D.A., Sivitz, W.I., and Mark, A.L. (2002). The concept of selective leptin resistance evidence from agouti yellow obese mice. Diabetes 51, 439–442.

Couturier, C., Sarkis, C., Séron, K., Belouzard, S., Chen, P., Lenain, A., Corset, L., Dam, J., Vauthier, V., Dubart, A., Mallet, J., Froguel, P., Rouille, Y., and Jockers, R. (2007). Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity. Proceedings of the National Academy of Sciences 104, 19476–19481.

Crespi, E.J., and Denver, R.J. (2006). Leptin (ob gene) of the South African clawed frog Xenopus laevis. Proceedings of the National Academy of Sciences 103, 10092– 10097.

128

Dagil, R., Knudsen, M.J., Olsen, J.G., O’Shea, C., Franzmann, M., Goffin, V., Teilum, K., Breinholt, J., and Kragelund, B.B. (2012). The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation: insight from structures of the prolactin receptor. Structure 20, 270–282.

Dalman, M.R., Deeter, A., Nimishakavi, G., and Duan, Z.-H. (2012). Fold change and p- value cutoffs significantly alter microarray interpretations. BMC Bioinformatics 13, S11.

Dalman, M.R., Liu, Q., King, M.D., Bagatto, B., and Londraville, R.L. (2013). Leptin expression affects metabolic rate in zebrafish embryos (D. rerio). Frontiers in Physiology 4.

Dardeno, T.A., Chou, S.H., Moon, H.-S., Chamberland, J.P., Fiorenza, C.G., and Mantzoros, C.S. (2010). Leptin in human physiology and therapeutics. Frontiers in Neuroendocrinology 31, 377–393.

De Luca, C., Kowalski, T.J., Zhang, Y., Elmquist, J.K., Lee, C., Kilimann, M.W., Ludwig, T., Liu, S.-M., Chua, S.C., and others (2005). Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. Journal of Clinical Investigation 115, 3484–3493.

Denver, R.J., Bonett, R.M., and Boorse, G.C. (2011). Evolution of leptin structure and function. Neuroendocrinology 94, 21–38.

Van Der Sar, A.M., Musters, R.J., Van Eeden, F.J., Appelmelk, B.J., Vandenbroucke- Grauls, C.M., and Bitter, W. (2003). Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections. Cellular Microbiology 5, 601–611.

Doring, H., Schwarzer, K., Nuesslein-Hildesheim, B., and Schmidt, I. (1998). Leptin selectively increases energy expenditure of food-restricted lean mice. International Journal of Obesity and Related Metabolic Disorders: Journal of the International Association for the Study of Obesity 22, 83–88.

Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A.F., Beil, F.T., Shen, J., Vinson, C., Rueger, J.M., and Karsenty, G. (2000). Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100, 197–207.

Dusserre, E., Moulin, P., and Vidal, H. (2000). Differences in mRNA expression of the proteins secreted by the adipocytes in human subcutaneous and visceral adipose tissues. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1500, 88–96.

Elefteriou, F., Takeda, S., Ebihara, K., Magre, J., Patano, N., Kim, C.A., Ogawa, Y., Liu, X., Ware, S.M., Craigen, W.J., Robert, J.J., Vinson, C., Nakao, V.K., Capeau, J.,

129

Karsenty, G.(2004). Serum leptin level is a regulator of bone mass. Proceedings of the National Academy of Sciences of the United States of America 101, 3258– 3263.

Elmquist, J.K., Maratos-Flier, E., Saper, C.B., and Flier, J.S. (1998). Unraveling the central nervous system pathways underlying responses to leptin. Nature Neuroscience 1, 445–450.

Enard, W., Khaitovich, P., Klose, J., Zöllner, S., Heissig, F., Giavalisco, P., Nieselt- Struwe, K., Muchmore, E., Varki, A., Ravid, R., Doxiadis, G.M., Bontrop, R.E., Paabo, S. (2002). Intra- and interspecific variation in primate gene expression patterns. Science 296, 340–343.

Eschmeyer, W.N. (2003). The catalog of fishes on-line. California Academy of Sciences, San Francisco, California.

Faggioni, R., Fantuzzi, G., Gabay, C., Moser, A., Dinarello, C.A., Feingold, K.R., and Grunfeld, C. (1999). Leptin deficiency enhances sensitivity to endotoxin-induced lethality. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 276, R136–R142.

Faggioni, R., Jones-Carson, J., Reed, D.A., Dinarello, C.A., Feingold, K.R., Grunfeld, C., and Fantuzzi, G. (2000). Leptin-deficient (ob/ob) mice are protected from T cell- mediated hepatotoxicity: role of tumor necrosis factor α and IL-18. Proceedings of the National Academy of Sciences 97, 2367–2372.

Faggioni, R., Cattley, R.C., Guo, J., Flores, S., Brown, H., Qi, M., Yin, S., Hill, D., Scully, S., Chen, C., et al. (2001). IL-18-binding protein protects against lipopolysaccharide-induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice. The Journal of Immunology 167, 5913–5920.

Fan, C.-Y., Pan, J., Chu, R., Lee, D., Kluckman, K.D., Usuda, N., Singh, I., Yeldandi, A.V., Rao, M.S., Maeda, N., and Reddy, J.K. (1996). Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene. Journal of Biological Chemistry 271, 24698–24710.

Fantuzzi, G., and Faggioni, R. (2000). Leptin in the regulation of immunity, inflammation, and hematopoiesis. Journal of Leukocyte Biology 68, 437–446.

Farooqi, I.S., Jebb, S.A., Langmack, G., Lawrence, E., Cheetham, C.H., Prentice, A.M., Hughes, I.A., McCamish, M.A., and O’Rahilly, S. (1999). Effects of recombinant leptin therapy in a child with congenital leptin deficiency. New England Journal of Medicine 341, 879–884.

130

Farooqi, I.S., Matarese, G., Lord, G.M., Keogh, J.M., Lawrence, E., Agwu, C., Sanna, V., Jebb, S.A., Perna, F., Fontana, S., et al. (2002). Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. The Journal of Clinical Investigation 110, 1093–1103.

Fearon, D.T., and Locksley, R.M. (1996). The instructive role of innate immunity in the acquired immune response. Science 272, 50–54.

Fei, H., Okano, H.J., Li, C., Lee, G.-H., Zhao, C., Darnell, R., and Friedman, J.M. (1997). Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues. Proceedings of the National Academy of Sciences 94, 7001–7005.

Férézou-Viala, J., Roy, A.-F., Sérougne, C., Gripois, D., Parquet, M., Bailleux, V., Gertler, A., Delplanque, B., Djiane, J., Riottot, M., and Taouis, M. (2007). Long- term consequences of maternal high-fat feeding on hypothalamic leptin sensitivity and diet-induced obesity in the offspring. American Journal of Physiology- Regulatory, Integrative and Comparative Physiology 293, R1056–R1062.

Ferguson, D.P., Dangott, L.J., Schmitt, E.E., Vellers, H.L., and Lightfoot, J.T. (2014). Differential skeletal muscle proteome of high-and low-active mice. Journal of Applied Physiology 116, 1057–1067.

Fernández-Riejos, P., Najib, S., Santos-Alvarez, J., Martín-Romero, C., Pérez-Pérez, A., González-Yanes, C., and Sánchez-Margalet, V. (2010). Role of leptin in the activation of immune cells. Mediators of Inflammation 20

Ferreira-Dias, G., Claudino, F., Carvalho, H., Agrícola, R., Alpoim-Moreira, J., and Robalo Silva, J. (2005). Seasonal reproduction in the mare: possible role of plasma leptin, body weight and immune status. Domestic Animal Endocrinology 29, 203–213.

Ferron, M., Wei, J., Yoshizawa, T., Del Fattore, A., DePinho, R.A., Teti, A., Ducy, P., and Karsenty, G. (2010). Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism. Cell 142, 296–308.

Figueiro, M.G., Plitnick, B., and Rea, M.S. (2012). Light modulates leptin and ghrelin in sleep-restricted adults. International Journal of Endocrinology 2012.

Florant, G.L., Porst, H., Peiffer, A., Hudachek, S.F., Pittman, C., Summers, S.A., Rajala, M.W., and Scherer, P.E. (2004). Fat-cell mass, serum leptin and adiponectin changes during weight gain and loss in yellow-bellied marmots (Marmota flaviventris). Journal of Comparative Physiology B 174, 633–639.

131

Fong, T.M., Huang, R.-R.C., Tota, M.R., Mao, C., Smith, T., Varnerin, J., Karpitskiy, V.V., Krause, J.E., and Van der Ploeg, L.H. (1998). Localization of leptin binding domain in the leptin receptor. Molecular Pharmacology 53, 234–240.

Fraisl, P., Mazzone, M., Schmidt, T., and Carmeliet, P. (2009). Regulation of angiogenesis by oxygen and metabolism. Developmental Cell 16, 167–179.

Fraser, D.A., Thoen, J., Reseland, J.E., Førre, Ø., and Kjeldsen-Kragh, J. (1999). Decreased CD4+ lymphocyte activation and increased interleukin-4 production in peripheral blood of rheumatoid arthritis patients after acute starvation. Clinical Rheumatology 18, 394–401.

Friedman, J.M., and Halaas, J.L. (1998). Leptin and the regulation of body weight in mammals. Nature 395, 763–770.

Froiland, E., Murashita, K., Jørgensen, E.H., and Kurokawa, T. (2010). Leptin and ghrelin in anadromous Arctic charr: cloning and change in expressions during a seasonal feeding cycle. General and Comparative Endocrinology 165, 136–143.

Frühbeck, G. (1999). Pivotal role of nitric oxide in the control of blood pressure after leptin administration. Diabetes 48, 903–908.

Fu, S., Yang, L., Li, P., Hofmann, O., Dicker, L., Hide, W., Lin, X., Watkins, S.M., Ivanov, A.R., and Hotamisligil, G.S. (2011). Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity. Nature 473, 528–531.

Fuentes, E.N., Safian, D., Einarsdottir, I.E., Valdés, J.A., Elorza, A.A., Molina, A., and Björnsson, B.T. (2013). Nutritional status modulates plasma leptin, AMPK and TOR activation, and mitochondrial biogenesis: implications for cell metabolism and growth in skeletal muscle of the fine flounder. General and Comparative Endocrinology 186, 172–180.

Fujita, A., Sato, J.R., Rodrigues, L.O., Ferreira, C.E., and Sogayar, M.C. (2006). Evaluating different methods of microarray data normalization. BMC Bioinformatics 7, 469.

Galindo-Villegas, J., García-Moreno, D., de Oliveira, S., Meseguer, J., and Mulero, V. (2012). Regulation of immunity and disease resistance by commensal microbes and chromatin modifications during zebrafish development. Proceedings of the National Academy of Sciences 109, E2605–E2614.

Ghilardi, N., Ziegler, S., Wiestner, A., Stoffel, R., Heim, M.H., and Skoda, R.C. (1996). Defective STAT signaling by the leptin receptor in diabetic mice. Proceedings of the National Academy of Sciences 93, 6231–6235.

132

Goïot, H., Attoub, S., Kermorgant, S., Laigneau, J.-P., Lardeux, B., Lehy, T., Lewin, M.J., and Bado, A. (2001). Antral mucosa expresses functional leptin receptors coupled to STAT-3 signaling, which is involved in the control of gastric secretions in the rat. Gastroenterology 121, 1417–1427.

Goldstein, J.L., Austin, S.L., Boyette, K., Kanaly, A., Veerapandiyan, A., Rehder, C., Kishnani, P.S., and Bali, D.S. (2010). Molecular analysis of the AGL gene: identification of 25 novel mutations and evidence of genetic heterogeneity in patients with Glycogen Storage Disease Type III. Genetics in Medicine 12, 424– 430.

Gorissen, M., Bernier, N.J., Nabuurs, S.B., Flik, G., and Huising, M.O. (2009). Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution. Journal of Endocrinology 201, 329–339.

Gracey, A.Y., Lee, T.-H., Higashi, R.M., and Fan, T. (2011). Hypoxia-induced mobilization of stored triglycerides in the euryoxic goby Gillichthys mirabilis. The Journal of Experimental Biology 214, 3005–3012.

Grim, J.M., Miles, D.R.B., and Crockett, E.L. (2010). Temperature acclimation alters oxidative capacities and composition of membrane lipids without influencing activities of enzymatic antioxidants or susceptibility to lipid peroxidation in fish muscle. The Journal of Experimental Biology 213, 445–452.

Gruen, M.L., Hao, M., Piston, D.W., and Hasty, A.H. (2007). Leptin requires canonical migratory signaling pathways for induction of monocyte and macrophage chemotaxis. American Journal of Physiology-Cell Physiology 293, C1481– C1488.

Grunfeld, C., Zhao, C., Fuller, J., Pollack, A., Moser, A., Friedman, J., and Feingold, K.R. (1996). Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. Journal of Clinical Investigation 97, 2152.

Grunwald, D.J., and Eisen, J.S. (2002). Headwaters of the zebrafish—emergence of a new model vertebrate. Nature Reviews Genetics 3, 717–724.

Guo, Z., Su, W., Allen, S., Pang, H., Daugherty, A., Smart, E., and Gong, M.C. (2005). COX-2 up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovascular Research 67, 723–735.

Halaas, J.L., and Friedman, J.M. (1997). Leptin and its receptor. Journal of Endocrinology 155, 215–216.

Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, S.L., Chait, B.T., Rabinowitz, D., Lallone, R.L., Burley, S.K., and Friedman, J.M. (1995). Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269, 543–546.

133

Halaas, J.L., Boozer, C., Blair-West, J., Fidahusein, N., Denton, D.A., and Friedman, J.M. (1997). Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proceedings of the National Academy of Sciences 94, 8878–8883.

Hamilton, B.S., Paglia, D., Kwan, A.Y., and Deitel, M. (1995). Increased obese mRNA expression in omental fat cells from massively obese humans. Nature Medicine 1, 953–956.

Hamrick, M.W., and Ferrari, S.L. (2008). Leptin and the sympathetic connection of fat to bone. Osteoporosis International 19, 905–912.

Hamrick, M.W., Pennington, C., Newton, D., Xie, D., and Isales, C. (2004). Leptin deficiency produces contrasting phenotypes in bones of the limb and spine. Bone 34, 376–383.

Hamrick, M.W., Della-Fera, M.A., Choi, Y.-H., Pennington, C., Hartzell, D., and Baile, C.A. (2005). Leptin treatment induces loss of bone marrow adipocytes and increases bone formation in leptin-deficient ob/ob mice. Journal of Bone and Mineral Research 20, 994–1001.

Hansen, C.A., and Sidell, B.D. (1983). Atlantic hagfish cardiac muscle: metabolic basis of tolerance to anoxia. American Journal of Physiology 244, R356–R362.

Harada, S., and Rodan, G.A. (2003). Control of osteoblast function and regulation of bone mass. Nature 423, 349–355.

Harding, H.P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Molecular Cell 6, 1099–1108.

Harvey, J., Shanley, L.J., O’Malley, D., and Irving, A.J. (2005). Leptin: a potential cognitive enhancer? Biochemical Society Transactions 33, 1029–1032.

Hausman, G.J., Barb, C.R., and Lents, C.A. (2012). Leptin and reproductive function. Biochimie 94, 2075–2081.

Havel, P.J., Kasim-Karakas, S., Mueller, W., Johnson, P.R., Gingerich, R.L., and Stern, J.S. (1996). Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sustained weight loss. The Journal of Clinical Endocrinology & Metabolism 81, 4406– 4413.

134

Haynes, W.G., Morgan, D.A., Walsh, S.A., Mark, A.L., and Sivitz, W.I. (1997). Receptor-mediated regional sympathetic nerve activation by leptin. Journal of Clinical Investigation 100, 270.

Van Heek, M., Compton, D.S., France, C.F., Tedesco, R.P., Fawzi, A.B., Graziano, M.P., Sybertz, E.J., Strader, C.D., and Davis Jr, H.R. (1997). Diet-induced obese mice develop peripheral, but not central, resistance to leptin. Journal of Clinical Investigation 99, 385.

Herbomel, P., Thisse, B., and Thisse, C. (1999). Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126, 3735–3745.

Hermann, A.C., Millard, P.J., Blake, S.L., and Kim, C.H. (2004). Development of a respiratory burst assay using zebrafish kidneys and embryos. Journal of Immunological Methods 292, 119–129.

Heymsfield, S.B., Greenberg, A.S., Fujioka, K., Dixon, R.M., Kushner, R., Hunt, T., Lubina, J.A., Patane, J., Self, B., Hunt, P., and McCamish, M. (1999). Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. Jama 282, 1568–1575.

Hodgkin, A.L., and Huxley, A.F. (1939). Action potentials recorded from inside a nerve fibre. Nature 144, 710–711.

Hoggard, N., Hunter, L., Duncan, J.S., Williams, L.M., Trayhurn, P., and Mercer, J.G. (1997). Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proceedings of the National Academy of Sciences 94, 11073– 11078.

Hoggard, N., Hunter, L., Lea, R.G., Trayhurn, P., and Mercer, J.G. (2000). Ontogeny of the expression of leptin and its receptor in themurine fetus and placenta. British Journal of Nutrition 83, 317–326.

Holness, M.J., Munns, M.J., and Sugden, M.C. (1999). Current concepts concerning the role of leptin in reproductive function. Molecular and Cellular Endocrinology 157, 11–20.

Holzel, M., Grimm, T., Rohrmoser, M., Malamoussi, A., Harasim, T., Gruber-Eber, A., Kremmer, E., and Eick, D. (2007). The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing. Nucleic Acids Research 35, 789–800.

Hotamisligil, G.S., Shargill, N.S., and Spiegelman, B.M. (1993). Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259, 87–91.

135

Howard, J.K., Lord, G.M., Matarese, G., Vendetti, S., Ghatei, M.A., Ritter, M.A., Lechler, R.I., and Bloom, S.R. (1999). Leptin protects mice from starvation- induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. The Journal of Clinical Investigation 104, 1051–1059.

Howe, L.R., Subbaramaiah, K., Hudis, C.A., and Dannenberg, A.J. (2013). Molecular pathways: adipose inflammation as a mediator of obesity-associated cancer. Clinical Cancer Research 19, 6074–6083.

Hsu, A., Aronoff, D.M., Phipps, J., Goel, D., and Mancuso, P. (2007). Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Clinical & Experimental Immunology 150, 332–339.

Hu, N., Sedmera, D., Yost, H.J., and Clark, E.B. (2000). Structure and function of the developing zebrafish heart. The Anatomical Record 260, 148–157.

Hube, F., Lietz, U., Igel, M., Jensen, P.B., Tornqvist, H., Joost, H.-G., and Hauner, H. (1996). Difference in leptin mRNA levels between omental and subcutaneous abdominal adipose tissue from obese humans. Hormone and Metabolic Research 28, 690–693.

Hudson, A.W., and Birnbaum, M.J. (1995). Identification of a nonneuronal isoform of synaptotagmin. Proceedings of the National Academy of Sciences 92, 5895–5899.

Huising, M.O., Geven, E.J., Kruiswijk, C.P., Nabuurs, S.B., Stolte, E.H., Spanings, F.T., Verburg-van Kemenade, B.L., and Flik, G. (2006). Increased leptin expression in common carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation. Endocrinology 147, 5786–5797.

Iacobas, D.A., Fan, C., Iacobas, S., and Haddad, G.G. (2008). Integrated transcriptomic response to cardiac chronic hypoxia: translation regulators and response to stress in cell survival. Functional & Integrative Genomics 8, 265–275.

Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264.

Isaacs, J.S., Xu, W., and Neckers, L. (2003). Heat shock protein 90 as a molecular target for cancer therapeutics. Cancer Cell 3, 213–217.

Jacob, E., Drexel, M., Schwerte, T., and Pelster, B. (2002). Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 283, R911–R917.

136

Jaquet, D., Leger, J., Levy-Marchal, C., Oury, J.F., and Czernichow, P. (1998). Ontogeny of leptin in human fetuses and newborns: effect of intrauterine growth retardation on serum leptin concentrations. The Journal of Clinical Endocrinology & Metabolism 83, 1243–1246.

Jeffery, I.B., Higgins, D.G., and Culhane, A.C. (2006). Comparison and evaluation of methods for generating differentially expressed gene lists from microarray data. BMC Bioinformatics 7, 359.

Jimeno, C.D. (2008). A transcriptomic approach toward understanding PAMP-driven macrophage activation and dietary immunostimulant in fish. PhD Thesis, 1-222, Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Ciències, Universitat Autòmona de Barcelona, Barcelona, Spain

Johnson, R.M., Johnson, T.M., and Londraville, R.L. (2000). Evidence for leptin expression in fishes. The Journal of Experimental Zoology 286, 718.

Johnson, S.C. (1967). Hierarchical clustering schemes. Psychometrika 32, 241–254.

Jovanović, B., Ji, T., and Palić, D. (2011). Gene expression of zebrafish embryos exposed to titanium dioxide nanoparticles and hydroxylated fullerenes. Ecotoxicology and Environmental Safety 74, 1518–1525.

Kaji, T., and Nonogaki, K. (2012). Role of homeobox genes in the hypothalamic development and energy balance. Frontiers in Bioscience (Landmark Edition) 18, 740–747.

Kalinka, A.T., Varga, K.M., Gerrard, D.T., Preibisch, S., Corcoran, D.L., Jarrells, J., Ohler, U., Bergman, C.M., and Tomancak, P. (2010). Gene expression divergence recapitulates the developmental hourglass model. Nature 468, 811–814.

Karlsson, E.A., and Beck, M.A. (2010). The burden of obesity on infectious disease. Experimental Biology and Medicine 235, 1412–1424.

Kau, A.L., Ahern, P.P., Griffin, N.W., Goodman, A.L., and Gordon, J.I. (2011). Human nutrition, the gut microbiome and the immune system. Nature 474, 327–336.

Keen-Rhinehart, E., Ondek, K., and Schneider, J.E. (2013). Neuroendocrine regulation of appetitive ingestive behavior. Frontiers in Neuroscience 7.

Keim, N.L., Stern, J.S., and Havel, P.J. (1998). Relation between circulating leptin concentrations and appetite during a prolonged, moderate energy deficit in women. The American Journal of Clinical Nutrition 68, 794–801.

137

Khan, S.N., and Lane, J.M. (2004). The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in orthopaedic applications. Expert Opinion in Biolgy Therapy 4, 741–748.

Kim, S., Sohn, I., Ahn, J.-I., Lee, K.-H., Lee, Y.S., and Lee, Y.S. (2004). Hepatic gene expression profiles in a long-term high-fat diet-induced obesity mouse model. Gene 340, 99–109.

Kim, K.-H., Rosen, A., Bruneau, B.G., Hui, C., and Backx, P.H. (2012). Iroquois homeodomain transcription factors in heart development and function. Circulation Research 110, 1513–1524.

Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T.F. (1995). Stages of embryonic development of the zebrafish. Developmental Dynamics 203, 253–310.

Kinoshita, Y., Jarell, A.D., Flaman, J.M., Foltz, G., Schuster, J., Sopher, B.L., Irvin, D.K., Kanning, K., Kornblum, H.I., Nelson, P.SHieter, P., Morrison, R.S. (2001). Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells. Journal of Biological Chemistry 276, 6656–6665.

Kling, P., Rønnestad, I., Stefansson, S.O., Murashita, K., Kurokawa, T., and Björnsson, B.T. (2009). A homologous salmonid leptin radioimmunoassay indicates elevated plasma leptin levels during fasting of rainbow trout. General and Comparative Endocrinology 162, 307–312.

Kobayashi, Y., Quiniou, S., Booth, N.J., and Peterson, B.C. (2011). Expression of leptin- like peptide (LLP) mRNA in channel catfish (Ictalurus punctatus) is induced by exposure to Edwardsiella ictaluri but is independent of energy status. General and Comparative Endocrinology 173, 411–418.

Kronfeld-Schor, N., Richardson, C., Silvia, B.A., Kunz, T.H., and Widmaier, E.P. (2000). Dissociation of leptin secretion and adiposity during prehibernatory fattening in little brown bats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 279, R1277–R1281.

Kurokawa, T., and Murashita, K. (2009). Genomic characterization of multiple leptin genes and a leptin receptor gene in the Japanese medaka, Oryzias latipes. General and Comparative Endocrinology 161, 229–237.

Kurokawa, T., Uji, S., and Suzuki, T. (2005). Identification of cDNA coding for a homologue to mammalian leptin from pufferfish, Takifugu rubripes. Peptides 26, 745–750.

138

Schwartz, L.T., Sachdeva, S., and M Stahl, S. (2012). Genetic data supporting the NMDA glutamate receptor hypothesis for schizophrenia. Current Pharmaceutical Design 18, 1580–1592.

Laharrague, P., Larrouy, D., Fontanilles, A., Truel, N., Campfield, A., Tenenbaum, R., Galitzky, J., Corberand, J.X., Pénicaud, L., and Casteilla, L. (1998). High expression of leptin by human bone marrow adipocytes in primary culture. The FASEB Journal 12, 747–752.

Lam, Q.L., and Lu, L. (2007). Role of leptin in immunity. Cell and Molecular Immunology 4, 1–13.

Lam, C.-W., James, J.T., McCluskey, R., and Hunter, R.L. (2004). Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicological Sciences 77, 126–134.

Lammert, A., Kiess, W., Bottner, A., Glasow, A., and Kratzsch, J. (2001). Soluble leptin receptor represents the main leptin binding activity in human blood. Biochemical and Biophysical Research Communications 283, 982–988.

Langheinrich, U. (2003). Zebrafish: a new model on the pharmaceutical catwalk. Bioessays 25, 904–912.

Lee, A.W., Hengstler, H., Schwald, K., Berriel-Diaz, M., Loreth, D., Kirsch, M., Kretz, O., Haas, C.A., de Angelis, M.H., Herzig, S., Brummendorf, T., Klingenspor, M., Rathjen, F.G., Rozman J., Nicholson, G., Cox, R.D., Schafer, M.K.E. (2012). Functional inactivation of the genome-wide association study obesity gene neuronal growth regulator 1 in mice causes a body mass phenotype. PloS One 7, e41537.

Lee, G.-H., Proenca, R., Montez, J.M., Carroll, K.M., Darvishzadeh, J.G., Lee, J.I., and Friedman, J.M. (1996). Abnormal splicing of the leptin receptor in diabetic mice. Nature 379(6566) 632-5.

Lee, F.-Y.J., Li, Y., Yang, E.K., Yang, S.Q., Lin, H.Z., Trush, M.A., Dannenberg, A.J., and Diehl, A.M. (1999). Phenotypic abnormalities in macrophages from leptin- deficient, obese mice. American Journal of Physiology-Cell Physiology 276, C386–C394.

Leggio, L., Ferrulli, A., Cardone, S., Nesci, A., Miceli, A., Malandrino, N., Capristo, E., Canestrelli, B., Monteleone, P., Kenna, G.A., Swift, R.M., and Addolorato, G. (2012). Ghrelin system in alcohol-dependent subjects: role of plasma ghrelin levels in alcohol drinking and craving. Addiction Biology 17, 452–464.

Levraud, J.-P., Boudinot, P., Colin, I., Benmansour, A., Peyrieras, N., Herbomel, P., and Lutfalla, G. (2007). Identification of the zebrafish IFN receptor: implications for

139

the origin of the vertebrate IFN system. The Journal of Immunology 178, 4385– 4394.

Li, J., Li, F., and Zhao, A. (2006). Inflammation and leptin. Drug Discovery Today: Disease Mechanisms 3, 387–393.

Li, J., Yu, L., Zhang, H., Wu, J., Yuan, J., Li, X., and Li, M. (2009). Down-regulation of pescadillo inhibits proliferation and tumorigenicity of breast cancer cells. Cancer Science 100, 2255–2260.

Liang, C.-P., and Tall, A.R. (2001). Transcriptional profiling reveals global defects in energy metabolism, lipoprotein, and bile acid synthesis and transport with reversal by leptin treatment in ob/ob mouse liver. Journal of Biological Chemistry 276, 49066–49076.

Lieschke, G.J., Oates, A.C., Crowhurst, M.O., Ward, A.C., and Layton, J.E. (2001). Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood 98, 3087–3096.

Lim, G., Kim, H., McCabe, M.F., Chou, C.-W., Wang, S., Chen, L.L., Marota, J.J., Blood, A., Breiter, H.C., and Mao, J. (2014). A leptin-mediated central mechanism in analgesia-enhanced opioid reward in rats. The Journal of Neuroscience 34, 9779–9788.

Lin, W.-J., Hsueh, H.-M., and Chen, J.J. (2010). Power and sample size estimation in microarray studies. BMC Bioinformatics 11, 48.

Ling, J., and Söll, D. (2010). Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site. Proceedings of the National Academy of Sciences 107, 4028–4033.

Litman, R., Gupta, R., Brosh Jr, R.M., and Cantor, S.B. (2008). BRCA-FA pathway as a target for anti-tumor drugs. Anti-Cancer Agents in Medicinal Chemistry 8, 426.

Little, A.G., Kunisue, T., Kannan, K. and Seebacher, F. (2013) Thyroid hormone actions are temperature-specific and regulate thermal acclimation in zebrafish (Danio rerio). BMC Biology. 11, 26.

Liu, Q., Chen, Y., Copeland, D., Ball, H., Duff, R.J., Rockich, B., and Londraville, R.L. (2010). Expression of leptin receptor gene in developing and adult zebrafish. General and Comparative Endocrinology 166, 346–355.

Liu, Q., Dalman, M., Chen, Y., Akhter, M., Brahmandam, S., Patel, Y., Lowe, J., Thakkar, M., Gregory, A.-V., Phelps, D., et al. (2012). Knockdown of leptin A expression dramatically alters zebrafish development. General and Comparative Endocrinology 178, 562–572.

140

Lo, D., Feng, L., Li, L., Carson, M.J., Crowley, M., Pauza, M., Nguyen, A., and Reilly, C.R. (1999). Integrating innate and adaptive immunity in the whole animal. Immunological Reviews 169, 225–239.

Loffreda, S., Yang, S.Q., Lin, H.Z., Karp, C.L., Brengman, M.L., Wang, D.J., Klein, A.S., Bulkley, G.B., Bao, C., Noble, P.W., Lane, M.D., Diehl, A.M.. (1998). Leptin regulates proinflammatory immune responses. The FASEB Journal 12, 57–65.

Londraville, R.L., and Duvall, C.S. (2002). Murine leptin injections increase intracellular fatty acid-binding protein in green sunfish ( Lepomis cyanellus). General and Comparative Endocrinology 129, 56–62.

Londraville, R.L., and Niewiarowski, P.H. (2010). Leptin signaling systems in reptiles and amphibians. Leptin in Non-Mammalian Vertebrates 25–36.

Londraville, R.L., Macotela, Y., Duff, R.J., Easterling, M.R., Liu, Q., and Crespi, E.J. (2014). Comparative endocrinology of leptin: Assessing function in a phylogenetic context. General and Comparative Endocrinology 203C: 146-157.

López, I.P., Marti, A., Milagro, F.I., Zulet, M. de los A., Moreno-Aliaga, M.J., Martinez, J.A., and Miguel, C. (2003). DNA microarray analysis of genes differentially expressed in diet-induced (cafeteria) obese rats. Obesity Research 11, 188–194.

Lord, G.M., Matarese, G., Howard, J.K., Baker, R.J., Bloom, S.R., and Lechler, R.I. (1998). Leptin modulates the T-cell immune response and reverses starvation- induced immunosuppression. Nature 394, 897–901.

Lynch, W.C., and Libby, L. (1983). Naloxone suppresses intake of highly preferred saccharin solutions in food deprived and sated rats. Life Sciences 33, 1909–1914.

MacDougald, O.A., Hwang, C.-S., Fan, H., and Lane, M.D. (1995). Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes. Proceedings of the National Academy of Sciences 92, 9034–9037.

Macia, L., Delacre, M., Abboud, G., Ouk, T.-S., Delanoye, A., Verwaerde, C., Saule, P., and Wolowczuk, I. (2006). Impairment of dendritic cell functionality and steady- state number in obese mice. The Journal of Immunology 177, 5997–6006.

Madej, T., Boguski, M.S., and Bryant, S.H. (1995). Threading analysis suggests that the obese gene product may be a helical cytokine. FEBS Letters 373, 13–18.

Maffei, M., Halaas, J., Ravussin, E., Pratley, R.E., Lee, G.H., Zhang, Y., Fei, H., Kim, S., Lallone, R., Ranganathan, S., Kern., P.A., and Friedman, J.M.. (1995). Leptin

141

levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nature Medicine 1, 1155–1161.

Magnadóttir, B. (2006). Innate immunity of fish (overview). Fish & Shellfish Immunology 20, 137–151.

Mah, N., Thelin, A., Lu, T., Nikolaus, S., Kühbacher, T., Gurbuz, Y., Eickhoff, H., Klöppel, G., Lehrach, H., Mellgard, B., Costello, C.M., Schreiber, S.. (2004). A comparison of oligonucleotide and cDNA-based microarray systems. Physiological Genomics 16, 361–370.

Makimura, H., Mizuno, T.M., Yang, X.-J., Silverstein, J., Beasley, J., and Mobbs, C.V. (2001). Cerulenin mimics effects of leptin on metabolic rate, food intake, and body weight independent of the melanocortin system, but unlike leptin, cerulenin fails to block neuroendocrine effects of fasting. Diabetes 50, 733–739.

Makky, K., Duvnjak, P., Pramanik, K., Ramchandran, R., and Mayer, A.N. (2008). A whole-animal microplate assay for metabolic rate using zebrafish. Journal of Biomolecular Screening. 10: 960-7.

Mancour, L.V., Daghestani, H.N., Dutta, S., Westfield, G.H., Schilling, J., Oleskie, A.N., Herbstman, J.F., Chou, S.Z., and Skiniotis, G. (2012). Ligand-induced architecture of the leptin receptor signaling complex. Molecular Cell 48, 655–661.

Mancuso, P., Gottschalk, A., Phare, S.M., Peters-Golden, M., Lukacs, N.W., and Huffnagle, G.B. (2002). Leptin-deficient mice exhibit impaired host defense in Gram-negative pneumonia. The Journal of Immunology 168, 4018–4024.

Mancuso, P., Canetti, C., Gottschalk, A., Tithof, P.K., and Peters-Golden, M. (2004). Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity and enhancing group IVC iPLA2 (cPLA2γ) protein expression. American Journal of Physiology-Lung Cellular and Molecular Physiology 287, L497–L502.

Mancuso, P., Huffnagle, G.B., Olszewski, M.A., Phipps, J., and Peters-Golden, M. (2006). Leptin corrects host defense defects after acute starvation in murine pneumococcal pneumonia. American Journal of Respiratory and Critical Care Medicine 173, 212–218.

Mancuso, P., Myers, M.G., Goel, D., Serezani, C.H., O’Brien, E., Goldberg, J., Aronoff, D.M., and Peters-Golden, M. (2012). Ablation of leptin receptor-mediated ERK activation impairs host defense against Gram-negative pneumonia. The Journal of Immunology 189, 867–875.

Mandel, M.A., and Mahmoud, A.A. (1978). Impairment of cell-mediated immunity in mutation diabetic mice (db/db). The Journal of Immunology 120, 1375–1377.

142

Margetic, S., Gazzola, C., Pegg, G.G., and Hill, R.A. (2002). Leptin: a review of its peripheral actions and interactions. International Journal of Obesity and Related Metabolic Disorders: Journal of the International Association for the Study of Obesity 26, 1407–1433.

Mariano, G., Terrazzano, G., Coccia, E., Vito, P., Varricchio, E., and Paolucci, M. (2013). Effects of recombinant trout leptin in superoxide production and NF- κB/MAPK phosphorylation in blood leukocytes. Peptides 48, 59–69.

Marinoni, E., Corona, G., Ciardo, F., Letizia, C., Moscarini, M., Di Iorio, R. (2010). Changes in the interrelationship between leptin, resistin and adiponectin in early neonatal life. Frontiers in Bioscience 2: 52–58.

Mark, A.L., Shaffer, R.A., Correia, M.L., Morgan, D.A., Sigmund, C.D., and Haynes, W.G. (1999). Contrasting blood pressure effects of obesity in leptin-deficient ob/ob mice and agouti yellow obese mice. Journal of Hypertension 17, 1949– 1953.

Markert, C.L., and Faulhaber, I. (1965). Lactate dehydrogenase isozyme patterns of fish. Journal of Experimental Zoology 159, 319–332.

Marques, I.J., Leito, J.T., Spaink, H.P., Testerink, J., Jaspers, R.T., Witte, F., van den Berg, S., and Bagowski, C.P. (2008). Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts. Journal of Comparative Physiology B 178, 77–92.

Martín-Romero, C., Santos-Alvarez, J., Goberna, R., and Sánchez-Margalet, V. (2000). Human leptin enhances activation and proliferation of human circulating T lymphocytes. Cellular Immunology 199, 15–24.

Al Maskari, M.Y., and Alnaqdy, A.A. (2006). Correlation between serum leptin levels, body mass index and obesity in omanis. Sultan Qaboos University Medical Journal 6, 27.

Masuzaki, H., Ogawa, Y., Sagawa, N., Hosoda, K., Matsumoto, T., Mise, H., Nishimura, H., Yoshimasa, Y., Tanaka, I., Mori, T., and Nakao, K. (1997). Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nature Medicine 3, 1029–1033.

Matarese, G., Di Giacomo, A., Sanna, V., Lord, G.M., Howard, J.K., Di Tuoro, A., Bloom, S.R., Lechler, R.I., Zappacosta, S., and Fontana, S. (2001). Requirement for leptin in the induction and progression of autoimmune encephalomyelitis. The Journal of Immunology 166, 5909–5916.

143

Mathavan, S., Lee, S.G., Mak, A., Miller, L.D., Murthy, K.R.K., Govindarajan, K.R., Tong, Y., Wu, Y.L., Lam, S.H., Yang, H., Ruan, Y., Korzh, V., Gong, Z., Liu, E.T., Lufkin, T. (2005). Transcriptome analysis of zebrafish embryogenesis using microarrays. PLoS Genetics 1, e29.

Matsuda, J., Yokota, I., Tsuruo, Y., Murakami, T., Ishimura, K., Shima, K., and Kuroda, Y. (1999). Developmental changes in long-form leptin receptor expression and localization in rat brain. Endocrinology 140, 5233–5238.

De Matteis, R., and Cinti, S. (1998). Ultrastructural immunolocalization of leptin receptor in mouse brain. Neuroendocrinology 68, 412–419.

McCarthy, D.J., and Smyth, G.K. (2009). Testing significance relative to a fold-change threshold is a TREAT. Bioinformatics 25, 765–771.

McClintock, J.M., Carlson, R., Mann, D.M., and Prince, V.E. (2001). Consequences of Hox gene duplication in the vertebrates: an investigation of the zebrafish Hox paralogue group 1 genes. Development 128, 2471–2484.

McGaffin, K., Witham, W., Yester, K., and O’Donnell, C. (2011). Leptin modulates cardiac glucose metabolism and attenuates injury in acute myocardial infarction. In Circulation (Lippincott Williams and Wilkins Philadelphia, PA19106-3621 USA).

Meeker, N.D., and Trede, N.S. (2008). Immunology and zebrafish: spawning new models of human disease. Developmental & Comparative Immunology 32, 745–757.

Mendelsohn, B.A., Kassebaum, B.L., and Gitlin, J.D. (2008). The zebrafish embryo as a dynamic model of anoxia tolerance. Dev Dyn 237, 1780–1788.

Mercer, J.G., Moar, K.M., Ross, A.W., Hoggard, N., and Morgan, P.J. (2000). Photoperiod regulates arcuate nucleus POMC, AGRP, and leptin receptor mRNA in Siberian hamster hypothalamus. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 278, R271–R281.

Meyer, A., and Schartl, M. (1999). Gene and genome duplications in vertebrates: the one- to-four (-to-eight in fish) rule and the evolution of novel gene functions. Current Opinion in Cell Biology 11, 699–704.

Miller, R.A., Galecki, A., and Shmookler-Reis, R.J. (2001). Interpretation, design, and analysis of gene array expression experiments. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56, B52–B57.

Mirzoyan, Z., and Pandur, P. (2013). The iroquois complex is required in the dorsal mesoderm to ensure normal heart development in Drosophila. PloS One 8, e76498.

144

Mistry, A.M., Swick, A.G., and Romsos, D.R. (1997). Leptin rapidly lowers food intake and elevates metabolic rates in lean and ob/ob mice. The Journal of Nutrition 127, 2065–2072.

Mistry, A.M., Swick, A., and Romsos, D.R. (1999). Leptin alters metabolic rates before acquisition of its anorectic effect in developing neonatal mice. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 277, R742– R747.

Mito, N., Yoshino, H., Hosoda, T., and Sato, K. (2004). Analysis of the effect of leptin on immune function in vivo using diet-induced obese mice. Journal of Endocrinology 180, 167–173.

Montague, C.T., Prins, J.B., Sanders, L., Digby, J.E., and O’Rahilly, S. (1997). Depot- and sex-specific differences in human leptin mRNA expression: implications for the control of regional fat distribution. Diabetes 46, 342–347.

Moore, F.B., Hosey, M., and Bagatto, B. (2006). Cardiovascular system in larval zebrafish responds to developmental hypoxia in a family specific manner. Frontiers in Zoology 3.

Moraes, R.C., Blondet, A., Birkenkamp-Demtröder, K., Tirard, J., Orntoft, T.F., Gertler, A., Durand, P., Naville, D., and Begeot, M. (2003). Study of the alteration of gene expression in adipose tissue of diet-induced obese mice by microarray and reverse transcription-polymerase chain reaction analyses. Endocrinology 144, 4773–4782.

Morais, S., Knoll-Gellida, A., André, M., Barthe, C., and Babin, P.J. (2007). Conserved expression of alternative splicing variants of peroxisomal acyl-CoA oxidase 1 in vertebrates and developmental and nutritional regulation in fish. Physiological Genomics 28, 239–252.

Moran, O., and Phillip, M. (2003). Leptin: obesity, diabetes and other peripheral effects– a review. Pediatric Diabetes 4, 101–109.

Morash, B., Li, A., Murphy, P.R., Wilkinson, M., and Ur, E. (1999). Leptin gene expression in the brain and pituitary gland. Endocrinology 140, 5995–5998.

Morgan, T.H. (1910). Chromosomes and heredity. American Naturalist 449–496.

Morton, G.J., Cummings, D.E., Baskin, D.G., Barsh, G.S., and Schwartz, M.W. (2006). Central nervous system control of food intake and body weight. Nature 443, 289– 295.

Murashita, K., Uji, S., Yamamoto, T., Rønnestad, I., and Kurokawa, T. (2008). Production of recombinant leptin and its effects on food intake in rainbow trout

145

(Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 150, 377–384.

Murashita, K., Jordal, A.-E.O., Nilsen, T.O., Stefansson, S.O., Kurokawa, T., Björnsson, B.T., Moen, A.-G.G., and Rønnestad, I. (2011). Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 158, 79–86.

Myers, M.G., Cowley, M.A., and Münzberg, H. (2008). Mechanisms of leptin action and leptin resistance. Annual Reviews in Physiology 70, 537–556.

Nadon, R., and Shoemaker, J. (2002). Statistical issues with microarrays: processing and analysis. Trends in Genetics 18, 265–271.

Nasevicius, A., and Ekker, S.C. (2000). Effective targeted gene “knockdown”in zebrafish. Nature Genetics 26, 216–220.

Neely, M.N., Pfeifer, J.D., and Caparon, M. (2002). Streptococcus-zebrafish model of bacterial pathogenesis. Infection and Immunity 70, 3904–3914.

Nickola, M.W., Wold, L.E., Colligan, P.B., Wang, G.-J., Samson, W.K., and Ren, J. (2000). Leptin attenuates cardiac contraction in rat ventricular myocytes role of NO. Hypertension 36, 501–505.

Nieman, D.C., Trone, G.A., and Austin, M.D. (2003). A new handheld device for measuring resting metabolic rate and oxygen consumption. Journal of the American Dietetic Association 103, 588–593.

Niimi, M., Sato, M., Yokote, R., Tada, S., and Takahara, J. (1999). Effects of central and peripheral injection of leptin on food intake and on brain Fos expression in the Otsuka Long-Evans Tokushima Fatty rat with hyperleptinaemia. Journal of Neuroendocrinology 11, 605–612.

Niv-Spector, L., Raver, N., Friedman-Einat, M., Grosclaude, J., Gussakovsky, E. x00a0e, Livnah, O., and Gertler, A. (2005). Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor. Journal of Biochemistry. J 390, 475–484.

Novoa, B., and Figueras, A. (2012). Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases. In Current Topics in Innate Immunity II, (Springer), pp. 253–275.

Novoa, B., Figueras, A., Ashton, I., and Secombes, C.J. (1996). In vitro studies on the regulation of rainbow trout (Oncorhynchus mykiss) macrophage respiratory burst activity. Developmental & Comparative Immunology 20, 207–216.

146

Nusslein-Volhard, C., and Dahm, R. (2002). Zebrafish, A practical approach (Oxford University Press, New York, NY)

O’Brien, S.M., Scully, P., Fitzgerald, P., Scott, L.V., and Dinan, T.G. (2007). Plasma cytokine profiles in depressed patients who fail to respond to selective serotonin reuptake inhibitor therapy. Journal of Psychiatric Research 41, 326–331.

O’Connor, K.I., Taylor, A.C., and Metcalfe, N.B. (2000). The stability of standard metabolic rate during a period of food deprivation in juvenile Atlantic salmon. Journal of Fish Biology 57, 41–51.

O’Mahony, F.C., O’Donovan, C., Hynes, J., Moore, T., Davenport, J., and Papkovsky, D.B. (2005). Optical oxygen microrespirometry as a platform for environmental toxicology and animal model studies. Environmental Science & Technology 39, 5010–5014.

O’Rahilly, S. (2009). Human genetics illuminates the paths to metabolic disease. Nature 462, 307.

Ong, K.K., Ahmed, M.L., Sherriff, A., Woods, K.A., Watts, A., Golding, J., and Dunger, D.B. (1999). Cord blood leptin is associated with size at birth and predicts infancy weight gain in humans. The Journal of Clinical Endocrinology & Metabolism 84, 1145–1148.

Ozata, M., Ozdemir, I.C., and Licinio, J. (1999). Human leptin deficiency caused by a missense mutation: multiple endocrine defects, decreased sympathetic tone, and immune system dysfunction indicate new targets for leptin action, greater central than peripheral resistance to the effects of leptin, and spontaneous correction of leptin-mediated defects. The Journal of Clinical Endocrinology & Metabolism 84, 3686–3695.

Paily, O., and Agans, R. (2012). Application of phylogenetic microarrays to interrogation of human microbiota. FEMS Microbiology Ecology 79, 2–11.

Pallares, P., Garcia-Fernandez, R.A., Criado, L.M., Letelier, C.A., Fernandez-Toro, J.M., Esteban, D., Flores, J.M., and Gonzalez-Bulnes, A. (2010). Substantiation of ovarian effects of leptin by challenging a mouse model of obesity/type 2 diabetes. Theriogenology 73, 1088–1095.

Papathanassoglou, E., El-Haschimi, K., Li, X.C., Matarese, G., Strom, T., and Mantzoros, C. (2006). Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice. The Journal of Immunology 176, 7745–7752.

147

Papaioannou, E., Utari, P.D., and Quax, W.J. (2013). Choosing an appropriate infection model to study quorum sensing inhibition in Pseudomonas infections. International Journal of Molecular Sciences 14, 19309–19340.

Du Pasquier, L. (1982). Antibody diversity in lower vertebrates—why is it so restricted? Nature296, 311-313.

Pawitan, Y., Michiels, S., Koscielny, S., Gusnanto, A., and Ploner, A. (2005). False discovery rate, sensitivity and sample size for microarray studies. Bioinformatics 21, 3017–3024.

Paz-Filho, G., Wong, M.-L., and Licinio, J. (2010). The procognitive effects of leptin in the brain and their clinical implications. International Journal of Clinical Practice 64, 1808–1812.

De Pedro, N., Martinez-Alvarez, R., and Delgado, M.J. (2006). Acute and chronic leptin reduces food intake and body weight in goldfish (Carassius auratus). Journal of Endocrinology 188, 513–520.

Peelman, F., Waelput, W., Iserentant, H., Lavens, D., Eyckerman, S., Zabeau, L., and Tavernier, J. (2004). Leptin: linking adipocyte metabolism with cardiovascular and autoimmune diseases. Progress in Lipid Research 43, 283–301.

Pounds, S.B. (2006). Estimation and control of multiple testing error rates for microarray studies. Briefings in Bioinformatics 7, 25–36.

Portner, H.O. and Knust, R. (2007) Climate change affects marine fishes through the oxygen limitation of thermal tolerance. Science 315, 95–97.

Procaccini, C., Lourenco, E.V., Matarese, G., and La Cava, A. (2009). Leptin signaling: a key pathway in immune responses. Current Signal Transduction Therapy 4, 22.

Procaccini, C., De Rosa, V., Galgani, M., Abanni, L., Calì, G., Porcellini, A., Carbone, F., Fontana, S., Horvath, T.L., La Cava, A., and Matarese, G. (2010). An oscillatory switch in mTOR kinase activity sets regulatory T cell responsiveness. Immunity 33, 929–941.

Procaccini, C., Jirillo, E., and Matarese, G. (2012). Leptin as an immunomodulator. Molecular Aspects of Medicine 33, 35–45.

Prokop, J.W., Duff, R.J., Ball, H.C., Copeland, D.L., and Londraville, R.L. (2012). Leptin and leptin receptor: analysis of a structure to function relationship in interaction and evolution from humans to fish. Peptides 38, 326–336.

Prokop, J.W., Schmidt, C., Gasper, D., Duff, R.J., Milsted, A., Ohkubo, T., Ball, H.C., Shawkey, M.D., Mays Jr, H.L., Cogburn, L.A., Londraville., R.L.. (2014).

148

Discovery of the elusive leptin in birds: identification of several “missing links” in the evolution of leptin and its receptor. PloS One 9, e92751.

Proulx, K., Richard, D., and Walker, C.-D. (2002). Leptin regulates appetite-related neuropeptides in the hypothalamus of developing rats without affecting food intake. Endocrinology 143, 4683–4692.

Ptak, A., Kolaczkowska, E., and Gregoraszczuk, E.L. (2013). Leptin stimulation of cell cycle and inhibition of apoptosis gene and protein expression in OVCAR-3 ovarian cancer cells. Endocrine 43, 394–403.

Rauta, P.R., Nayak, B., and Das, S. (2012). Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunology Letters 148, 23–33.

Rawls, J.F., Mahowald, M.A., Ley, R.E., and Gordon, J.I. (2006). Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127, 423–433.

Renn, S.C., Aubin-Horth, N., and Hofmann, H.A. (2004). Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray. BMC Genomics 5, 42.

Robertson, S.A., Leinninger, G.M., and Myers Jr, M.G. (2008). Molecular and neural mediators of leptin action. Physiology & Behavior 94, 637–642.

Robinson, J.M. (2009). Phagocytic leukocytes and reactive oxygen species. Histochemistry and Cell Biology 131, 465–469.

Ronnestad, I., Nilsen, T.O., Murashita, K., Angotzi, A.R., Gamst Moen, A.-G., Stefansson, S.O., Kling, P., Thrandur Björnsson, B., and Kurokawa, T. (2010). Leptin and leptin receptor genes in Atlantic salmon: cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status. General and Comparative Endocrinology 168, 55–70.

De Rosa, V., Procaccini, C., Calì, G., Pirozzi, G., Fontana, S., Zappacosta, S., La Cava, A., and Matarese, G. (2007). A key role of leptin in the control of regulatory T cell proliferation. Immunity 26, 241–255.

Rowell, M.J. (1995). Colorimetric method for CO2 measurement in soils. Soil Biology and Biochemistry 27, 373–375.

Rubin, G.M., and Lewis, E.B. (2000). A brief history of Drosophila’s contributions to genome research. Science 287, 2216–2218.

149

Rubinstein, A.L. (2006). Zebrafish assays for drug toxicity screening. Expert Opinion on Drug Metabolism and Toxicology. 2(2), 231-240.

Rummel, C., Inoue, W., Poole, S., and Luheshi, G.N. (2009). Leptin regulates leukocyte recruitment into the brain following systemic LPS-induced inflammation. Molecular Psychiatry 15, 523–534.

Ruxton, G.D. (2006). The unequal variance t-test is an underused alternative to Student’s t-test and the Mann–Whitney U test. Behavioral Ecology 17, 688–690.

Sáinz, N., Rodríguez, A., Catalán, V., Becerril, S., Ramírez, B., Gómez-Ambrosi, J., and Frühbeck, G. (2009). Leptin administration favors muscle mass accretion by decreasing FoxO3a and increasing PGC-1α in ob/ob mice. PLoS One 4, e6808.

Salomonis, N., Hanspers, K., Zambon, A.C., Vranizan, K., Lawlor, S.C., Dahlquist, K.D., Doniger, S.W., Stuart, J., Conklin, B.R., and Pico, A.R. (2007). GenMAPP 2: new features and resources for pathway analysis. BMC Bioinformatics 8, 217.

Sanchez-pozo, C., Rodriquez-Bano, J., Dominquez- Castellano, A., Muniain, M.A., Goberna, R., and Sanchez-Margalet, V. (2003). Leptin stimulates the oxidative burst in control monocytes but attenuates the oxidative burst in monocytes from HIV-infected patients. Clinical & Experimental Immunology 134, 464–469.

Sauberlich, H.E. (1984). Implications of nutritional status on human biochemistry, physiology, and health. Clinical Biochemistry 17, 132–142.

Scarpace, P.J., and Zhang, Y. (2009). Leptin resistance: a prediposing factor for diet- induced obesity. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 296, R493–R500.

Scarpace, P.J., Matheny, M., Pollock, B.H., and Tumer, N. (1997). Leptin increases uncoupling protein expression and energy expenditure. American Journal of Physiology-Endocrinology And Metabolism 273, E226–E230.

Scarth, J.P. (2006). Modulation of the growth hormone-insulin-like growth factor (GH- IGF) axis by pharmaceutical, nutraceutical and environmental xenobiotics: an emerging role for xenobiotic-metabolizing enzymes and the transcription factors regulating their expression. A review. Xenobiotica 36, 119–218.

Schmid, P.M., Heid, I., Buechler, C., Steege, A., Resch, M., Birner, C., Endemann, D.H., Riegger, G.A., and Luchner, A. (2012). Expression of fourteen novel obesity- related genes in zucker diabetic fatty rats. Cardiovasc Diabetol 11, 48.

Schmid, S.M., Hallschmid, M., Jauch- Chara, K., Born, J., and Schultes, B. (2008). A single night of sleep deprivation increases ghrelin levels and feelings of hunger in normal-weight healthy men. Journal of Sleep Research 17, 331–334.

150

Schneeberger, M., Gomis, R., and Claret, M. (2014). Hypothalamic and brainstem neuronal circuits controlling homeostatic energy balance. Journal of Endocrinology 220, T25–T46.

Schneider, J.E., Zhou, D., and Blum, R.M. (2000). Leptin and metabolic control of reproduction. Hormones and Behavior 37, 306–326.

Schneider, T.J., Fischer, G.M., Donohoe, T.J., Colarusso, T.P., and Rothstein, T.L. (1999). A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes. The Journal of Experimental Medicine 189, 949–956.

Schwartz, M.W., Peskind, E., Raskind, M., Boyko, E.J., and Porte, D. (1996). Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. Nature Medicine 2, 589–593.

Sena, A., Sarliève, L.L., and Rebel, G. (1985). Brain myelin of genetically obese mice. Journal of the Neurological Sciences 68, 233–244.

Serkova, N.J., Jackman, M., Brown, J.L., Liu, T., Hirose, R., Roberts, J.P., Maher, J.J., and Niemann, C.U. (2006). Metabolic profiling of livers and blood from obese Zucker rats. Journal of Hepatology 44, 956–962.

Seron, K., Couturier, C., Belouzard, S., Bacart, J., Monté, D., Corset, L., Bocquet, O., Dam, J., Vauthier, V., Lecøeur, C, Bailleul, B., Hoflack, B., Froguel, P., Jockers, R., Rouille, Y. (2011). Endospanins regulate a postinternalization step of the leptin receptor endocytic pathway. Journal of Biological Chemistry 286, 17968– 17981.

Sharma, D., Wang, J., Fu, P.P., Sharma, S., Nagalingam, A., Mells, J., Handy, J., Page, A.J., Cohen, C., Anania, F.A., Saxena, N.K. (2010). Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis. Hepatology 52, 1713–1722.

Shimabukuro, M., Koyama, K., Chen, G., Wang, M.-Y., Trieu, F., Lee, Y., Newgard, C.B., and Unger, R.H. (1997). Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proceedings of the National Academy of Sciences 94, 4637–4641.

Sieger, D., Stein, C., Neifer, D., van der Sar, A.M., and Leptin, M. (2009). The role of gamma interferon in innate immunity in the zebrafish embryo. Disease Models & Mechanisms 2, 571–581.

151

Siegmund, B., Lear-Kaul, K.C., Faggioni, R., and Fantuzzi, G. (2002). Leptin deficiency, not obesity, protects mice from Con A-induced hepatitis. European Journal of Immunology 32, 552–560.

Siegrist-Kaiser, C.A., Pauli, V., Juge-Aubry, C.E., Boss, O., Pernin, A., Chin, W.W., Cusin, I., Rohner-Jeanrenaud, F., Burger, A.G., Zapf, J., and Meijer, C.A. (1997). Direct effects of leptin on brown and white adipose tissue. Journal of Clinical Investigation 100, 2858.

Sierra-Honigmann, M.R., Nath, A.K., Murakami, C., Garcı́a-Cardeña, G., Papapetropoulos, A., Sessa, W.C., Madge, L.A., Schechner, J.S., Schwabb, M.B., Polverini, P.J., et al. (1998). Biological action of leptin as an angiogenic factor. Science 281, 1683–1686.

Silverstein, J.T., and Plisetskaya, E.M. (2000). The effects of NPY and insulin on food intake regulation in fish. American Zoologist 40, 296–308.

Smith, S.E., Li, J., Garbett, K., Mirnics, K., and Patterson, P.H. (2007). Maternal immune activation alters fetal brain development through interleukin-6. The Journal of Neuroscience 27, 10695–10702.

Van Soest, J.J., Stockhammer, O.W., Ordas, A., Bloemberg, G.V., Spaink, H.P., and Meijer, A.H. (2011). Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda. BMC Immunology 12, 58.

Sole, C., Dolcet, X., Segura, M.F., Gutierrez, H., Diaz-Meco, M.-T., Gozzelino, R., Sanchis, D., Bayascas, J.R., Gallego, C., Moscat, J., Davies, A.M, Comella, J.X. (2004). The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-κB signaling. The Journal of Cell Biology 167, 479–492.

Soukas, A., Cohen, P., Socci, N.D., and Friedman, J.M. (2000). Leptin-specific patterns of gene expression in white adipose tissue. Genes & Development 14, 963–980.

Spence, R., Gerlach, G., Lawrence, C., and Smith, C. (2008). The behaviour and ecology of the zebrafish, Danio rerio. Biological Reviews 83, 13–34.

Spiegel, K., Leproult, R., L’Hermite-Balériaux, M., Copinschi, G., Penev, P.D., and Van Cauter, E. (2004a). Leptin levels are dependent on sleep duration: relationships with sympathovagal balance, carbohydrate regulation, cortisol, and thyrotropin. The Journal of Clinical Endocrinology & Metabolism 89, 5762–5771.

Spiegel, K., Tasali, E., Penev, P., and Van Cauter, E. (2004b). Brief communication: Sleep curtailment in healthy young men is associated with decreased leptin levels,

152

elevated ghrelin levels, and increased hunger and appetite. Ann. Intern. Med. 141, 846–850.

Stackley, K.D., Beeson, C.C., Rahn, J.J., and Chan, S.S. (2011). Bioenergetic profiling of zebrafish embryonic development. PloS One 6, e25652.

Stafford, J., Neumann, N.F., and Belosevic, M. (1999). Inhibition of macrophage activity by mitogen-induced goldfish leukocyte deactivating factor. Developmental & Comparative Immunology 23, 585–596.

Stehling, O., Doring, H., Ertl, J., Preibisch, G., and Schmidt, I. (1996). Leptin reduces juvenile fat stores by altering the circadian cycle of energy expenditure. American Journal of Physiology-Regulatory Integrative and Comparative Physiology 40, R1770.

Stehling, O., Döring, H., Nuesslein-Hildesheim, B., Olbort, M., and Schmidt, I. (1997). Leptin does not reduce body fat content but augments cold defense abilities in thermoneutrally reared rat pups. Pflügers Archiv 434, 694–697.

Stein, C., Caccamo, M., Laird, G., and Leptin, M. (2007). Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish. Genome Biol 8, R251.

Steppan, C.M., and Swick, A.G. (1999). A role for leptin in brain development. Biochemical and Biophysical Research Communications 256, 600–602.

Stice, E., Figlewicz, D.P., Gosnell, B.A., Levine, A.S., and Pratt, W.E. (2013). The contribution of brain reward circuits to the obesity epidemic. Neuroscience & Biobehavioral Reviews 37, 2047–2058.

St-Pierre, J., and Tremblay, M.L. (2012). Modulation of leptin resistance by protein tyrosine phosphatases. Cell Metabolism 15, 292–297.

Streisinger, G., Walker, C., Dower, N., Knauber, D., and Singer, F. (1981). Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 291, 293– 296.

Takeda, S., Elefteriou, F., Levasseur, R., Liu, X., Zhao, L., Parker, K.L., Armstrong, D., Ducy, P., and Karsenty, G. (2002). Leptin regulates bone formation via the sympathetic nervous system. Cell 111, 305–317.

Tartaglia, L.A. (1997). The leptin receptor. Journal of Biological Chemistry 272, 6093– 6096.

Tartaglia, L.A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Richards, G.J., Campfield, L.A., Clark, F.T., Deeds, J., Muir., C., Sanker, S., Moriarty, A.,

153

Moore, K.J., Smutko, J.S., Mays, G.G., Wool, E.A., Monroe, C.A., Tepper., R.I. (1995). Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263–1271.

Tataranni, P.A., Monroe, M.B., Dueck, C.A., Traub, S.A., Nicolson, M., Manore, M.M., Matt, K.S., and Ravussin, E. (1997). Adiposity, plasma leptin concentration and reproductive function in active and sedentary females. International Journal of Obesity and Related Metabolic Disorders: Journal of the International Association for the Study of Obesity 21, 818–821.

Taylor, J.S., and Raes, J. (2004). Duplication and divergence: the evolution of new genes and old ideas. Annu. Rev. Genet. 38, 615–643.

Taylor, J.S., Van de Peer, Y., Braasch, I., and Meyer, A. (2001). Comparative genomics provides evidence for an ancient genome duplication event in fish. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 356, 1661–1679.

Tinoco, A.B., Nisembaum, L.G., Isorna, E., Delgado, M.J., and de Pedro, N. (2012). Leptins and leptin receptor expression in the goldfish (Carassius auratus). Regulation by food intake and fasting/overfeeding conditions. Peptides 34, 329– 335.

Torraca, V., Masud, S., Spaink, H.P., and Meijer, A.H. (2014). Macrophage-pathogen interactions in infectious diseases: new therapeutic insights from the zebrafish host model. Disease Models & Mechanisms 7, 785–797.

Trede, N.S., Langenau, D.M., Traver, D., Look, A.T., and Zon, L.I. (2004). The use of zebrafish to understand immunity. Immunity 20, 367–379.

Trombley, S., Maugars, G., Kling, P., Björnsson, B.T., and Schmitz, M. (2012). Effects of long-term restricted feeding on plasma leptin, hepatic leptin expression and leptin receptor expression in juvenile Atlantic salmon (Salmo salar). General and Comparative Endocrinology 175, 92–99.

Vaisse, C., Halaas, J.L., Horvath, C.M., Darnell, J.E., Stoffel, M., and Friedman, J.M. (1996). Leptin activation of Stat3 in the hypothalamus of wild–type and ob/ob mice but not db/db mice. Nature Genetics 14, 95–97.

Volkoff, H., Joy Eykelbosh, A., and Ector Peter, R. (2003). Role of leptin in the control of feeding of goldfish Carassius auratus: interactions with cholecystokinin, neuropeptide Y and orexin A, and modulation by fasting. Brain Research 972, 90–109.

Waddington, C.H. (1942). Canalization of development and the inheritance of acquired characters. Nature 150 (3811): 563–565.

154

Wagner, M., Noguera, D.R., Juretschko, S., Rath, G., Koops, H.-P., and Schleifer, K.-H. (1998). Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia- oxidizing bacteria in activated sludge. Water Science and Technology 37, 441– 449.

Walley, A.J., Jacobson, P., Falchi, M., Bottolo, L., Andersson, J.C., Petretto, E., Bonnefond, A., Vaillant, E., Lecoeur, C., Vatin, V., Jernas, M., Balding, D., Petteni, M., Park, Y.S., Aitman, T., Richardson, S., Sjostrom, L., Carlsson, L.M., Froguel., P. (2011). Differential coexpression analysis of obesity-associated networks in human subcutaneous adipose tissue. International Journal of Obesity 36, 137–147.

Wang, J., Liu, R., Hawkins, M., Barzilai, N., and Rossetti, L. (1998). A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature 393, 684–688.

Wang, J., Duncan, D., Shi, Z., and Zhang, B. (2013). WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013. Nucleic Acids Research gkt439.

Wauman, J., and Tavernier, J. (2010). Leptin receptor signaling: pathways to leptin resistance. Frontiers in Bioscience (Landmark Edition) 16, 2771–2793.

Weigle, D.S. (1994). Appetite and the regulation of body composition. The FASEB Journal 8, 302–310.

Wells, P., and Pinder, A. (1996). The respiratory development of Atlantic salmon. II. Partitioning of oxygen uptake among gills, yolk sac and body surfaces. Journal of Experimental Biology 199, 2737–2744.

Westerfield, M. (1994). The zebrafish book: a guide for the laboratory use of zebrafish Danio (Brachydanio rerio) (University of Oregon Press, Institute of Neuroscience).

Westerfield, M. (2000). The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio) (University of Oregon Press).

Van der Weyden, L., and Adams, D.J. (2007). The Ras-association domain family (RASSF) members and their role in human tumourigenesis. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1776, 58–85.

White, D.W., Kuropatwinski, K.K., Devos, R., Baumann, H., and Tartaglia, L.A. (1997). Leptin receptor (OB-R) signaling Cytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization. Journal of Biological Chemistry 272, 4065–4071.

155

Winnicki, M., Phillips, B.G., Accurso, V., Van De Borne, P., Shamsuzzaman, A., Patil, K., Narkiewicz, K., and Somers, V.K. (2001). Independent association between plasma leptin levels and heart rate in heart transplant recipients. Circulation 104, 384–386.

Witten, D., and Tibshirani, R. (2007). A comparison of fold-change and the t-statistic for microarray data analysis. Technical Report, Stanford University. 1-13.

Woese, C.R., Olsen, G.J., Ibba, M., and Söll, D. (2000). Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process. Microbiology and Molecular Biology Reviews 64, 202–236.

Won, E.T., Baltzegar, D.A., Picha, M.E., and Borski, R.J. (2012). Cloning and characterization of leptin in a Perciform fish, the striped bass (Morone saxatilis): Control of feeding and regulation by nutritional state. General and Comparative Endocrinology 178, 98–107.

Zanetta, C., Riboldi, G., Nizzardo, M., Simone, C., Faravelli, I., Bresolin, N., Comi, G.P., and Corti, S. (2014). Molecular, genetic and stem cell-mediated therapeutic strategies for spinal muscular atrophy (SMA). Journal of Cellular and Molecular Medicine 18, 187–196.

Zarkesh-Esfahani, H., Pockley, G., Metcalfe, R.A., Bidlingmaier, M., Wu, Z., Ajami, A., Weetman, A.P., Strasburger, C.J., and Ross, R.J. (2001). High-dose leptin activates human leukocytes via receptor expression on monocytes. The Journal of Immunology 167, 4593–4599.

Zarkesh-Esfahani, H., Pockley, A.G., Wu, Z., Hellewell, P.G., Weetman, A.P., and Ross, R.J. (2004). Leptin indirectly activates human neutrophils via induction of TNF-α. The Journal of Immunology 172, 1809–1814.

Zhang, X.-Y., and Wang, D.-H. (2006). Thermogenesis, food intake and serum leptin in cold-exposed lactating Brandt’s voles Lasiopodomys brandtii. Journal of Experimental Biology 210, 512–521.

Zhang, B., Kirov, S., and Snoddy, J. (2005). WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Research 33, W741–W748.

Zhang, F., Basinski, M.B., Beals, J.M., Briggs, S.L., Churgay, L.M., Clawson, D.K., DiMarchi, R.D., Furman, T.C., Hale, J.E., Hsiung, H.M., Schoner, B.E., Smith, D.P., Zhang, X.Y., Wery, J.P., Schevitz R.W. (1997). Crystal structure of the obese protein Ieptin-E100. Nature 387, 206-209.

Zhang, H., Chen, H., Zhang, Y., Li, S., Lu, D., Zhang, H., Meng, Z., Liu, X., and Lin, H. (2013a). Molecular cloning, characterization and expression profiles of multiple

156

leptin genes and a leptin receptor gene in orange-spotted grouper ( Epinephelus coioides). General and Comparative Endocrinology 181, 295–305.

Zhang, J., Li, T., Xu, L., Li, W., Cheng, M., Zhuang, J., Chen, Y., and Xu, W. (2013b). Leptin promotes ossification through multiple ways of bone metabolism in osteoblast: a pilot study. Gynecological Endocrinology 29, 758–762.

Zhang, M., Gosnell, B.A., and Kelley, A.E. (1998). Intake of high-fat food is selectively enhanced by muopioid receptor stimulation within the nucleus accumbens. Journal of Pharmacology and Experimental Therapeutics 285, 908–914.

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J.M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425–432.

Zhang, Y., Wilsey, J.T., Frase, C.D., Matheny, M.M., Bender, B.S., Zolotukhin, S., Scarpace, P.J. (2002). Peripheral but not central leptin prevents the immunosuppression associated with hypoleptinemia in rats. Journal of Endocrinology 174, 455–461.

Zhao, T., Zhang, Z.-N., Rong, Z., and Xu, Y. (2011). Immunogenicity of induced pluripotent stem cells. Nature 474, 212–215.

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APPENDIX

Table A1 - All significantly regulated genes involved in leptin A knockdown in the developing zebrafish embryo at 72 hours post fertilization (hpf). Gene assignment and symbol is taken directly from microarray data generated by PARTEK software.

Gene Assignment Gene Adj P Fold Symbol value Change XM_003200280 // LOC100537029 // immunoglobulin superfamily LOC1005370 6.66E-08 35.8 DCC subclass member 3-like / 29 --- 1.65E-08 32.6 XM_693882 // LOC570404 // uncharacterized LOC570404 // --- // LOC570404 2.13E-06 20.2 570404 /// ENSDART0000005 --- 1.76E-08 18.7 XM_001339896 // LOC799595 // immunoglobulin superfamily DCC LOC799595 4.55E-08 17.1 subclass member 3-like // - --- 1.76E-09 17.0 --- 7.38E-07 11.8 BC151913 // si:dkey-204l11.1 // si:dkey-204l11.1 // --- // 100006301 si:dkey- 2.84E-07 10.2 204l11.1 ENSDART00000062845 // mmp9 // matrix metalloproteinase 9 // --- mmp9 0.000245 9.9 // 406397 /// NM_213123 524 NM_001200012 // LOC562935 // heat shock cognate 70 kDa protein LOC562935 2.28E-05 9.0 // --- // 562935 /// ENS ENSDART00000055623 // hbbe3 // hemoglobin beta embryonic-3 // hbbe3 2.46E-06 8.6 --- // 30596 /// NM_00101 NM_001161552 // fosl1a // FOS-like antigen 1a // --- // 564241 /// fosl1a 4.85E-05 8.5 ENSDART00000008373 / NM_001113589 // hsp70l // heat shock cognate 70-kd protein, like // hsp70l 6.51E-05 7.7 --- // 560210 /// N NM_131099 // foxn4 // forkhead box N4 // --- // 30315 /// foxn4 2.71E-08 7.3 ENSDART00000008994 // foxn4 / --- 3.26E-06 7.2 ENSDART00000003646 // optc // opticin // --- // 445189 /// optc 8.84E-05 7.0 NM_001003583 // optc // opti ENSDART00000033848 // brf1a // BRF1 homolog, subunit of RNA brf1a 5.59E-09 6.8 polymerase III transcriptio NM_001173501 // whsc2 // Wolf-Hirschhorn syndrome candidate 2 whsc2 1.17E-06 6.7 // --- // 559677 /// ENSD --- 0.000680 6.7 485 0.000507 6.4 418 NM_199896 // iars // isoleucyl-tRNA synthetase // --- // 334393 /// iars 6.06E-07 6.3 ENSDART00000004423 --- 2.53E-07 6.3 ENSDART00000006180 // dla // deltaA // --- // 30131 /// NM_130954 dla 1.31E-06 6.3 // dla // deltaA // -

158

NM_001045353 // hes2.2 // hairy and enhancer of split 2.2 // --- // hes2.2 3.73E-06 6.1 751634 /// ENSDART0 --- 7.80E-06 6.0 ENSDART00000122681 // nes // nestin // --- // 100150939 /// nes 2.81E-08 6.0 XM_001919887 // nes // nest NM_131441 // notch1a // notch homolog 1a // --- // 30718 /// notch1a 6.98E-07 5.9 ENSDART00000129224 // notc --- 7.26E-07 5.9 XM_682888 // wu:fi04f09 // wu:fi04f09 // --- // 559540 wu:fi04f09 1.29E-07 5.8 ENSDART00000111598 // cxcl-c1c // chemokine (C-X-C motif) cxcl-c1c 4.09E-06 5.6 ligand C1c // --- // 795785 / NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 1.67E-06 5.6 --- 6.63E-06 5.5 --- 9.59E-08 5.4 --- 8.96E-08 5.4 XM_690545 // wu:fc14a10 // wu:fc14a10 // --- // 567253 /// wu:fc14a10 0.000278 5.3 ENSDART00000112838 // wu:fc1 174 XM_001340105 // LOC799825 // protein lin-28 homolog A-like // --- LOC799825 0.000129 5.2 // 799825 /// ENSDART 481 --- 0.000957 5.2 809 ENSDART00000055428 // cbx7a // chromobox homolog 7a // --- // cbx7a 0.000179 5.2 550551 /// NM_001017853 / 022 NM_001045073 // hsp90aa1.2 // heat shock protein 90, alpha hsp90aa1.2 0.000118 5.1 (cytosolic), class A member 315 --- 1.33E-05 5.1 ENSDART00000078563 // neurog1 // neurogenin 1 // --- // 30239 /// neurog1 1.57E-06 5.0 NM_131041 // neurog1 BC124167 // azi1 // 5-azacytidine induced gene 1 // --- // 563066 azi1 7.07E-06 5.0 NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 9.13E-06 4.8 ENSDART00000019259 // dlb // deltaB // --- // 30141 /// NM_130958 dlb 1.05E-08 4.8 // dlb // deltaB // - --- 1.20E-06 4.7 ENSDART00000151494 // wu:fj64h06 // wu:fj64h06 // --- // 336342 wu:fj64h06 1.15E-05 4.7 /// XM_002665873 // wu: NM_153673 // unc45b // unc-45 homolog B (C. elegans) // --- // unc45b 0.000520 4.6 266640 /// ENSDART000000 767 XM_001339301 // LOC798927 // uncharacterized LOC798927 // --- LOC798927 0.000987 4.6 // 798927 799 --- 0.000437 4.5 418 --- 2.16E-06 4.5 DQ360116 // prtgb // protogenin homolog b (Gallus gallus) // --- // prtgb 7.33E-07 4.5 572241 --- 5.51E-08 4.4 --- 7.33E-06 4.4 --- 2.90E-05 4.4 ENSDART00000025428 // epha2 // eph receptor A2 // --- // 30689 /// epha2 4.81E-07 4.3 NM_131415 // epha2 / --- 0.000323 4.3 872 --- 1.61E-08 4.2

159

XM_001919922 // LOC557824 // heat shock protein 105 kDa-like // - LOC557824 1.68E-07 4.2 -- // 557824 /// ENSDA --- 2.87E-06 4.1 ENSDART00000021299 // nmd3 // NMD3 homolog (S. cerevisiae) // nmd3 6.19E-05 4.1 --- // 541444 /// NM_0010 ENSDART00000122353 // LOC553492 // uncharacterized LOC553492 4.12E-06 4.0 LOC553492 // --- // 553492 /// ENSDA ENSDART00000091158 // irg1l // immunoresponsive gene 1, like // - irg1l 0.000630 4.0 -- // 562007 /// NM_00 192 --- 1.67E-06 4.0 XM_682888 // wu:fi04f09 // wu:fi04f09 // --- // 559540 wu:fi04f09 0.000156 4.0 145 --- 9.55E-07 3.9 ENSDART00000099224 // dld // deltaD // --- // 30138 /// NM_130955 dld 2.72E-07 3.9 // dld // deltaD // - ENSDART00000110219 // zgc:171476 // zgc:171476 // --- // 334271 /// zgc:171476 0.000291 3.9 NM_001114886 // zgc 494 NM_001044310 // aars // alanyl-tRNA synthetase // --- // 324940 /// aars 6.37E-06 3.9 NM_001040035 // aar --- 1.27E-07 3.8 BC117615 // arhgef1b // Rho guanine nucleotide exchange factor arhgef1b 3.84E-05 3.8 (GEF) 1b // --- // 55798 ENSDART00000038727 // DDX47 (1 of 2) // DEAD (Asp-Glu-Ala- DDX47 5.31E-05 3.7 Asp) box polypeptide 47 // -- ENSDART00000014127 // si:dkey-3j24.1 // si:dkey-3j24.1 // --- // si:dkey- 4.83E-08 3.7 557055 /// ENSDART0000 3j24.1 NM_131531 // hoxc1a // homeo box C1a // --- // 58046 /// hoxc1a 0.000517 3.7 ENSDART00000103131 // hoxc1a / 812 --- 0.000861 3.7 983 NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 1.01E-06 3.7 NM_199820 // eif3s10 // eukaryotic translation initiation factor 3, eif3s10 0.000411 3.7 subunit 10 (theta) 755 ENSDART00000109892 // SAMD9 // sterile alpha motif domain SAMD9 3.43E-07 3.6 containing 9 // --- // --- NM_001172556 // bxdc2 // brix domain containing 2 // --- // 402823 bxdc2 2.82E-06 3.6 /// ENSDART000000330 NM_001039636 // smyd1b // SET and MYND domain containing 1b smyd1b 0.000595 3.6 // --- // 569027 /// BC1630 933 --- 0.000425 3.6 446 --- 0.000425 3.6 571 DQ851840 // prdm1b // PR domain containing 1b, with ZNF domain prdm1b 0.000254 3.6 // --- // 569677 113 NM_001258317 // im:7137886 // im:7137886 // --- // 449866 /// im:7137886 0.000248 3.6 HM114349 // im:7137886 // 451 NM_001145786 // dnttip2 // deoxynucleotidyltransferase, terminal, dnttip2 0.000208 3.5 interacting protein 2 522 ENSDART00000056005 // ascl1a // achaete-scute complex-like 1a ascl1a 1.54E-05 3.5 (Drosophila) // --- // 30 XM_002665422 // LOC100334443 // zinc finger protein 36, C3H1 LOC1003344 0.000131 3.5 type-like 1-like // --- // 43 722 XM_002666924 // LOC100333070 // uncharacterized LOC1003330 0.000415 3.4 LOC100333070 // --- // 100333070 /// EN 70 257 --- 0.000392 3.4 152 ENSDART00000073440 // DNAJA4 // DnaJ (Hsp40) homolog, DNAJA4 0.000223 3.4 subfamily A, member 4 // --- // - 849

160

ENSDART00000074499 // olig4 // oligodendrocyte transcription olig4 5.09E-05 3.3 factor 4 // --- // 324857 NM_200631 // srfl // serum response factor like // --- // 393604 /// srfl 7.83E-07 3.3 BC057414 // srfl / XM_688618 // LOC565341 // uncharacterized LOC565341 // --- // LOC565341 4.81E-05 3.3 565341 NM_199658 // insm1b // insulinoma-associated 1b // --- // 323882 /// insm1b 4.81E-06 3.3 ENSDART00000075331 ENSDART00000084411 // LOC568788 // novel protein similar to LOC568788 6.33E-07 3.3 vertebrate ADAM metallopept --- 0.000310 3.3 252 XM_691849 // si:ch73-21g5.7 // si:ch73-21g5.7 // --- // 568516 /// si:ch73- 3.07E-05 3.3 ENSDART00000142921 / 21g5.7 ENSDART00000109394 // her13 // hairy-related 13 // --- // 550600 /// her13 3.58E-05 3.3 NM_001017901 // he ENSDART00000014822 // coe2 // coe2 // --- // 30692 /// NM_131418 coe2 0.000135 3.3 // coe2 // coe2 // --- 409 BC135096 // zgc:163061 // zgc:163061 // --- // 100037379 /// zgc:163061 0.000630 3.3 ENSDART00000109720 // zgc: 401 BC049436 // mphosph10 // M-phase phosphoprotein 10 (U3 small mphosph10 3.16E-05 3.2 nucleolar ribonucleoprotei --- 0.000155 3.2 384 NM_200573 // onecut1 // one cut domain, family member 1 // --- // onecut1 3.30E-06 3.2 393545 /// ENSDART000 --- 0.000155 3.2 828 --- 0.000155 3.2 979 --- 0.000360 3.2 841 ENSDART00000022060 // atf3 // activating transcription factor 3 // atf3 2.69E-05 3.2 --- // 393939 /// NM NM_214716 // hspa4a // heat shock protein 4a // --- // 335865 /// hspa4a 0.000225 3.2 ENSDART00000021037 // 059 --- 9.67E-07 3.2 NM_001034986 // lama1 // laminin, alpha 1 // --- // 569971 /// lama1 3.16E-06 3.2 DQ131910 // lama1 // lam BC068429 // zgc:85936 // zgc:85936 // --- // 406563 /// zgc:85936 1.59E-05 3.2 ENSDART00000123163 // zgc:85936 ENSDART00000129223 // PHF21B (1 of 2) // PHD finger protein PHF21B 2.77E-05 3.1 21B // --- // --- ENSDART00000126063 // TTF2 // transcription termination factor, TTF2 1.31E-06 3.1 RNA polymerase II // -- --- 0.000173 3.1 071 ENSDART00000146005 // gtpbp1 // GTP binding protein 1 // --- // gtpbp1 5.41E-06 3.1 378721 /// NM_213475 // XM_678195 // LOC555628 // inositol 1,4,5-triphosphate receptor- LOC555628 9.99E-05 3.1 interacting protein-like --- 3.90E-05 3.1 ENSDART00000129888 // LOC100537256 // 5-azacytidine-induced LOC1005372 0.000204 3.0 protein 1-like // --- // 10 56 197 --- 3.01E-05 3.0 --- 0.000100 3.0 128 BC058295 // tab1 // TGF-beta activated kinase 1/MAP3K7 binding tab1 3.17E-07 3.0 protein 1 // --- // 4030 XM_001343253 // LOC100003830 // uncharacterized LOC1000038 0.000133 3.0

161

LOC100003830 // --- // 100003830 /// EN 30 833 NM_001020590 // nip7 // nuclear import 7 homolog (S. cerevisiae) // nip7 0.000429 3.0 --- // 553616 /// E 013 ENSDART00000050855 // notch1b // notch homolog 1b // --- // notch1b 1.60E-05 3.0 794892 /// NM_131302 // not --- 0.000734 3.0 627 ENSDART00000015688 // invs // inversin // --- // 245946 /// invs 2.57E-06 3.0 NM_152970 // invs // invers ENSDART00000123251 // zgc:63779 // zgc:63779 // --- // 393647 /// zgc:63779 2.44E-06 3.0 NM_200674 // zgc:6377 ENSDART00000122898 // PHF21B (2 of 2) // PHD finger protein PHF21B 5.45E-05 3.0 21B // --- // ------2.74E-06 3.0 NM_131181 // hoxb8b // homeo box B8b // --- // 30420 /// hoxb8b 0.000101 3.0 ENSDART00000076154 // hoxb8b / 992 --- 9.42E-06 2.9 ENSDART00000060729 // zgc:162301 // zgc:162301 // --- // 797343 /// zgc:162301 0.000150 2.9 NM_001083867 // zgc 834 NM_131462 // vsx2 // visual system homeobox 2 // --- // 796163 /// vsx2 6.68E-06 2.9 ENSDART00000030448 / ENSDART00000128937 // ipo8 // importin 8 // --- // 555520 /// ipo8 7.94E-06 2.9 NM_001044334 // ipo8 // i NM_001080577 // celsr2 // cadherin, EGF LAG seven-pass G-type celsr2 2.50E-05 2.9 receptor 2 // --- // 5604 BC168513 // gcn1l1 // GCN1 general control of amino-acid synthesis gcn1l1 2.02E-06 2.9 1-like 1 (yeast) // BC055618 // ftsj // FtsJ homolog 3 (E. coli) // --- // 321247 ftsj 0.000264 2.9 09 ENSDART00000130626 // rars // arginyl-tRNA synthetase // --- // rars 8.20E-05 2.9 337070 /// ENSDART00000 --- 4.68E-05 2.9 NM_200533 // zgc:65772 // zgc:65772 // --- // 393505 /// zgc:65772 0.000208 2.9 ENSDART00000101091 // zgc:6577 348 NM_131884 // cebpb // CCAAT/enhancer binding protein (C/EBP), cebpb 0.000543 2.9 beta // --- // 140814 /// 014 BC090774 // si:dkey-40h20.1 // si:dkey-40h20.1 // --- // 100526805 /// si:dkey- 6.58E-05 2.9 ENSDART000001345 40h20.1 NM_001080577 // celsr2 // cadherin, EGF LAG seven-pass G-type celsr2 8.07E-05 2.9 receptor 2 // --- // 5604 ENSDART00000047503 // usp36 // ubiquitin specific peptidase 36 // usp36 7.23E-05 2.9 --- // 327239 /// ENS --- 0.000111 2.9 694 ENSDART00000132659 // si:ch211-163l21.4 // si:ch211-163l21.4 // -- si:ch211- 0.000274 2.9 - // 567844 /// ENSDA 163l21.4 085 NM_001123245 // LOC559147 // novel protein similar to vertebrate LOC559147 5.63E-05 2.9 hairy and enhancer of ENSDART00000102888 // cdk6 // cyclin-dependent kinase 6 // --- // cdk6 0.000329 2.8 100034507 /// ENSDART 34 --- 0.000252 2.8 983 NM_001037370 // tsr1 // TSR1, 20S rRNA accumulation, homolog tsr1 8.11E-05 2.8 (yeast) // --- // 325037 / --- 0.000185 2.8 967 ENSDART00000126038 // tcp1 // t-complex polypeptide 1 // --- // tcp1 6.79E-05 2.8 30477 /// NM_131230 // --- 0.000192 2.8 761

162

XM_001335100 // LOC100000783 // histone H2A-like // --- // LOC1000007 0.000405 2.8 100000783 /// ENSDART0000014 83 809 NM_001045013 // chaf1a // chromatin assembly factor 1, subunit A chaf1a 0.000272 2.8 (p150) // --- // 56321 893 XM_003200002 // LOC100537178 // e3 ubiquitin-protein ligase LOC1005371 0.000490 2.8 RNF14-like // --- // 100537 78 168 BC045402 // rrp1 // ribosomal RNA processing 1 homolog (S. rrp1 0.000640 2.8 cerevisiae) // --- // 321059 587 ENSDART00000063923 // znf259 // zinc finger protein 259 // --- // znf259 0.000644 2.8 406382 /// NM_213108 483 NM_131158 // dbx1a // developing brain homeobox 1a // --- // 30394 dbx1a 9.87E-05 2.8 /// ENSDART000000224 ENSDART00000105292 // polr3e // polymerase (RNA) III (DNA polr3e 0.000233 2.8 directed) polypeptide E // -- 938 --- 4.48E-05 2.8 --- 0.000579 2.8 3 ENSDART00000066446 // heatr1 // HEAT repeat containing 1 // --- heatr1 3.38E-05 2.8 // 334446 /// NM_199900 XM_694292 // LOC570775 // protein argonaute-1-like // --- // 570775 LOC570775 0.000133 2.8 /// ENSDART00000138 395 NM_001159975 // lyrm5b // LYR motif containing 5b // --- // 561760 lyrm5b 5.45E-06 2.8 /// ENSDART000001338 BC116487 // si:ch211-160d20.3 // si:ch211-160d20.3 // --- // 692267 /// si:ch211- 1.33E-05 2.8 ENSDART00000004 160d20.3 ENSDART00000113284 // im:7137453 // im:7137453 // --- // im:7137453 4.61E-06 2.7 100003232 NM_001079975 // sesn2 // sestrin 2 // --- // 100149745 /// sesn2 8.12E-05 2.7 ENSDART00000102384 // sesn2 XM_001332478 // LOC792891 // insulinoma-associated protein 1- LOC792891 3.85E-06 2.7 like // --- // 792891 /// --- 3.95E-05 2.7 ENSDART00000030946 // prdm8 // PR domain containing 8 // --- // prdm8 0.000440 2.7 406719 /// NM_213410 // 393 ENSDART00000061365 // cad // carbamoyl-phosphate synthetase 2, cad 6.42E-05 2.7 aspartate transcarbamyla ENSDART00000022840 // minal // MYC induced nuclear antigen- minal 1.67E-05 2.7 like // --- // 393308 /// NM NM_201089 // exosc10 // exosome component 10 // --- // 394064 /// exosc10 9.23E-05 2.7 ENSDART00000137410 // ENSDART00000111052 // olig3 // oligodendrocyte transcription olig3 0.000652 2.7 factor 3 // --- // 566728 827 --- 0.000102 2.7 644 BC091690 // im:7152348 // im:7152348 // --- // 559250 im:7152348 0.000131 2.7 48 --- 0.000385 2.7 116 ENSDART00000121998 // grwd1 // glutamate-rich WD repeat grwd1 9.66E-05 2.6 containing 1 // --- // 445115 / XM_002666310 // LOC100333582 // zinc finger protein GLIS2-like LOC1003335 0.000113 2.6 // --- // 100333582 /// 82 386 ENSDART00000008277 // pibf1 // progesterone immunomodulatory pibf1 9.67E-07 2.6 binding factor 1 // --- // --- 0.000985 2.6 315 NM_001077735 // bop1 // block of proliferation 1 // --- // 777627 /// bop1 0.000707 2.6 ENSDART0000013702 588 --- 7.45E-05 2.6 ENSDART00000124833 // pdcd11 // programmed cell death 11 // --- pdcd11 0.000352 2.6

163

// 794079 /// ENSDART00 043 --- 3.41E-05 2.6 --- 0.000845 2.6 581 NM_001013336 // slc25a32b // solute carrier family 25, member 32b slc25a32b 0.000234 2.6 // --- // 503758 /// 7 ENSDART00000055709 // her2 // hairy-related 2 // --- // 30300 /// her2 0.000772 2.6 NM_131089 // her2 // 672 NM_001127335 // ddx21 // DEAD (Asp-Glu-Ala-Asp) box ddx21 4.23E-05 2.6 polypeptide 21 // --- // 799650 /// --- 2.65E-05 2.6 BC114311 // depdc1b // DEP domain containing 1B // --- // depdc1b 0.000202 2.6 100006170 813 XM_001919998 // LOC100150699 // uncharacterized LOC1001506 9.65E-06 2.6 LOC100150699 // --- // 100150699 99 --- 0.000124 2.6 861 --- 2.53E-05 2.6 BC090774 // si:dkey-40h20.1 // si:dkey-40h20.1 // --- // 100526805 /// si:dkey- 0.000340 2.6 ENSDART000001345 40h20.1 409 ENSDART00000124657 // LOC564151 // claspin-like // --- // 564151 LOC564151 0.000480 2.6 /// XM_002665601 // LO 917 ENSDART00000001201 // bysl // bystin-like // --- // 394081 /// bysl 0.000435 2.5 NM_201106 // bysl // bys 02 NM_001118895 // rrp9 // ribosomal RNA processing 9, small rrp9 2.89E-05 2.5 subunit (SSU) processome comp ENSDART00000029387 // ppan // peter pan homolog (Drosophila) // ppan 7.23E-06 2.5 --- // 317739 /// NM_20 --- 0.000408 2.5 65 NM_001204248 // eprs // glutamyl-prolyl-tRNA synthetase // --- // eprs 0.000139 2.5 562037 979 ENSDART00000003648 // wdr3 // WD repeat domain 3 // --- // wdr3 0.000103 2.5 321058 /// NM_198873 // wdr3 906 ENSDART00000029313 // cdkal1 // CDK5 regulatory subunit cdkal1 2.22E-06 2.5 associated protein 1-like 1 // ENSDART00000036703 // pfdn2 // prefoldin subunit 2 // --- // pfdn2 0.000230 2.5 323012 /// NM_001045289 // 525 ENSDART00000112007 // nin // ninein (GSK3B interacting protein) nin 2.06E-05 2.5 // --- // 571879 /// BC --- 0.000491 2.5 249 NM_001045030 // prtga // protogenin homolog a (Gallus gallus) // --- prtga 7.03E-06 2.5 // 563834 /// DQ36 NM_001204169 // isg15 // ISG15 ubiquitin-like modifier // --- // isg15 0.000426 2.5 558956 /// ENSDART0000 782 --- 0.000298 2.5 599 ENSDART00000043210 // pax2b // paired box gene 2b // --- // 60638 pax2b 3.74E-07 2.5 /// NM_131640 // pax2 AY960153 // celsr1b // cadherin EGF LAG seven-pass G-type celsr1b 3.71E-05 2.5 receptor 1b // --- // 556520 ENSDART00000047851 // jag1a // jagged 1a // --- // 140421 /// jag1a 7.72E-06 2.5 NM_131861 // jag1a // jag --- 1.40E-05 2.5 --- 0.000336 2.5 79 --- 1.62E-05 2.5

164

--- 0.000609 2.5 009 ENSDART00000045677 // atoh1b // atonal homolog 1b // --- // atoh1b 5.19E-09 2.5 493915 /// NM_001128679 // ENSDART00000029771 // ahsa1l // AHA1, activator of heat shock ahsa1l 0.000568 2.5 protein ATPase homolog 1, 354 ENSDART00000020834 // LOC100334245 // protein pellino LOC1003342 0.000643 2.5 homolog 2-like // --- // 10033424 45 962 NM_001128255 // mthfr // 5,10-methylenetetrahydrofolate mthfr 0.000778 2.5 reductase (NADPH) // --- // 567 245 NM_001171028 // lppr3a // lipid phosphate phosphatase-related lppr3a 3.57E-05 2.5 protein type 3a // --- // XM_001336913 // LOC100004336 // uncharacterized LOC1000043 0.000123 2.5 LOC100004336 // --- // 100004336 36 642 ENSDART00000131319 // ctps1a // CTP synthase 1a // --- // 322089 ctps1a 0.000140 2.5 /// ENSDART00000017330 254 --- 0.000118 2.4 755 NM_001172398 // im:7162084 // im:7162084 // --- // 567411 /// im:7162084 0.000175 2.4 ENSDART00000103502 // im: 952 --- 0.000272 2.4 751 ENSDART00000142244 // dnajb1b // DnaJ (Hsp40) homolog, dnajb1b 4.94E-07 2.4 subfamily B, member 1b // --- // --- 0.000293 2.4 911 BC054616 // zgc:64090 // zgc:64090 // --- // 393383 /// zgc:64090 7.49E-06 2.4 ENSDART00000057052 // zgc:64090 ENSDART00000138972 // si:ch73-386h18.1 // si:ch73-386h18.1 // --- si:ch73- 0.000253 2.4 // 563768 /// ENSDART 386h18.1 276 ENSDART00000054441 // zgc:111991 // zgc:111991 // --- // 553667 /// zgc:111991 0.000993 2.4 NM_001020640 // zgc 675 NM_001003831 // nop14 // NOP14 nucleolar protein homolog nop14 0.000212 2.4 (yeast) // --- // 321133 /// B 318 --- 6.84E-05 2.4 --- 6.86E-05 2.4 --- 6.85E-05 2.4 ENSDART00000151494 // wu:fj64h06 // wu:fj64h06 // --- // 336342 wu:fj64h06 1.84E-05 2.4 --- 0.000181 2.4 762 --- 2.46E-05 2.4 --- 8.09E-05 2.4 ENSDART00000059345 // eed // embryonic ectoderm development eed 2.42E-05 2.4 // --- // 550463 /// NM_001 ENSDART00000140499 // LOC100536110 // zinc finger protein 658- LOC1005361 0.000820 2.4 like // --- // 100536110 10 147 ENSDART00000077642 // atoh1a // atonal homolog 1a // --- // 30303 atoh1a 3.43E-07 2.4 /// NM_131091 // atoh --- 7.98E-05 2.4 --- 0.000162 2.4 865 ENSDART00000040812 // nuf2 // NUF2, NDC80 kinetochore nuf2 0.000638 2.4 complex component, homolog // --- 926 --- 0.000120 2.4 756 ENSDART00000130641 // bcl3 // B-cell CLL/lymphoma 3 // --- // bcl3 0.000109 2.4 565656 /// XM_688922 // b 093 --- 0.000723 2.4

165

093 ENSDART00000101537 // zgc:158350 // zgc:158350 // --- // 569582 /// zgc:158350 3.75E-07 2.4 NM_001080993 // zgc ENSDART00000025214 // tsr2 // TSR2, 20S rRNA accumulation, tsr2 2.07E-05 2.4 homolog (S. cerevisiae) // - BC122127 // si:dkeyp-84a8.8 // si:dkeyp-84a8.8 // --- // 797099 /// si:dkeyp- 4.28E-06 2.3 ENSDART00000105776 84a8.8 ENSDART00000014945 // hcfc1a // host cell factor C1a // --- // hcfc1a 0.000164 2.3 564853 /// NM_001045064 085 NM_173285 // ttc27 // tetratricopeptide repeat domain 27 // --- // ttc27 9.39E-05 2.3 317640 /// ENSDART00 ENSDART00000015681 // dbx1b // developing brain homeobox 1b // dbx1b 0.000122 2.3 --- // 30416 /// NM_1311 99 NM_001163312 // lbx1b // ladybird homeobox 1b // --- // 793810 /// lbx1b 0.000278 2.3 ENSDART00000007770 / 665 ENSDART00000013063 // LOC796505 // putative ATP-dependent LOC796505 0.000984 2.3 RNA helicase DHX33-like // -- 18 XM_001341900 // masp1 // mannan-binding lectin serine peptidase masp1 8.07E-05 2.3 1 (C4/C2 activating com XM_002667560 // im:7141335 // im:7141335 // --- // 570945 /// im:7141335 3.40E-05 2.3 ENSDART00000092389 // im: --- 2.35E-06 2.3 BC133161 // zgc:158803 // zgc:158803 // --- // 100038767 /// zgc:158803 8.35E-06 2.3 ENSDART00000100619 // zgc: NM_001082551 // whsc1 // Wolf-Hirschhorn syndrome candidate 1 whsc1 0.000202 2.3 // --- // 100000709 /// E 061 NM_001201398 // wnt1 // wingless-type MMTV integration site wnt1 1.06E-07 2.3 family, member 1 // --- // NM_214748 // rpf2 // ribosome production factor 2 homolog (S. rpf2 4.87E-05 2.3 cerevisiae) // --- // 406 ENSDART00000132660 // polr3gla // polymerase (RNA) III (DNA polr3gla 0.000436 2.3 directed) polypeptide G lik 237 ENSDART00000115032 // hsp47 // heat shock protein 47 // --- // hsp47 1.61E-05 2.3 30449 /// NM_131204 // h BC090304 // im:7150454 // im:7150454 // --- // 553298 im:7150454 0.000282 2.3 08 NM_001025165 // metap1 // methionyl aminopeptidase 1 // --- // metap1 0.000952 2.3 503783 /// ENSDART000001 602 ENSDART00000058971 // lrrc20 // leucine rich repeat containing 20 lrrc20 0.000347 2.3 // --- // 492485 /// 991 --- 0.000510 2.3 222 --- 0.000172 2.3 011 XM_002666920 // LOC100332815 // uncharacterized LOC1003328 0.000226 2.3 LOC100332815 // --- // 100332815 15 063 NM_001214908 // wu:fc13c02 // wu:fc13c02 // --- // 561837 /// wu:fc13c02 2.43E-05 2.3 ENSDART00000134418 // wu: ENSDART00000141068 // sox11b // SRY-box containing gene 11b // sox11b 2.24E-05 2.3 --- // 30603 /// NM_1313 ENSDART00000073930 // notch3 // notch homolog 3 // --- // 58066 /// notch3 1.67E-05 2.3 NM_131549 // notch3 --- 1.62E-06 2.3 ENSDART00000098970 // lin28a // lin-28 homolog A (C. elegans) // - lin28a 9.27E-06 2.3 -- // 394066 /// NM_2 --- 7.13E-05 2.3 --- 7.13E-05 2.3 --- 7.13E-05 2.3

166

--- 7.13E-05 2.3 --- 7.13E-05 2.3 ENSDART00000062459 // rbm19 // RNA binding motif protein 19 // rbm19 8.40E-06 2.3 --- // 387255 /// NM_198 --- 0.000152 2.3 083 --- 2.54E-05 2.3 ENSDART00000018514 // dlc // deltaC // --- // 30120 /// NM_130944 dlc 0.000149 2.3 // dlc // deltaC // - 775 ENSDART00000112754 // LOC100001904 // cyclin-J-like // --- // LOC1000019 0.000411 2.3 100001904 /// XM_00133746 04 776 NM_131025 // ccnd1 // cyclin D1 // --- // 30222 /// ccnd1 0.000407 2.3 ENSDART00000051868 // ccnd1 // cycl 192 --- 2.87E-06 2.3 ENSDART00000019976 // zgc:110266 // zgc:110266 // --- // 550277 /// zgc:110266 0.000384 2.3 NM_001017614 // zgc 936 BC129132 // si:dkey-39a18.1 // si:dkey-39a18.1 // --- // 557637 /// si:dkey- 0.000463 2.3 ENSDART00000067151 39a18.1 736 ENSDART00000111184 // utp18 // UTP18 small subunit (SSU) utp18 6.22E-05 2.3 processome component homolog ( ENSDART00000126277 // zgc:110758 // zgc:110758 // --- // 503603 /// zgc:110758 4.36E-06 2.2 NM_001013290 // zgc NM_001045299 // crb2a // crumbs homolog 2a // --- // 723994 /// crb2a 8.99E-05 2.2 ENSDART00000084378 // c ENSDART00000049323 // LOC100534934 // POU domain, class 2, LOC1005349 0.000626 2.2 transcription factor 2-like 34 919 --- 6.34E-05 2.2 --- 2.68E-05 2.2 --- 0.000176 2.2 028 ENSDART00000111348 // zgc:76871 // zgc:76871 // --- // 406376 /// zgc:76871 0.000103 2.2 NM_213103 // zgc:7687 226 NM_001080635 // ldlrap1b // low density lipoprotein receptor ldlrap1b 0.000521 2.2 adaptor protein 1b // --- 194 ENSDART00000110269 // DDX24 // DEAD (Asp-Glu-Ala-Asp) box DDX24 7.39E-05 2.2 polypeptide 24 // --- // --- ENSDART00000111189 // gar1 // GAR1 ribonucleoprotein homolog gar1 4.35E-05 2.2 (yeast) // --- // 393950 / --- 0.000192 2.2 2 CU638789 // igdcc3 // immunoglobulin superfamily, DCC subclass, igdcc3 8.90E-07 2.2 member 3 // --- // 5559 --- 0.000186 2.2 786 ENSDART00000128435 // pinx1 // pin2/trf1-interacting protein 1 // - pinx1 0.000814 2.2 -- // 368253 /// NM_ 24 ENSDART00000142306 // xpot // exportin, tRNA (nuclear export xpot 3.74E-05 2.2 receptor for tRNAs) // --- ENSDART00000145072 // neurod4 // neurogenic differentiation 4 // neurod4 1.96E-05 2.2 --- // 266958 /// ENSD ENSDART00000148636 // trim71 // tripartite motif-containing 71 // trim71 2.91E-06 2.2 --- // 561754 /// ENS ENSDART00000131342 // zgc:172142 // zgc:172142 // --- // zgc:172142 1.22E-06 2.2 100136846 /// ENSDART000000788 ENSDART00000024778 // robo3 // roundabout homolog 3 // --- // robo3 5.58E-08 2.2 30770 /// NM_131482 // ro --- 0.000229 2.2 948

167

ENSDART00000082325 // aamp // angio-associated, migratory cell aamp 4.08E-05 2.2 protein // --- // 405874 --- 7.05E-05 2.2 ENSDART00000074174 // tbl3 // transducin (beta)-like 3 // --- // tbl3 1.07E-05 2.2 492761 /// NM_00100740 --- 0.000243 2.2 663 --- 0.000287 2.2 165 --- 2.64E-06 2.2 --- 0.000261 2.2 583 ENSDART00000055878 // rcl1 // RNA terminal phosphate cyclase- rcl1 9.93E-06 2.2 like 1 // --- // 445388 // ENSDART00000109211 // gemin5 // gem (nuclear organelle) gemin5 7.77E-06 2.2 associated protein 5 // --- // BC090774 // si:dkey-40h20.1 // si:dkey-40h20.1 // --- // 100526805 /// si:dkey- 0.000554 2.2 ENSDART000001345 40h20.1 715 --- 4.67E-05 2.2 ENSDART00000125785 // ddx10 // DEAD (Asp-Glu-Ala-Asp) box ddx10 8.16E-07 2.2 polypeptide 10 // --- // 5691 XM_003199510 // LOC100537878 // low-density lipoprotein LOC1005378 0.000366 2.1 receptor class A domain-contain 78 751 XM_003200284 // LOC100534700 // WD repeat-containing protein LOC1005347 0.000333 2.1 25-like // --- // 10053470 00 914 ENSDART00000016791 // eif3c // eukaryotic translation initiation eif3c 0.000843 2.1 factor 3, subunit C // 959 XM_682568 // si:dkey-15i8.3 // si:dkey-15i8.3 // --- // 559247 /// si:dkey- 2.08E-05 2.1 ENSDART00000102823 / 15i8.3 BC151938 // LOC570430 // similar to AFG3(ATPase family gene 3)- LOC570430 4.51E-05 2.1 like 1 // --- // 570430 ENSDART00000092679 // DHX35 // DEAH (Asp-Glu-Ala-His) box DHX35 1.53E-05 2.1 polypeptide 35 // --- // --- ENSDART00000102134 // zgc:158297 // zgc:158297 // --- // 791138 /// zgc:158297 0.000229 2.1 NM_001080620 // zgc 616 NM_198357 // tcerg1a // transcription elongation regulator 1a tcerg1a 9.25E-05 2.1 (CA150) // --- // 323071 XM_697961 // wu:fd16e03 // wu:fd16e03 // --- // 555585 wu:fd16e03 3.31E-05 2.1 NM_001099983 // arl14 // ADP-ribosylation factor-like 14 // --- // arl14 8.72E-06 2.1 556921 /// ENSDART00 BC124626 // zgc:153041 // zgc:153041 // --- // 768161 zgc:153041 0.000448 2.1 573 --- 0.000243 2.1 868 ENSDART00000145210 // ankle2 // ankyrin repeat and LEM ankle2 1.96E-05 2.1 domain containing 2 // --- // 55 ENSDART00000057752 // slit1b // slit homolog 1b (Drosophila) // --- slit1b 7.73E-06 2.1 // 561685 /// NM_00 ENSDART00000019446 // ascl1b // achaete-scute complex-like 1b ascl1b 0.000624 2.1 (Drosophila) // --- // 30 122 ENSDART00000142189 // LOC100536821 // myelin transcription LOC1005368 0.000153 2.1 factor 1-like // --- // 1005 21 118 --- 5.41E-05 2.1 ENSDART00000004379 // nol10 // nucleolar protein 10 // --- // nol10 0.000928 2.1 393206 /// NM_200237 // n 537 ENSDART00000138849 // zgc:56699 // zgc:56699 // --- // 405758 /// zgc:56699 4.24E-05 2.1 ENSDART00000000992 // ENSDART00000104616 // lepr // leptin receptor // --- // 567241 /// lepr 0.000394 2.1 NM_001113376 // lepr 959

168

XM_001334580 // si:ch211-156j22.4 // si:ch211-156j22.4 // --- // si:ch211- 1.87E-05 2.1 795944 156j22.4 NM_001045076 // hspa14 // heat shock protein 14 // --- // 565232 /// hspa14 0.000655 2.1 ENSDART00000080829 27 ENSDART00000128602 // tfdp2 // transcription factor Dp-2 // --- // tfdp2 0.000102 2.1 338204 /// NM_198208 961 --- 0.000167 2.1 111 ENSDART00000125555 // nup205 // nucleoporin 205 // --- // 445382 nup205 5.00E-07 2.1 /// NM_001003859 // nu ENSDART00000013409 // prmt3 // protein arginine prmt3 0.000202 2.1 methyltransferase 3 // --- // 550348 // 669 ENSDART00000121440 // INCENP // inner centromere protein INCENP 6.86E-05 2.1 antigens 135/155kDa // --- // NM_001143920 // wu:fi41d10 // wu:fi41d10 // --- // 556702 /// wu:fi41d10 0.000623 2.1 BC055576 // wu:fi41d10 // 617 --- 0.000445 2.1 478 XM_690528 // si:dkeyp-20g2.3 // si:dkeyp-20g2.3 // --- // 567236 /// si:dkeyp- 1.91E-05 2.1 ENSDART00000141028 20g2.3 ENSDART00000031486 // lyrm1 // LYR motif containing 1 // --- // lyrm1 0.000292 2.1 436779 /// ENSDART00000 825 ENSDART00000036147 // irx5b // iroquois homeobox protein 5b // - irx5b 0.000352 2.1 -- // 405792 /// NM_001 291 ENSDART00000110270 // pwp2h // PWP2 periodic tryptophan pwp2h 0.000373 2.1 protein homolog (yeast) // --- 324 NM_001045202 // pfas // phosphoribosylformylglycinamidine pfas 0.000439 2.0 synthase // --- // 570437 /// 005 --- 0.000458 2.0 25 ENSDART00000040083 // raver1 // ribonucleoprotein, PTB-binding raver1 5.40E-05 2.0 1 // --- // 553693 /// N NM_001003883 // ect2 // epithelial cell transforming sequence 2 ect2 0.000205 2.0 oncogene // --- // 4454 685 ENSDART00000105273 // im:7139382 // im:7139382 // --- // 572258 im:7139382 0.000878 2.0 /// XM_003199668 // im: 931 BC124432 // zgc:153702 // zgc:153702 // --- // 767652 /// zgc:153702 0.000332 2.0 ENSDART00000083807 // zgc:153 697 --- 5.03E-05 2.0 NM_001007285 // p4ha2 // procollagen-proline, 2-oxoglutarate 4- p4ha2 4.13E-05 2.0 dioxygenase (proline 4-h ENSDART00000046840 // exosc3 // exosome component 3 // --- // exosc3 0.000296 2.0 565000 /// NM_001029961 / 702 NM_001013516 // msi1 // musashi homolog 1 (Drosophila) // --- // msi1 0.000368 2.0 541389 /// ENSDART0000 016 ENSDART00000125452 // LOC100005923 // gastrula zinc finger LOC1000059 5.09E-05 2.0 protein XlCGF57.1-like // -- 23 BC083291 // zgc:101814 // zgc:101814 // --- // 450027 /// zgc:101814 8.34E-05 2.0 ENSDART00000020743 // zgc:101 ENSDART00000087480 // baz1b // bromodomain adjacent to zinc baz1b 4.04E-05 2.0 finger domain, 1B // --- // ENSDART00000134514 // smarca4 // SWI/SNF related, matrix smarca4 6.31E-06 2.0 associated, actin dependent re ENSDART00000022625 // nrarpb // notch-regulated ankyrin repeat nrarpb 0.000504 2.0 protein b // --- // 3532 883 --- 0.000979 2.0 294 BC056513 // larp1 // La ribonucleoprotein domain family, member larp1 2.59E-05 2.0 1 // --- // 327175 --- 3.47E-05 2.0

169

--- 3.78E-05 2.0 NM_001001820 // zic2b // zic family member 2 (odd-paired zic2b 4.96E-05 2.0 homolog, Drosophila) b // ------1.54E-05 2.0 --- 1.54E-05 2.0 BC139709 // LOC563828 // similar to complement factor B/C2B // -- LOC563828 0.000111 2.0 - // 563828 953 --- 0.000483 2.0 482 XM_002661208 // LOC100004261 // mitogen-activated protein LOC1000042 0.000662 2.0 kinase kinase kinase 14-like 61 909 ENSDART00000002961 // rcor2 // REST corepressor 2 // --- // rcor2 1.71E-05 2.0 402934 /// NM_205638 // rco ENSDART00000101661 // zgc:56039 // zgc:56039 // --- // 335889 /// zgc:56039 2.24E-06 2.0 NM_199984 // zgc:5603 ENSDART00000031752 // rfx4 // regulatory factor X, 4 // --- // rfx4 2.51E-05 2.0 403016 /// NM_205712 // --- 1.66E-05 2.0 --- 2.83E-06 2.0 NM_200627 // cdkal1 // CDK5 regulatory subunit associated protein cdkal1 4.50E-07 2.0 1-like 1 // --- // 39 ENSDART00000073555 // ctu2 // cytosolic thiouridylase subunit 2 ctu2 0.000829 2.0 homolog (S. pombe) // - 444 BC128790 // si:ch211-261f7.2 // si:ch211-261f7.2 // --- // 567341 /// si:ch211- 0.000106 2.0 ENSDART0000008442 261f7.2 636 ENSDART00000103307 // cyp24a1 // cytochrome P450, family 24, cyp24a1 0.000357 2.0 subfamily A, polypeptide 1 933 NM_001003888 // polr1d // polymerase (RNA) I polypeptide D // --- polr1d 4.95E-05 2.0 // 445412 /// ENSDART ENSDART00000082466 // LOC100334928 // TGF-beta receptor LOC1003349 0.000266 2.0 type-2-like // --- // 100334928 28 407 ENSDART00000045071 // foxk2 // forkhead box K2 // --- // 324141 foxk2 3.67E-05 2.0 /// NM_001197257 // fox ENSDART00000062576 // thyn1 // thymocyte nuclear protein 1 // --- thyn1 8.42E-08 2.0 // 393630 /// NM_2006 ENSDART00000098394 // wnt3a // wingless-type MMTV wnt3a 7.24E-05 2.0 integration site family, member 3A // BC150244 // chd7 // chromodomain helicase DNA binding protein 7 chd7 0.000109 2.0 // --- // 569471 363 ENSDART00000023101 // pes // pescadillo // --- // 30228 /// pes 3.79E-06 2.0 NM_131030 // pes // pescadi --- 0.000903 2.0 811 DQ535893 // urp2 // urotensin II-related peptide // --- // 100001008 urp2 0.000139 2.0 /// ENSDART0000009 766 ENSDART00000127444 // taf1a // TATA box binding protein taf1a 2.11E-05 2.0 (TBP)-associated factor, RNA po --- 3.96E-05 2.0 --- 0.000188 2.0 123 XM_001334721 // LOC797129 // uncharacterized LOC797129 // --- LOC797129 5.13E-05 1.9 // 797129 NM_001114564 // plekhs1 // pleckstrin homology domain plekhs1 0.000491 1.9 containing, family S member 1 // 756 --- 0.000343 1.9 717 ENSDART00000099389 // zgc:158228 // zgc:158228 // --- // 791166 /// zgc:158228 8.33E-05 1.9 NM_001080648 // zgc

170

NM_001002160 // rpf1 // ribosome production factor 1 homolog (S. rpf1 0.000698 1.9 cerevisiae) // --- // 837 --- 5.56E-05 1.9 NM_001253811 // chrng // cholinergic receptor, nicotinic, gamma // chrng 0.000168 1.9 --- // 325080 /// JN 987 BC090426 // vars // valyl-tRNA synthetase // --- // 114427 /// vars 0.000775 1.9 ENSDART00000004832 // va 582 NM_001080192 // oc90 // otoconin 90 // --- // 568941 oc90 1.16E-05 1.9 --- 2.91E-05 1.9 NM_001115059 // tgfbr1b // transforming growth factor, beta tgfbr1b 0.000475 1.9 receptor 1 b // --- // 7929 426 --- 0.000261 1.9 319 ENSDART00000060998 // nme5 // non-metastatic cells 5, protein nme5 4.20E-05 1.9 expressed in (nucleoside- --- 0.000495 1.9 873 --- 3.65E-05 1.9 ENSDART00000136660 // GREB1L (2 of 2) // growth regulation by GREB1L 9.07E-05 1.9 estrogen in breast cancer NM_001199738 // topbp1 // topoisomerase (DNA) II binding protein topbp1 4.36E-06 1.9 1 // --- // 407679 /// ENSDART00000141005 // si:dkey-56k23.2 // si:dkey-56k23.2 // --- // si:dkey- 0.000195 1.9 100003476 /// NM_001 56k23.2 683 --- 0.000162 1.9 841 --- 0.000406 1.9 251 ENSDART00000065331 // sall4 // sal-like 4 (Drosophila) // --- // sall4 0.000762 1.9 572527 /// NM_00108060 998 NM_131134 // fzd5 // frizzled homolog 5 // --- // 30364 /// fzd5 8.55E-05 1.9 ENSDART00000031761 // fzd5 --- 0.000680 1.9 087 NM_001100951 // bcl11ab // B-cell CLL/lymphoma 11Ab // --- // bcl11ab 0.000428 1.9 566491 /// ENSDART0000014 159 ENSDART00000142467 // si:ch211-199g17.2 // si:ch211-199g17.2 // - si:ch211- 9.83E-05 1.9 -- // 566074 199g17.2 XM_001920057 // LOC100151027 // ankyrin repeat domain- LOC1001510 0.000117 1.9 containing protein 1-like // --- 27 878 --- 0.000346 1.9 217 ENSDART00000103132 // hoxc4a // homeo box C4a // --- // 30345 /// hoxc4a 0.000287 1.9 NM_131122 // hoxc4a / 058 JN416859 // dicp2.2 // diverse immunoglobulin domain-containing dicp2.2 1.20E-07 1.9 protein 2.2 // --- // 1 ENSDART00000140961 // si:ch1073-55a19.2 // si:ch1073-55a19.2 // - si:ch1073- 0.000634 1.9 -- // 100317326 /// XM 55a19.2 628 NM_001044949 // daxx // death-associated protein 6 // --- // 561006 daxx 0.000622 1.9 /// ENSDART00000123 581 ENSDART00000017393 // traip // TRAF-interacting protein // --- // traip 0.000488 1.9 402900 /// NM_205607 NM_001083004 // dhx37 // DEAH (Asp-Glu-Ala-His) box dhx37 0.000289 1.9 polypeptide 37 // --- // 100009635 091 ENSDART00000036280 // ggt1a // gamma-glutamyltransferase 1a // ggt1a 0.000378 1.9 --- // 393387 /// ENSDAR 089 BC096971 // baz1a // bromodomain adjacent to zinc finger domain, baz1a 0.000258 1.9 1A // --- // 334173 803 NM_205703 // atad3b // ATPase family, AAA domain containing 3B atad3b 0.000115 1.9 // --- // 403004 /// ENS 52

171

--- 0.000441 1.9 974 --- 0.000441 1.9 936 --- 0.000276 1.9 633 XM_680337 // LOC557301 // cocaine- and amphetamine-regulated LOC557301 0.000201 1.9 transcript protein-like // 995 ENSDART00000109247 // ANAPC1 // anaphase promoting complex ANAPC1 3.63E-05 1.9 subunit 1 // --- // --- /// ENSDART00000075941 // plekhh1 // pleckstrin homology domain plekhh1 0.000420 1.9 containing, family H (with 467 --- 0.000699 1.9 966 BC162433 // shroom4 // shroom family member 4 // --- // 559035 shroom4 2.54E-06 1.9 ENSDART00000092110 // lin54 // lin-54 homolog // --- // 560688 /// lin54 0.000635 1.9 NM_001076567 // lin5 916 --- 7.60E-06 1.9 ENSDART00000028017 // mad2l1 // MAD2 mitotic arrest deficient- mad2l1 0.000486 1.9 like 1 (yeast) // --- // 056 XM_002662078 // LOC100332577 // pumilio domain-containing LOC1003325 0.000367 1.9 protein C14orf21-like // --- 77 418 --- 4.65E-05 1.9 ENSDART00000019144 // arid2 // AT rich interactive domain 2 arid2 9.22E-05 1.9 (ARID, RFX-like) // --- // ENSDART00000142870 // tyw1 // tRNA-yW synthesizing protein 1 tyw1 6.38E-05 1.9 homolog (S. cerevisiae) // NM_001118893 // vps13a // vacuolar protein sorting 13 homolog A vps13a 1.98E-07 1.9 (S. cerevisiae) // ------0.000422 1.8 717 --- 2.63E-05 1.8 NM_199711 // nsun2 // NOL1/NOP2/Sun domain family, member 2 nsun2 0.000980 1.8 // --- // 325292 /// ENSDAR 197 NM_001100089 // setd8b // SET domain containing (lysine setd8b 1.59E-05 1.8 methyltransferase) 8b // --- // AF538326 // robo4 // roundabout homolog 4 // --- // 560765 /// robo4 1.11E-05 1.8 ENSDART00000004127 // ro NM_001082825 // ddit3 // DNA-damage-inducible transcript 3 // --- ddit3 0.000261 1.8 // 561924 /// ENSDART 178 ENSDART00000084184 // aimp1 // aminoacyl tRNA synthetase aimp1 2.44E-05 1.8 complex-interacting multifunct ENSDART00000142218 // xpo5 // exportin 5 // --- // 558662 /// xpo5 3.33E-05 1.8 ENSDART00000114091 // xpo NM_001193539 // arhgap11a // Rho GTPase activating protein 11A arhgap11a 0.000365 1.8 // --- // 406608 /// ENS 458 ENSDART00000002225 // dnajb1a // DnaJ (Hsp40) homolog, dnajb1a 4.57E-06 1.8 subfamily B, member 1a // --- // ENSDART00000066721 // ngdn // neuroguidin, EIF4E binding ngdn 2.60E-05 1.8 protein // --- // 386642 /// N ENSDART00000148393 // LOC571089 // nucleoprotein TPR // --- // LOC571089 2.58E-05 1.8 571089 /// ENSDART000001 NM_198072 // mtr // 5-methyltetrahydrofolate-homocysteine mtr 0.000533 1.8 methyltransferase // --- // 3 192 ENSDART00000082866 // foxj1a // forkhead box J1a // --- // 767737 foxj1a 1.37E-05 1.8 /// NM_001076706 // f ENSDART00000139068 // zgc:114130 // zgc:114130 // --- // 570332 /// zgc:114130 0.000705 1.8 ENSDART00000059660 199 ENSDART00000104718 // KIF20B // kinesin family member 20B // - KIF20B 0.000406 1.8 -- // --- 337

172

--- 0.000823 1.8 442 --- 0.000363 1.8 859 ENSDART00000102952 // suz12a // suppressor of zeste 12 homolog suz12a 1.71E-05 1.8 (Drosophila) a // --- // ENSDART00000018159 // si:ch211-262e15.1 // si:ch211-262e15.1 // - si:ch211- 0.000944 1.8 -- // 798356 /// XM_00 262e15.1 122 ENSDART00000128587 // ddx54 // DEAD (Asp-Glu-Ala-Asp) box ddx54 0.000294 1.8 polypeptide 54 // --- // 2867 386 ENSDART00000067161 // tnpo3 // transportin 3 // --- // 394062 /// tnpo3 0.000620 1.8 NM_201087 // tnpo3 // 822 ENSDART00000031524 // utp23 // UTP23, small subunit (SSU) utp23 0.000812 1.8 processome component, homolog 802 ENSDART00000115117 // brd4 // bromodomain containing 4 // --- // brd4 0.000268 1.8 570531 /// ENSDART0000 024 ENSDART00000067056 // ticrr // TopBP1-interacting, checkpoint, ticrr 4.42E-05 1.8 and replication regulato ENSDART00000079945 // tut1 // terminal uridylyl transferase 1, U6 tut1 2.51E-05 1.8 snRNA-specific // ------8.20E-05 1.8 BC133085 // zgc:158345 // zgc:158345 // --- // 100009644 zgc:158345 0.000306 1.8 639 ENSDART00000024104 // mycn // v-myc myelocytomatosis viral mycn 0.000188 1.8 related oncogene, neuroblast 4 NM_212765 // med14 // mediator complex subunit 14 // --- // 336923 med14 1.63E-06 1.8 /// BC045931 // med1 NM_001100074 // bcl6ab // B-cell CLL/lymphoma 6a, genome bcl6ab 9.34E-06 1.8 duplicate b // --- // 10000193 NM_001113615 // ehmt2 // euchromatic histone-lysine N- ehmt2 8.94E-05 1.8 methyltransferase 2 // --- // 569 ENSDART00000051614 // tchp // trichoplein, keratin filament tchp 0.000583 1.8 binding // --- // 678595 // 575 BC044365 // gtf3c5 // general transcription factor IIIC, polypeptide gtf3c5 0.000505 1.8 5 // --- // 565198 817 NM_131289 // brn1.2 // brain POU domain gene 1.2 // --- // 30547 /// brn1.2 0.000512 1.8 ENSDART00000110243 096 --- 7.40E-05 1.8 ENSDART00000079385 // si:ch211-107m4.1 // si:ch211-107m4.1 // -- si:ch211- 0.000350 1.8 - // 569278 107m4.1 068 --- 2.11E-05 1.8 NM_001100015 // chfr // checkpoint with forkhead and ring finger chfr 7.71E-05 1.8 domains, E3 ubiquitin NM_001007761 // fgf7 // fibroblast growth factor 7 // --- // 493181 /// fgf7 1.76E-05 1.8 ENSDART00000082 ENSDART00000074796 // npr3 // natriuretic peptide receptor 3 // -- npr3 0.000500 1.8 - // 569395 /// ENSDA 549 ENSDART00000139113 // SMARCAD1 (2 of 2) // SWI/SNF-related, SMARCAD1 8.26E-05 1.8 matrix-associated actin-dep ENSDART00000135026 // si:ch211-136a13.1 // si:ch211-136a13.1 // - si:ch211- 0.000571 1.8 -- // 559014 136a13.1 516 ENSDART00000125062 // LOC792623 // uncharacterized LOC792623 5.18E-05 1.8 LOC792623 // --- // 792623 BC134967 // zgc:162344 // zgc:162344 // --- // 563289 /// zgc:162344 0.000730 1.8 ENSDART00000102463 // zgc:162 125 BC124176 // zgc:152925 // zgc:152925 // --- // 767754 /// zgc:152925 4.48E-05 1.8 ENSDART00000092584 // zgc:152 XM_690108 // si:dkey-20n3.1 // si:dkey-20n3.1 // --- // 566820 /// si:dkey- 0.000422 1.8 ENSDART00000139241 / 20n3.1 85 ENSDART00000026178 // zgc:66125 // zgc:66125 // --- // 393796 /// zgc:66125 8.55E-05 1.8

173

NM_200823 // zgc:6612 ENSDART00000055845 // thoc5 // THO complex 5 // --- // 325064 /// thoc5 0.000655 1.8 NM_212692 // thoc5 // 504 ENSDART00000099265 // ddx46 // DEAD (Asp-Glu-Ala-Asp) box ddx46 0.000232 1.8 polypeptide 46 // --- // 3219 407 ENSDART00000013961 // mycl1a // v-myc myelocytomatosis viral mycl1a 2.18E-05 1.7 oncogene homolog 1, lung c BC092807 // zgc:110224 // zgc:110224 // --- // 550335 /// zgc:110224 0.000264 1.7 ENSDART00000040443 // zgc:110 823 ENSDART00000051473 // ddx31 // DEAD (Asp-Glu-Ala-Asp) box ddx31 0.000185 1.7 polypeptide 31 // --- // 3252 623 --- 0.000353 1.7 757 ENSDART00000099315 // zgc:112104 // zgc:112104 // --- // 550440 /// zgc:112104 1.51E-05 1.7 NM_001017745 // zgc --- 4.36E-05 1.7 NM_001256204 // elac2 // elaC homolog 2 (E. coli) // --- // 492652 /// elac2 0.000756 1.7 ENSDART000001225 777 NM_001128298 // zgc:173506 // zgc:173506 // --- // 563236 zgc:173506 0.000296 1.7 088 NM_001079982 // aggf1 // angiogenic factor with G patch and FHA aggf1 0.000356 1.7 domains 1 // --- // 559 892 --- 1.68E-05 1.7 ENSDART00000047212 // cpsf2 // cleavage and polyadenylation cpsf2 0.000100 1.7 specific factor 2 // --- // 133 ENSDART00000079173 // lepr // leptin receptor // --- // 567241 lepr 0.000165 1.7 713 --- 0.000359 1.7 455 ENSDART00000140545 // LOC100534840 // gastrula zinc finger LOC1005348 0.000794 1.7 protein XlCGF57.1-like // -- 40 381 XM_682226 // dhx32 // DEAH (Asp-Glu-Ala-His) box polypeptide dhx32 1.45E-06 1.7 32 // --- // 558937 /// EN NM_001007383 // snrpa1 // small nuclear ribonucleoprotein snrpa1 0.000194 1.7 polypeptide A' // --- // 4925 463 ENSDART00000034705 // ntmt1 // N-terminal Xaa-Pro-Lys N- ntmt1 2.04E-05 1.7 methyltransferase 1 // --- // 4 BC093128 // dhps // deoxyhypusine synthase // --- // 406329 dhps 0.000678 1.7 571 --- 0.000258 1.7 961 NM_001202459 // jarid2b // jumonji, AT rich interactive domain 2b jarid2b 0.000491 1.7 // --- // 558456 /// 332 --- 0.000156 1.7 293 ENSDART00000006538 // otx1a // orthodenticle homolog 1a // --- // otx1a 0.000365 1.7 30462 /// NM_131215 / 2 --- 0.000234 1.7 218 --- 0.000429 1.7 842 BC155189 // rrbp1b // ribosome binding protein 1 homolog b (dog) rrbp1b 0.000400 1.7 // --- // 567029 /// A 337 --- 0.000718 1.7 985 --- 0.000220 1.7 246 XM_002664026 // ascc3 // activating signal cointegrator 1 complex ascc3 0.000156 1.7 subunit 3 // --- // 1 852 --- 0.000198 1.7

174

574 ENSDART00000121477 // ASCC3 // activating signal cointegrator 1 ASCC3 0.000287 1.7 complex subunit 3 // -- 303 NM_001172565 // ak7a // adenylate kinase 7a // --- // 402854 /// ak7a 8.88E-06 1.7 ENSDART00000048716 // --- 0.000143 1.7 361 --- 0.000303 1.7 366 ENSDART00000124716 // LOC100330611 // uncharacterized LOC1003306 0.000498 1.7 LOC100330611 // --- // 100330611 11 028 --- 6.16E-05 1.7 NM_131191 // smn1 // survival motor neuron 1 // --- // 30432 /// smn1 5.71E-05 1.7 ENSDART00000028099 // ENSDART00000124542 // DIDO1 (2 of 2) // death inducer- DIDO1 0.000125 1.7 obliterator 1 // --- // --- 076 ENSDART00000033878 // znf668 // zinc finger protein 668 // --- // znf668 0.000566 1.7 797423 /// ENSDART000 418 --- 0.000218 1.7 51 ENSDART00000049151 // gli1 // GLI-Kruppel family member 1 // -- gli1 0.000390 1.7 - // 352930 /// ENSDART0 265 NM_001109869 // kdm8 // lysine (K)-specific demethylase 8 // --- // kdm8 1.77E-05 1.7 436936 /// ENSDART0 ENSDART00000115182 // nup214 // nucleoporin 214 // --- // nup214 0.000232 1.7 100529857 /// BC142901 // nup 632 --- 0.000229 1.7 137 ENSDART00000006463 // phf6 // PHD finger protein 6 // --- // phf6 0.000230 1.7 327070 /// NM_199765 // ph 266 --- 4.56E-05 1.7 XR_117678 // LOC562819 // novel NACHT domain containing LOC562819 6.02E-06 1.7 protein // --- // 562819 NM_200204 // zgc:56178 // zgc:56178 // --- // 393173 /// BC045972 // zgc:56178 3.65E-05 1.7 zgc:56178 // zgc:5 XM_001923613 // LOC100002916 // kinetochore-associated protein LOC1000029 0.000237 1.7 DSN1 homolog // --- // 1 16 954 XM_678081 // mcm9 // minichromosome maintenance complex mcm9 1.04E-05 1.7 component 9 // --- // 555610 // --- 0.000947 1.7 715 --- 0.000878 1.7 017 ENSDART00000144415 // cdk13 // cyclin-dependent kinase 13 // --- cdk13 0.000283 1.7 // 559027 /// ENSDART0 676 ENSDART00000037515 // msto1 // misato homolog 1 (Drosophila) // msto1 0.000233 1.7 --- // 334306 /// NM_19 228 BC091950 // igsf9b // immunoglobulin superfamily, member 9b // --- igsf9b 0.000744 1.7 // 553348 316 --- 0.000861 1.7 22 ENSDART00000113907 // sbno1 // strawberry notch homolog 1 sbno1 6.88E-05 1.7 (Drosophila) // --- // 100005 ENSDART00000129911 // klhl31 // kelch-like 31 (Drosophila) // --- // klhl31 0.000154 1.7 407079 75 --- 0.000305 1.7 364 ENSDART00000133379 // LOC564732 // histone H3.2-like // --- // LOC564732 0.000543 1.7 564732 /// ENSDART000001 077 ENSDART00000022498 // tti1 // Tel2 interacting protein 1 homolog tti1 0.000219 1.7

175

(S. pombe) // --- // 5 63 ENSDART00000023568 // kif18a // kinesin family member 18A // --- kif18a 9.42E-05 1.7 // 393209 /// NM_20023 NM_131116 // hoxb2a // homeo box B2a // --- // 30338 /// hoxb2a 5.16E-06 1.7 ENSDART00000007226 // hoxb2a / --- 0.000178 1.7 387 ENSDART00000125891 // fam193b // family with sequence fam193b 0.000208 1.7 similarity 193, member B // --- / 806 ENSDART00000029457 // sh2d3ca // SH2 domain containing 3Ca // sh2d3ca 1.70E-05 1.7 --- // 558860 ENSDART00000125067 // plce1 // phospholipase C, epsilon 1 // --- // plce1 0.000970 1.7 568288 /// NM_00116 646 ENSDART00000080740 // dph1 // DPH1 homolog (S. cerevisiae) // -- dph1 0.000195 1.6 - // 550559 /// NM_0010 151 NM_001001948 // nup54 // nucleoporin 54 // --- // 335750 /// nup54 0.000183 1.6 BC066517 // nup54 // nucle 538 NM_212586 // ccnt2b // cyclin T2b // --- // 321464 /// ccnt2b 0.000656 1.6 ENSDART00000053045 // ccnt2b // 785 ENSDART00000126525 // fam98a // family with sequence similarity fam98a 1.40E-05 1.6 98, member A // --- // ENSDART00000127719 // KDM6B (1 of 2) // lysine (K)-specific KDM6B 0.000161 1.6 demethylase 6B // --- // -- 793 ENSDART00000125836 // LOC559362 // carabin-like // --- // 559362 LOC559362 0.000202 1.6 /// XM_682696 // LOC55 984 NM_001044858 // chd4a // chromodomain helicase DNA binding chd4a 0.000122 1.6 protein 4a // --- // 558344 014 --- 0.000229 1.6 888 NM_001126416 // rexo1 // REX1, RNA exonuclease 1 homolog (S. rexo1 1.73E-05 1.6 cerevisiae) // --- // 5645 --- 0.000923 1.6 053 --- 0.000262 1.6 779 --- 0.000603 1.6 508 ENSDART00000065649 // LOC797032 // growth arrest and DNA LOC797032 0.000167 1.6 damage-inducible protein GADD4 855 ENSDART00000006215 // hira // HIR histone cell cycle regulation hira 0.000186 1.6 defective homolog A (S. 726 ENSDART00000081350 // slc9a3.1 // solute carrier family 9 slc9a3.1 7.34E-05 1.6 (sodium/hydrogen exchanger), ENSDART00000012241 // kif23 // kinesin family member 23 // --- // kif23 9.01E-06 1.6 30627 /// NM_131355 / ENSDART00000121684 // nat8l // N-acetyltransferase 8-like // --- // nat8l 1.40E-05 1.6 564754 /// NM_00108 ENSDART00000020923 // mdn1 // midasin homolog (yeast) // --- // mdn1 0.000132 1.6 555704 31 BC139688 // zgc:163014 // zgc:163014 // --- // 100038766 /// zgc:163014 1.09E-05 1.6 ENSDART00000122378 // CEP1 ENSDART00000032277 // ddx51 // DEAD (Asp-Glu-Ala-Asp) box ddx51 6.43E-05 1.6 polypeptide 51 // --- // 4453 NM_131161 // pou3f1 // POU class 3 homeobox 1 // --- // 30398 /// pou3f1 0.000253 1.6 ENSDART00000013148 // 534 NM_199900 // heatr1 // HEAT repeat containing 1 // --- // 334446 heatr1 0.000722 1.6 037 NM_001004596 // gtf2h1 // general transcription factor II H, gtf2h1 0.000436 1.6 polypeptide 1 // --- // 44 989 XM_003199700 // LOC100536294 // thyroid adenoma-associated LOC1005362 2.57E-07 1.6 protein homolog // --- // 10 94

176

--- 0.000451 1.6 399 ENSDART00000123436 // zgc:66306 // zgc:66306 // --- // 393635 /// zgc:66306 0.000495 1.6 NM_200662 // zgc:6630 57 NM_001004596 // gtf2h1 // general transcription factor II H, gtf2h1 1.73E-05 1.6 polypeptide 1 // --- // 44 ENSDART00000109262 // brip1 // BRCA1 interacting protein C- brip1 0.000393 1.6 terminal helicase 1 // --- / 942 ENSDART00000002085 // nkx1.2lb // NK1 transcription factor nkx1.2lb 5.46E-05 1.6 related 2-like,b // --- // 4 --- 0.000973 1.6 747 NM_001034975 // slit1b // slit homolog 1b (Drosophila) // --- // slit1b 0.000175 1.6 561685 /// BC163568 // 346 --- 0.000247 1.6 721 --- 0.000975 1.6 92 BC134067 // uggt2 // UDP-glucose glycoprotein glucosyltransferase uggt2 0.000329 1.6 2 // --- // 497542 // 631 --- 1.54E-05 1.6 XM_002663420 // LOC100332907 // uncharacterized LOC1003329 0.000112 1.6 LOC100332907 // --- // 100332907 /// EN 07 394 ENSDART00000146415 // samd11 // sterile alpha motif domain samd11 0.000840 1.6 containing 11 // --- // 5691 429 ENSDART00000136408 // zranb3 // zinc finger, RAN-binding zranb3 0.000265 1.6 domain containing 3 // --- // 643 BC154002 // dlgap4a // discs, large (Drosophila) homolog-associated dlgap4a 0.000313 1.6 protein 4a // --- / 115 BC125843 // si:ch1073-192f24.1 // si:ch1073-192f24.1 // --- // si:ch1073- 0.000955 1.6 100307076 /// ENSDART000 192f24.1 508 XM_002665273 // LOC100334672 // uncharacterized LOC1003346 4.75E-05 1.6 LOC100334672 // --- // 100334672 72 ENSDART00000037846 // KIAA1797 // KIAA1797 // --- // --- KIAA1797 6.50E-05 1.6 --- 0.000347 1.6 792 BC139865 // ahctf1 // AT hook containing transcription factor 1 // -- ahctf1 6.51E-05 1.6 - // 553494 --- 7.35E-05 1.6 XM_687759 // LOC564421 // histone H2B 1/2-like // --- // 564421 /// LOC564421 2.15E-05 1.6 ENSDART00000122306 NM_001118892 // plk4 // polo-like kinase 4 (Drosophila) // --- // plk4 2.88E-05 1.6 368390 /// ENSDART000 NM_001001837 // zic6 // zic family member 6 // --- // 415097 /// zic6 5.74E-05 1.6 ENSDART00000105760 // NM_200866 // ctnnbl1 // catenin, beta like 1 // --- // 393840 /// ctnnbl1 0.000750 1.6 ENSDART00000015535 // 639 ENSDART00000109580 // xpo6 // exportin 6 // --- // 333980 /// xpo6 0.000680 1.6 NM_194374 // xpo6 // expo 061 NM_173238 // srrt // serrate RNA effector molecule homolog srrt 3.77E-05 1.6 (Arabidopsis) // --- // 1923 --- 0.000401 1.6 753 ENSDART00000058627 // epb4.1l4 // erythrocyte protein band 4.1- epb4.1l4 5.21E-05 1.6 like 4 // --- // 30470 / --- 0.000289 1.6 762 ENSDART00000007857 // mettl2a // methyltransferase like 2A // --- mettl2a 0.000497 1.6 // 100006618 /// NM_0 724 --- 0.000288 1.6

177

783 --- 0.000474 1.6 875 --- 0.000360 1.6 018 --- 0.000481 1.6 449 ENSDART00000140085 // LOC557043 // uncharacterized protein LOC557043 0.000446 1.6 C6orf150-like // --- // 5570 919 ENSDART00000101582 // pcgf6 // polycomb group ring finger 6 // -- pcgf6 4.19E-05 1.6 - // 555238 /// NM_001 NM_001126454 // fam222a // family with sequence similarity 222, fam222a 0.000125 1.6 member A // --- // 7939 156 --- 0.000151 1.6 366 ENSDART00000024256 // hoxb6a // homeo box B6a // --- // 30341 /// hoxb6a 0.000252 1.6 NM_131119 // hoxb6a / 516 ENSDART00000091850 // atrip // ATR interacting protein // --- // atrip 0.000394 1.6 558534 /// NM_00104486 307 ENSDART00000110794 // usp54a // ubiquitin specific peptidase 54a usp54a 0.000188 1.6 // --- // 563912 /// N 67 DQ377344 // rest // RE1-silencing transcription factor // --- // 564772 rest 0.000413 1.6 977 --- 0.000706 1.6 729 ENSDART00000060809 // smg5 // Smg-5 homolog, nonsense smg5 8.38E-05 1.6 mediated mRNA decay factor (C. el BC155614 // zglp1 // zinc finger, GATA-like protein 1 // --- // 751739 zglp1 0.000199 1.6 575 ENSDART00000141873 // zc3h18 // zinc finger CCCH-type zc3h18 0.000841 1.5 containing 18 // --- // 555770 // 405 ENSDART00000128056 // LOC100537185 // zinc finger Ran- LOC1005371 0.000628 1.5 binding domain-containing protein 85 736 XM_003198282 // si:ch211-265o23.1 // si:ch211-265o23.1 // --- // si:ch211- 6.40E-05 1.5 564715 /// ENSDART0000 265o23.1 --- 0.000241 1.5 836 --- 0.000203 1.5 667 XM_693706 // ptcd1 // pentatricopeptide repeat domain 1 // --- // ptcd1 0.000939 1.5 570247 /// ENSDART000 593 ENSDART00000013440 // gemin2 // gem (nuclear organelle) gemin2 0.000204 1.5 associated protein 2 // --- // 741 NM_001202452 // chd8 // chromodomain helicase DNA binding chd8 0.000425 1.5 protein 8 // --- // 568214 // 851 DQ017634 // kat2a // K(lysine) acetyltransferase 2A // --- // 555517 kat2a 0.000680 1.5 121 XM_679151 // LOC556362 // alkylated DNA repair protein alkB LOC556362 0.000545 1.5 homolog 8-like // --- // 55 159 BC091813 // si:ch211-175f11.3 // si:ch211-175f11.3 // --- // 338297 si:ch211- 0.000182 1.5 175f11.3 232 NM_001020686 // zgc:112350 // zgc:112350 // --- // 553715 /// zgc:112350 0.000541 1.5 BC095776 // zgc:112350 // 467 --- 3.14E-05 1.5 --- 0.000398 1.5 393 --- 0.000597 1.5 012 BC054913 // zgc:63566 // zgc:63566 // --- // 394144 /// zgc:63566 8.51E-05 1.5 ENSDART00000022646 // zgc:63566

178

ENSDART00000105322 // LOC566404 // erythroid differentiation- LOC566404 0.000220 1.5 related factor 1-like // - 215 ENSDART00000012470 // hoxb4a // homeo box B4a // --- // 30340 /// hoxb4a 0.000108 1.5 NM_131118 // hoxb4a / 825 NM_200503 // prmt7 // protein arginine N-methyltransferase 7 // --- prmt7 6.70E-05 1.5 // 393475 /// ENSDA --- 0.000633 1.5 668 ENSDART00000081064 // FBRSL1 // fibrosin-like 1 // --- // --- FBRSL1 3.82E-05 1.5 --- 0.000583 1.5 555 --- 0.000441 1.5 781 NM_001126398 // fam222ba // family with sequence similarity 222, fam222ba 0.000745 1.5 member Ba // --- // 56 105 --- 0.000482 1.5 566 ENSDART00000053267 // hnrnpa1 // heterogeneous nuclear hnrnpa1 0.000791 1.5 ribonucleoprotein A1 // --- // 3 384 --- 0.000923 1.5 043 ENSDART00000018743 // phf20a // PHD finger protein 20, a // --- // phf20a 0.000338 1.5 563332 /// ENSDART00 084 --- 0.000516 1.5 142 ENSDART00000106039 // ARHGEF37 // Rho guanine nucleotide ARHGEF37 0.000365 1.5 exchange factor (GEF) 37 // -- 34 ENSDART00000137768 // si:ch211-284g18.3 // si:ch211-284g18.3 // - si:ch211- 0.000267 1.5 -- // 567022 /// NM_00 284g18.3 276 NM_001161447 // nipblb // nipped-b homolog b (Drosophila) // --- // nipblb 0.000128 1.5 794108 /// ENSDART0 274 NM_001001501 // dzip1 // DAZ interacting protein 1 // --- // 402875 dzip1 0.000976 1.5 /// ENSDART00000083 61 NM_199542 // smc2 // structural maintenance of chromosomes 2 // -- smc2 1.26E-05 1.5 - // 321452 /// ENSDA ENSDART00000017229 // ncam1a // neural cell adhesion molecule ncam1a 0.000529 1.5 1a // --- // 30447 /// NM 511 ENSDART00000114005 // SECISBP2 // SECIS binding protein 2 // - SECISBP2 2.74E-05 1.5 -- // --- /// ENSDART0000 --- 1.04E-05 1.5 BC155053 // atad5a // ATPase family, AAA domain containing 5a // atad5a 0.000898 1.5 --- // 560591 /// ENSD 51 XM_683701 // LOC560304 // sushi domain-containing protein 4-like LOC560304 0.000223 0.7 // --- // 560304 /// E 664 ENSDART00000113089 // slc4a11 // solute carrier family 4, sodium slc4a11 0.000311 0.7 borate transporter, me 331 BC115129 // si:dkey-98p3.7 // si:dkey-98p3.7 // --- // 555526 /// si:dkey- 0.000263 0.7 ENSDART00000078647 // 98p3.7 66 BC117657 // zgc:136844 // zgc:136844 // --- // 724012 /// zgc:136844 0.000520 0.7 ENSDART00000126916 // zgc:136 163 --- 0.000525 0.7 061 NM_131825 // igf1 // insulin-like growth factor 1 // --- // 114433 /// igf1 0.000211 0.7 ENSDART000000047 883 --- 7.33E-06 0.7 ENSDART00000125253 // LOC100537717 // c-Jun-amino-terminal LOC1005377 0.000306 0.7 kinase-interacting protein 1 17 713 XM_001919130 // cdh8 // cadherin 8, type 2 // --- // 564205 /// cdh8 0.000468 0.7 ENSDART00000054552 // c 07 NM_001080701 // slc32a1 // solute carrier family 32 (GABA slc32a1 5.87E-05 0.7

179 vesicular transporter), membe XM_682063 // LOC558797 // interphotoreceptor matrix LOC558797 0.000343 0.7 proteoglycan 1-like // --- // 55879 216 --- 0.000773 0.7 291 NM_001030077 // slc9a7 // solute carrier family 9 (sodium/hydrogen slc9a7 0.000585 0.7 exchanger), member 7 811 --- 0.000677 0.7 353 ENSDART00000149964 // LOC793153 // anthrax toxin receptor 1- LOC793153 0.000681 0.7 like // --- // 793153 /// X 938 ENSDART00000091809 // shank3b // SH3 and multiple ankyrin shank3b 0.000861 0.7 repeat domains 3b // --- // 5 267 ENSDART00000058736 // wu:fi18a01 // wu:fi18a01 // --- // 567181 /// wu:fi18a01 0.000307 0.7 XM_690474 // wu:fi1 976 --- 0.000658 0.6 35 ENSDART00000078533 // kcnd3 // potassium voltage-gated kcnd3 0.000301 0.6 channel, Shal-related subfamily, 37 XR_084488 // si:dkeyp-51f11.3 // si:dkeyp-51f11.3 // --- // 100003458 si:dkeyp- 0.000166 0.6 /// ENSDART000001 51f11.3 451 ENSDART00000111680 // IQSEC2 // IQ motif and Sec7 domain 2 // IQSEC2 0.000436 0.6 --- // --- 343 --- 0.000298 0.6 808 ENSDART00000061794 // nr1h4 // nuclear receptor subfamily 1, nr1h4 0.000177 0.6 group H, member 4 // --- / 218 NM_001044879 // clk2a // CDC-like kinase 2a // --- // 558981 /// clk2a 0.000804 0.6 BC171570 // clk2a // C 63 ENSDART00000144175 // LOC100330186 // coiled-coil domain- LOC1003301 0.000399 0.6 containing protein 136-like // 86 142 BC129434 // tecpr1a // tectonin beta-propeller repeat containing 1a tecpr1a 0.000228 0.6 // --- // 556500 89 ENSDART00000088588 // kcnn1 // potassium intermediate/small kcnn1 0.000645 0.6 conductance calcium-activat 517 ENSDART00000135480 // LOC565449 // WSC domain-containing LOC565449 0.000416 0.6 protein 1-like // --- // 56544 89 ENSDART00000132366 // emp1 // epithelial membrane protein 1 // - emp1 0.000790 0.6 -- // 100318165 /// NM_ 701 ENSDART00000129471 // gucy1b3 // guanylate cyclase 1, soluble, gucy1b3 0.000176 0.6 beta 3 // --- // 1001503 431 XM_001340369 // LOC100000131 // novel protein similar to LOC1000001 0.000330 0.6 vertebrate adhesion molecule w 31 684 ENSDART00000147216 // LOC100535090 // uncharacterized LOC1005350 0.000290 0.6 LOC100535090 // --- // 100535090 90 165 --- 0.000144 0.6 7 ENSDART00000046431 // hsd17b4 // hydroxysteroid (17-beta) hsd17b4 0.000974 0.6 dehydrogenase 4 // --- // 393 911 ENSDART00000137310 // hpse2 // heparanase 2 // --- // 562573 /// hpse2 0.000137 0.6 ENSDART00000139873 // 017 ENSDART00000133143 // gpd1b // glycerol-3-phosphate gpd1b 0.000452 0.6 dehydrogenase 1b // --- // 325181 / 97 NM_001200058 // LOC100006857 // engulfment and cell motility LOC1000068 0.000506 0.6 protein 2 // --- // 100006 57 319 --- 0.000460 0.6 919 --- 0.000131 0.6 674 --- 1.35E-06 0.6

180

NM_001037241 // kctd8 // potassium channel tetramerisation kctd8 0.000770 0.6 domain containing 8 // --- / 719 NM_131086 // cdh4 // cadherin 4, retinal // --- // 30297 /// cdh4 0.000780 0.6 NM_001045273 // cdh4 // ca 966 --- 0.000301 0.6 955 ENSDART00000112302 // abca5 // ATP-binding cassette, sub- abca5 0.000580 0.6 family A (ABC1), member 5 // - 049 NM_001002299 // ptprub // protein tyrosine phosphatase, receptor ptprub 0.000363 0.6 type, U, b // --- // 3 275 --- 0.000628 0.6 229 --- 4.05E-05 0.6 ENSDART00000098435 // si:dkey-193b15.3 // si:dkey-193b15.3 // --- si:dkey- 1.27E-06 0.6 // 565533 /// ENSDART 193b15.3 --- 0.000275 0.6 607 BC134150 // zgc:162928 // zgc:162928 // --- // 557144 /// zgc:162928 0.000509 0.6 ENSDART00000139299 // zgc:162 794 NM_001030116 // scrn2 // secernin 2 // --- // 558306 /// scrn2 0.000575 0.6 ENSDART00000063857 // scrn2 // 318 ENSDART00000030811 // CABLES2 (2 of 2) // Cdk5 and Abl CABLES2 0.000293 0.6 enzyme substrate 2 // --- // --- 718 NM_001199730 // cacng2b // calcium channel, voltage-dependent, cacng2b 1.18E-07 0.6 gamma subunit 2b // --- ENSDART00000147683 // inpp4b // inositol polyphosphate-4- inpp4b 0.000131 0.6 phosphatase, type II // --- // 606 --- 0.000583 0.6 506 ENSDART00000077887 // oat // organic anion transporter // --- // oat 0.000827 0.6 404609 /// NM_207077 / 75 --- 0.000331 0.6 11 XM_687795 // grm7 // glutamate receptor, metabotropic 7 // --- // grm7 0.000531 0.6 564461 /// ENSDART000 771 --- 0.000261 0.6 817 --- 7.22E-05 0.6 ENSDART00000043795 // dlg1l // discs, large (Drosophila) homolog dlg1l 6.26E-05 0.6 1, like // --- // 4976 --- 0.000349 0.6 516 ENSDART00000017268 // eml2 // echinoderm microtubule eml2 0.000289 0.6 associated protein like 2 // --- / 491 ENSDART00000105753 // olfm3a // olfactomedin 3a // --- // 563182 olfm3a 0.000261 0.6 /// NM_001111172 // ol 255 --- 0.000564 0.6 893 --- 0.000364 0.6 624 ENSDART00000141377 // LOC556683 // protein FAM124A-like // -- LOC556683 5.52E-05 0.6 - // 556683 /// ENSDART000 ENSDART00000140388 // mcf2l // MCF.2 cell line derived mcf2l 4.08E-05 0.6 transforming sequence-like // -- --- 0.000449 0.6 405 ENSDART00000091513 // prkar1b // protein kinase, cAMP- prkar1b 1.55E-05 0.6 dependent, regulatory, type I, be ENSDART00000016936 // gucy1a3 // guanylate cyclase 1, soluble, gucy1a3 0.000130 0.6 alpha 3 // --- // 550420 466

181

ENSDART00000134222 // il15 // interleukin 15 // --- // 654826 /// il15 0.000875 0.6 NM_001039565 // il15 511 --- 0.000722 0.6 504 ENSDART00000085848 // pear1 // platelet endothelial aggregation pear1 0.000455 0.6 receptor 1 // --- // 57 25 ENSDART00000100681 // ncam2 // neural cell adhesion molecule 2 ncam2 0.000723 0.6 // --- // 114441 /// NM_ 695 --- 0.000878 0.6 179 ENSDART00000112946 // DOCK10 // dedicator of cytokinesis 10 // DOCK10 2.64E-05 0.6 --- // ------0.000873 0.6 985 ENSDART00000078838 // rab3aa // RAB3A, member RAS rab3aa 0.000409 0.6 oncogene family, a // --- // 445024 / 227 ENSDART00000001159 // mgat4b // mannosyl (alpha-1,3-)- mgat4b 2.05E-05 0.6 glycoprotein beta-1,4-N-acetylglu CU468746 // slitrk2 // SLIT and NTRK-like family, member 2 // --- slitrk2 2.30E-05 0.6 // 794165 BC116504 // zgc:136336 // zgc:136336 // --- // 692261 /// zgc:136336 0.000222 0.6 ENSDART00000097716 // zgc:136 7 --- 0.000459 0.6 234 --- 5.60E-05 0.6 ENSDART00000127936 // LOC100003615 // similar to novel zinc LOC1000036 0.000411 0.6 finger protein // --- // 10 15 154 ENSDART00000146151 // fbxo21 // F-box protein 21 // --- // 567738 fbxo21 9.66E-05 0.6 ENSDART00000145354 // tcirg1 // T-cell, immune regulator 1, tcirg1 3.76E-05 0.6 ATPase, H+ transporting, ly ENSDART00000109689 // GRAMD1B (1 of 2) // GRAM domain GRAMD1B 0.000785 0.6 containing 1B // --- // --- 486 ENSDART00000142958 // c2cd2l // c2cd2-like // --- // 566488 /// c2cd2l 1.65E-06 0.6 NM_001077383 // c2cd2l ENSDART00000077619 // LOC100332644 // LOC1003326 0.000383 0.6 galactosylgalactosylxylosylprotein 3-beta-glucuro 44 258 NM_001004576 // lingo1b // leucine rich repeat and Ig domain lingo1b 0.000716 0.6 containing 1b // --- // 44 644 AY934531 // smtnb // smoothelin b // --- // 664685 smtnb 0.000372 0.6 442 ENSDART00000059351 // ift20 // intraflagellar transport protein 20 ift20 0.000270 0.6 // --- // 414930 /// 214 NM_001201557 // si:dkey-33c12.3 // si:dkey-33c12.3 // --- // 335380 si:dkey- 0.000179 0.6 /// ENSDART00000080 33c12.3 013 ENSDART00000130891 // camk4 // calcium/calmodulin-dependent camk4 0.000975 0.6 protein kinase IV // --- // 77 ENSDART00000006619 // rbpms2 // RNA binding protein with rbpms2 0.000882 0.6 multiple splicing 2 // --- // 21 XM_001336516 // LOC100002173 // complement C1q-like protein 2- LOC1000021 0.000817 0.6 like // --- // 100002173 73 255 ENSDART00000012580 // LOC565776 // sodium-dependent LOC565776 0.000948 0.6 noradrenaline transporter-like // - 217 ENSDART00000109807 // EMILIN3 (1 of 2) // elastin microfibril EMILIN3 0.000393 0.6 interfacer 3 // --- // -- 409 --- 0.000625 0.6 037 ENSDART00000099157 // cdh18 // cadherin 18, type 2 // --- // cdh18 6.66E-05 0.6 767768 /// NM_001076735 // NM_001127515 // si:dkey-110k5.6 // si:dkey-110k5.6 // --- // 566657 si:dkey- 0.000338 0.6 /// ENSDART00000135 110k5.6 584

182

--- 0.000659 0.6 146 ENSDART00000139821 // LOC100331130 // f-box/LRR-repeat LOC1003311 0.000211 0.6 protein 20-like // --- // 100331 30 847 XM_680547 // LOC557467 // kelch repeat and BTB domain- LOC557467 1.46E-05 0.6 containing protein 11-like // ------0.000457 0.6 59 ENSDART00000122297 // LACTBL1 (2 of 2) // lactamase, beta-like LACTBL1 0.000714 0.6 1 // --- // --- 641 ENSDART00000105675 // ttc39c // tetratricopeptide repeat domain ttc39c 0.000521 0.6 39C // --- // 553591 // 116 ENSDART00000111759 // C9orf172 (1 of 2) // chromosome 9 open C9orf172 0.000265 0.6 reading frame 172 // --- / 718 ENSDART00000129866 // pcsk2 // proprotein convertase pcsk2 0.000314 0.6 subtilisin/kexin type 2 // --- // 307 ENSDART00000143759 // vipr1b // vasoactive intestinal peptide vipr1b 1.30E-05 0.6 receptor 1b // --- // 503 BC153402 // bada // BCL2-antagonist of cell death a // --- // 564921 bada 0.000611 0.6 /// ENSDART0000012 16 ENSDART00000057611 // oprm1 // opioid receptor, mu 1 // --- // oprm1 0.000895 0.6 65088 /// NM_131707 // o 521 XM_686460 // LOC563094 // novel protein similar to vertebrate LOC563094 0.000638 0.6 odz, odd Oz/ten-m (Drosop 165 --- 1.26E-06 0.6 NM_001201544 // wdr17 // WD repeat domain 17 // --- // 561804 /// wdr17 7.58E-05 0.6 ENSDART00000136300 // NM_001039679 // lgi3 // leucine-rich repeat LGI family, member 3 // lgi3 0.000479 0.6 --- // 654830 /// E 658 ENSDART00000147101 // galnt9 // UDP-N-acetyl-alpha-D- galnt9 2.70E-05 0.6 galactosamine:polypeptide N-acetyl CU468745 // flrt1b // fibronectin leucine rich transmembrane 1b // -- flrt1b 0.000146 0.6 - // 100144397 255 BC129328 // zgc:158624 // zgc:158624 // --- // 791190 /// zgc:158624 5.34E-06 0.6 ENSDART00000122936 // zgc:158 --- 0.000826 0.6 868 XM_001922148 // mcf2l // mcf.2 cell line derived transforming mcf2l 5.95E-05 0.6 sequence-like // --- // 1 ENSDART00000089934 // wu:fj40f01 // disintegrin and wu:fj40f01 0.000191 0.6 metalloproteinase domain-containing 403 --- 0.000880 0.6 893 NM_001199370 // oxtr // oxytocin receptor // --- // 100001530 /// oxtr 0.000899 0.6 ENSDART00000050114 // 493 XM_003201304 // atp8a2 // ATPase, aminophospholipid atp8a2 9.34E-06 0.6 transporter, class I, type 8A, memb NM_001077555 // zgc:153898 // zgc:153898 // --- // 561339 /// zgc:153898 0.000315 0.6 BC124768 // zgc:153898 // 501 ENSDART00000127299 // ptprb // protein tyrosine phosphatase, ptprb 0.000571 0.6 receptor type, b // --- // 573 --- 9.50E-05 0.6 ENSDART00000124399 // TRAK1 (1 of 3) // trafficking protein, TRAK1 0.000625 0.6 kinesin binding 1 // --- / 887 ENSDART00000014411 // kctd16a // potassium channel kctd16a 0.000892 0.6 tetramerisation domain containing 16 677 ENSDART00000148181 // inpp4aa // inositol polyphosphate-4- inpp4aa 0.000860 0.6 phosphatase, type Ia // --- / 017 XM_002665412 // gramd1b // GRAM domain containing 1B // --- // gramd1b 9.39E-05 0.6 100332318 /// ENSDART000

183

NM_001089420 // LOC568759 // novel protein similar to vertebrate LOC568759 0.000256 0.6 calcium channel voltag 733 XM_687812 // ip6k1 // inositol hexakisphosphate kinase 1 // --- // ip6k1 0.000443 0.6 564478 /// ENSDART00 132 NM_001080168 // trim3b // tripartite motif-containing 3b // --- // trim3b 0.000425 0.6 555391 /// ENSDART00 179 ENSDART00000127994 // palmdb // palmdelphin b // --- // palmdb 0.000561 0.6 100150681 /// ENSDART0000010998 416 ENSDART00000141103 // gfra4 // GDNF family receptor alpha 4 // gfra4 7.04E-05 0.6 --- // 100144564 /// NM_ NM_001045034 // kat2b // K(lysine) acetyltransferase 2B // --- // kat2b 0.000614 0.6 563942 /// ENSDART000 455 ENSDART00000078202 // PHKA2 // phosphorylase kinase, alpha 2 PHKA2 1.04E-05 0.6 (liver) // --- // --- /// ENSDART00000078232 // cdh10 // cadherin 10, type 2 (T2- cdh10 0.000788 0.6 cadherin) // --- // 568370 /// N 018 NM_214813 // zgc:85722 // zgc:85722 // --- // 407982 /// zgc:85722 0.000683 0.6 ENSDART00000030587 // zgc:8572 498 NM_213351 // calm1a // calmodulin 1a // --- // 406660 /// calm1a 2.25E-05 0.6 ENSDART00000034580 // calm1a --- 0.000243 0.6 898 NM_001014354 // kcnab1 // potassium voltage-gated channel, kcnab1 0.000661 0.6 shaker-related subfamily, be 412 ENSDART00000134261 // dnm1b // dynamin 1b // --- // 100333543 dnm1b 0.000736 0.6 /// NM_001256818 // dnm1b 818 --- 0.000490 0.6 29 ENSDART00000111380 // LOC100537785 // glycerophosphodiester LOC1005377 9.94E-07 0.6 phosphodiesterase domain-co 85 NM_001100037 // si:ch73-266o15.4 // si:ch73-266o15.4 // --- // 568862 si:ch73- 0.000991 0.6 /// ENSDART000001 266o15.4 785 ENSDART00000061547 // ltk // leukocyte tyrosine kinase // --- // ltk 0.000129 0.6 564470 /// NM_00100666 738 XM_685678 // LOC562282 // serine/threonine-protein phosphatase LOC562282 0.000820 0.6 2A 56 kDa regulatory sub 27 --- 0.000142 0.6 183 --- 0.000141 0.6 388 --- 7.71E-05 0.6 BC129395 // zgc:158703 // zgc:158703 // --- // 791157 /// zgc:158703 0.000855 0.6 ENSDART00000098764 // zgc:158 95 BC155338 // fam78a // family with sequence similarity 78, member fam78a 0.000723 0.5 A // --- // 100002878 447 --- 0.000217 0.5 344 ENSDART00000151392 // angptl5 // angiopoietin-like 5 // --- // angptl5 9.55E-05 0.5 566212 ENSDART00000110503 // adam11 // a disintegrin and adam11 0.000832 0.5 metalloproteinase domain 11 // --- // 214 --- 4.13E-05 0.5 ENSDART00000065567 // guca1d // guanylate cyclase activator 1d // guca1d 0.000696 0.5 --- // 494573 /// NM_ 217 NM_200831 // amph // amphiphysin // --- // 393804 /// amph 0.000662 0.5 ENSDART00000027689 // amph // amp 068 ENSDART00000114677 // CAMKK2 (2 of 2) // calcium/calmodulin- CAMKK2 4.63E-05 0.5 dependent protein kinase ki ENSDART00000038651 // LOC565294 // zinc finger protein 804A // LOC565294 0.000512 0.5 --- // 565294 /// NM_001 791

184

NM_205563 // trim9 // tripartite motif-containing 9 // --- // 336099 /// trim9 4.63E-05 0.5 ENSDART0000004 NM_001002463 // syt1a // synaptotagmin Ia // --- // 436736 /// syt1a 0.000385 0.5 ENSDART00000066896 // sy 874 ENSDART00000145669 // dtnbb // dystrobrevin, beta b // --- // dtnbb 0.000378 0.5 559786 /// ENSDART0000010 191 ENSDART00000148650 // stat2 // signal transducer and activator of stat2 0.000778 0.5 transcription 2 // -- 17 NM_001020721 // mtmr1a // myotubularin related protein 1a // --- // mtmr1a 2.81E-05 0.5 553750 /// ENSDART0 XM_001335344 // dnajc22 // DnaJ (Hsp40) homolog, subfamily C, dnajc22 3.91E-05 0.5 member 22 // --- // 79665 ENSDART00000081604 // adipor2 // adiponectin receptor 2 // --- // adipor2 0.000795 0.5 560140 /// NM_0010255 533 JQ649326 // wu:fb25h12 // wu:fb25h12 // --- // 571418 /// wu:fb25h12 1.95E-05 0.5 ENSDART00000129434 // OXR1 (3 XM_687176 // LOC563812 // tripartite motif-containing protein 35- LOC563812 0.000739 0.5 like // --- // 563812 791 --- 0.000191 0.5 721 ENSDART00000134922 // kctd7 // potassium channel kctd7 0.000668 0.5 tetramerisation domain containing 7 // 225 ENSDART00000019748 // lin7a // lin-7 homolog A (C. elegans) // --- lin7a 5.55E-05 0.5 // 393682 /// NM_200 --- 0.000415 0.5 926 --- 0.000752 0.5 706 ENSDART00000113289 // CASKIN1 (2 of 3) // CASK interacting CASKIN1 0.000476 0.5 protein 1 // --- // --- 399 --- 7.81E-06 0.5 BC155332 // cadpsb // Ca2+-dependent activator protein for cadpsb 0.000305 0.5 secretion b // --- // 100001 146 --- 0.000960 0.5 485 XM_002660888 // si:dkeyp-51f11.8 // si:dkeyp-51f11.8 // --- // si:dkeyp- 0.000768 0.5 100319971 51f11.8 959 NM_001020730 // zgc:109949 // zgc:109949 // --- // 553764 /// zgc:109949 0.000242 0.5 ENSDART00000038373 // zgc 486 BC076439 // rcan1 // regulator of calcineurin 1 // --- // 336348 rcan1 0.000232 0.5 312 XM_684615 // LOC561209 // sodium/potassium/calcium exchanger LOC561209 1.65E-07 0.5 3-like // --- // 561209 // NM_001130622 // slc1a7a // solute carrier family 1 (glutamate slc1a7a 6.43E-05 0.5 transporter), member 7a / ENSDART00000058552 // OSBPL10 (1 of 2) // oxysterol binding OSBPL10 5.81E-05 0.5 protein-like 10 // --- // - ENSDART00000112484 // nlgn2a // neuroligin 2a // --- // 100002407 nlgn2a 0.000709 0.5 /// NM_001166336 // n 307 --- 0.000971 0.5 819 NM_001003851 // negr1 // neuronal growth regulator 1 // --- // negr1 4.41E-06 0.5 445374 /// ENSDART000000 ENSDART00000121537 // FAM217B (2 of 2) // family with sequence FAM217B 0.000104 0.5 similarity 217, member B 956 ENSDART00000109713 // GLP2R // glucagon-like peptide 2 GLP2R 0.000421 0.5 receptor // --- // --- 681 ENSDART00000144624 // lppr4a // lipid phosphate phosphatase- lppr4a 0.000246 0.5 related protein type 4a // 741 ENSDART00000108629 // si:ch211-260b17.8 // si:ch211-260b17.8 // - si:ch211- 0.000120 0.5 -- // 560885 260b17.8 032

185

--- 0.000154 0.5 166 ENSDART00000073518 // GRAMD2 (1 of 2) // GRAM domain GRAMD2 0.000155 0.5 containing 2 // --- // --- /// XM_ 196 --- 0.000144 0.5 133 --- 0.000355 0.5 315 NM_001044796 // tmem244 // transmembrane protein 244 // --- // tmem244 0.000340 0.5 556137 /// ENSDART000000 663 ENSDART00000139827 // galnt13 // UDP-N-acetyl-alpha-D- galnt13 0.000362 0.5 galactosamine:polypeptide N-acety 428 --- 0.000705 0.5 251 ENSDART00000033819 // prkcda // protein kinase C, delta a // --- // prkcda 0.000104 0.5 334571 /// NM_21470 461 --- 1.87E-05 0.5 --- 1.87E-05 0.5 NM_001017733 // cacng8a // calcium channel, voltage-dependent, cacng8a 3.58E-05 0.5 gamma subunit 8a // --- NM_001105105 // prdm8b // PR domain containing 8b // --- // prdm8b 1.26E-05 0.5 557162 /// ENSDART000001214 ENSDART00000084381 // sybu // syntabulin (syntaxin-interacting) sybu 5.47E-05 0.5 // --- // 568207 /// NM ENSDART00000113266 // EFHA2 (1 of 2) // EF-hand domain EFHA2 5.88E-05 0.5 family, member A2 // --- // ------0.000703 0.5 309 ENSDART00000011946 // LOC100330554 // gamma-aminobutyric LOC1003305 0.000791 0.5 acid receptor subunit rho-1-li 54 959 --- 8.15E-05 0.5 --- 0.000749 0.5 151 XM_001340198 // LOC799930 // transmembrane prolyl 4- LOC799930 0.000580 0.5 hydroxylase-like // --- // 799930 / 996 --- 0.000316 0.5 517 --- 0.000451 0.5 472 ENSDART00000050445 // trim2a // tripartite motif-containing 2a // trim2a 1.75E-05 0.5 --- // 100003782 /// XM_002664863 // LOC100330359 // transmembrane protein 196- LOC1003303 7.74E-05 0.5 like // --- // 100330359 /// 59 ENSDART00000080166 // rnf157 // ring finger protein 157 // --- // rnf157 0.000298 0.5 555415 /// XM_677914 481 ENSDART00000041820 // lingo1a // leucine rich repeat and Ig lingo1a 0.000899 0.5 domain containing 1a // --- 71 --- 0.000936 0.5 811 NM_001002666 // slc1a1 // solute carrier family 1 slc1a1 3.77E-05 0.5 (neuronal/epithelial high affinity gl ENSDART00000134592 // lmbrd2b // LMBR1 domain containing lmbrd2b 0.000640 0.5 2b // --- // 335257 /// ENSDAR 917 NM_001080584 // mavs // mitochondrial antiviral signaling protein mavs 0.000406 0.5 // --- // 562867 /// 088 XM_003199736 // btbd3b // BTB (POZ) domain containing 3b // --- btbd3b 0.000410 0.5 // 562593 /// ENSDART00 4 --- 0.000537 0.5 253 ENSDART00000142919 // ssh1b // slingshot homolog 1b ssh1b 9.06E-05 0.5

186

(Drosophila) // --- // 567479 /// X --- 0.000829 0.5 506 ENSDART00000087643 // tesk2 // testis-specific kinase 2 // --- // tesk2 0.000865 0.5 568213 /// ENSDART000 622 ENSDART00000061007 // mt // metallothionein // --- // 30282 /// mt 7.22E-05 0.5 ENSDART00000061007 // m NM_001005392 // rcan3 // regulator of calcineurin family member 3 rcan3 0.000394 0.5 // --- // 325356 /// 706 --- 0.000702 0.5 635 CU468768 // sc:d0343 // sc:d0343 // --- // 100144400 sc:d0343 0.000136 0.5 489 ENSDART00000113715 // LOC100333333 // alpha-1,3-mannosyl- LOC1003333 0.000194 0.5 glycoprotein 4-beta-N-acetylgl 33 494 NM_001045010 // si:dkey-174m14.3 // si:dkey-174m14.3 // --- // si:dkey- 7.64E-06 0.5 563117 /// ENSDART000001 174m14.3 ENSDART00000101134 // khdrbs2 // KH domain containing, RNA khdrbs2 4.19E-05 0.5 binding, signal transduction ENSDART00000146255 // si:ch73-142c19.2 // si:ch73-142c19.2 // --- si:ch73- 0.000306 0.5 // 570928 /// ENSDART 142c19.2 965 XM_693062 // LOC569664 // calsyntenin-2-like // --- // 569664 /// LOC569664 0.000385 0.5 ENSDART00000085676 // 21 --- 0.000368 0.5 179 ENSDART00000115018 // LOC100329473 // girdin-like // --- // LOC1003294 0.000556 0.5 100329473 /// XM_002662271 73 844 ENSDART00000127502 // cdkl5 // cyclin-dependent kinase-like 5 // cdkl5 0.000290 0.5 --- // 559341 /// NM_0 982 --- 1.69E-05 0.5 ENSDART00000052912 // pcdh20 // protocadherin 20 // --- // pcdh20 0.000267 0.5 100007362 /// XM_001345807 / 395 ENSDART00000065143 // unc119b // unc-119 homolog b (C. unc119b 8.74E-05 0.5 elegans) // --- // 678653 /// NM --- 6.62E-05 0.5 NM_001002472 // atp2b3a // ATPase, Ca++ transporting, plasma atp2b3a 0.000774 0.5 membrane 3a // --- // 4367 099 --- 0.000171 0.5 054 ENSDART00000113599 // LOC559212 // 1,4-alpha-glucan- LOC559212 0.000496 0.5 branching enzyme-like // --- // 559 927 ENSDART00000112543 // LOC569706 // phosphorylase b kinase LOC569706 0.000444 0.5 regulatory subunit beta-like 532 --- 0.000633 0.5 668 ENSDART00000146321 // LOC567525 // novel protein similar to LOC567525 1.69E-05 0.5 vertebrate fibrinogen C dom ENSDART00000003939 // syngr1a // synaptogyrin 1a // --- // 450047 syngr1a 0.000490 0.5 /// NM_001006067 // s 806 ENSDART00000138448 // ttc7a // tetratricopeptide repeat domain ttc7a 1.86E-05 0.5 7A // --- // 559345 /// --- 0.000655 0.5 438 --- 0.000655 0.5 399 XM_001922407 // chrm4a // cholinergic receptor, muscarinic 4a // -- chrm4a 0.000437 0.5 - // 100150701 /// E 713 --- 0.000316 0.5 307 --- 0.000316 0.5

187

293 --- 0.000316 0.5 296 --- 0.000315 0.5 82 --- 0.000880 0.5 5 ENSDART00000075837 // LOC100331149 // oxysterol-binding LOC1003311 0.000888 0.5 protein-related protein 1-like 49 027 NM_001080589 // hs3st4 // heparan sulfate (glucosamine) 3-O- hs3st4 0.000783 0.5 sulfotransferase 4 // --- / 053 --- 0.000720 0.5 368 ENSDART00000137185 // LOC100334711 // cyclic nucleotide-gated LOC1003347 0.000642 0.5 cation channel alpha-3-li 11 063 --- 0.000547 0.5 566 --- 4.63E-05 0.5 NM_001004656 // reep6 // receptor accessory protein 6 // --- // reep6 5.09E-05 0.5 447918 /// BC081377 // NM_001017569 // cdk5r2a // cyclin-dependent kinase 5, regulatory cdk5r2a 0.000106 0.5 subunit 2a (p39) // -- 154 ENSDART00000148130 // acacb // acetyl-CoA carboxylase beta // -- acacb 0.000589 0.5 - // 556236 725 --- 0.000754 0.5 725 --- 0.000791 0.5 473 ENSDART00000077042 // zgc:153441 // zgc:153441 // --- // 751745 zgc:153441 0.000790 0.5 /// NM_001045455 // zgc 443 XM_686206 // LOC562831 // gamma-aminobutyric acid receptor LOC562831 0.000929 0.5 subunit pi-like // --- // 56 987 ENSDART00000015827 // tnr // tenascin R (restrictin, janusin) // --- tnr 0.000184 0.5 // 369191 /// NM_1 254 ENSDART00000108926 // PPEF2 // protein phosphatase, EF-hand PPEF2 0.000603 0.5 calcium binding domain 2 // 52 XM_695055 // LOC571463 // lipid phosphate phosphatase-related LOC571463 4.05E-05 0.5 protein type 5-like // -- NM_213221 // ccni // cyclin I // --- // 406239 /// ccni 0.000374 0.5 ENSDART00000129351 // ccni // cyclin 128 ENSDART00000147972 // dnm1a // dynamin 1a // --- // 100307098 dnm1a 0.000531 0.5 /// NM_001245965 // dnm1a 492 ENSDART00000112457 // samsn1b // SAM domain, SH3 domain samsn1b 0.000785 0.5 and nuclear localisation signal 77 ENSDART00000137779 // LOC563796 // uncharacterized protein LOC563796 2.81E-05 0.5 C6orf168-like // --- // 5637 ENSDART00000090790 // cadm2b // cell adhesion molecule 2b // --- cadm2b 0.000188 0.5 // 571698 /// NM_00111 395 --- 0.000100 0.5 738 ENSDART00000124642 // UGT2A3 (1 of 15) // UDP UGT2A3 0.000425 0.5 glucuronosyltransferase 2 family, polypep 764 --- 0.000742 0.5 438 --- 0.000738 0.5 789 ENSDART00000123044 // zgc:77222 // zgc:77222 // --- // 404631 /// zgc:77222 0.000409 0.5 NM_207099 // zgc:7722 568 NM_001115125 // hkdc1 // hexokinase domain containing 1 // --- // hkdc1 0.000844 0.5 321224 /// ENSDART000 007

188

ENSDART00000007333 // slc25a36a // solute carrier family 25, slc25a36a 0.000120 0.5 member 36a // --- // 43694 207 ENSDART00000125854 // gpr126 // G protein-coupled receptor 126 gpr126 8.33E-06 0.5 // --- // 561970 /// NM_ --- 0.000184 0.5 375 ENSDART00000144405 // LOC558729 // similar to copine IV // --- // LOC558729 0.000133 0.5 558729 /// ENSDART000 453 NM_001002642 // camk2n2 // calcium/calmodulin-dependent camk2n2 0.000932 0.5 protein kinase II inhibitor 2 / 26 NM_001045203 // si:dkeyp-84f11.5 // si:dkeyp-84f11.5 // --- // si:dkeyp- 0.000129 0.5 570476 /// ENSDART000001 84f11.5 958 ENSDART00000134519 // pde8b // phosphodiesterase 8B // --- // pde8b 0.000493 0.5 571011 /// ENSDART0000014 549 --- 0.000726 0.5 257 --- 5.08E-05 0.5 NM_001135043 // camk1db // calcium/calmodulin-dependent camk1db 0.000181 0.5 protein kinase 1Db // --- // 79 474 ENSDART00000125150 // LOC100537437 // ER lumen protein LOC1005374 0.000728 0.5 retaining receptor 3-like // --- 37 495 ENSDART00000110567 // LOC555627 // calsyntenin-3-like // --- // LOC555627 0.000325 0.5 555627 /// XM_678194 // 605 NM_200978 // prkcbb // protein kinase C, beta b // --- // 393953 /// prkcbb 8.70E-06 0.5 ENSDART00000029451 ENSDART00000090760 // si:ch211-195i6.4 // si:ch211-195i6.4 // --- si:ch211- 0.000998 0.5 // 555506 195i6.4 632 --- 9.91E-06 0.5 XM_002667840 // LOC100333967 // protein kinase C alpha type- LOC1003339 0.000591 0.5 like // --- // 100333967 // 67 1 NM_001076714 // grin1a // glutamate receptor, ionotropic, N- grin1a 0.000284 0.5 methyl D-aspartate 1a // -- 56 --- 0.000696 0.5 842 ENSDART00000045921 // frya // furry homolog a (Drosophila) // --- frya 0.000153 0.5 // 566822 /// XM_6901 621 NM_001012378 // dlg2 // discs, large (Drosophila) homolog 2 // --- // dlg2 0.000137 0.5 497638 /// AY8190 937 BC155646 // fam131a // family with sequence similarity 131, fam131a 0.000267 0.5 member A // --- // 10013686 776 --- 6.92E-05 0.5 NM_001258224 // opn4b // opsin 4b // --- // 100884131 /// GQ925717 opn4b 0.000304 0.5 // opn4b // opsin 4b 811 ENSDART00000056745 // acox3 // acyl-Coenzyme A oxidase 3, acox3 0.000566 0.5 pristanoyl // --- // 406421 / 918 ENSDART00000132919 // LOC569313 // neuronal pentraxin-1-like LOC569313 0.000725 0.5 // --- // 569313 /// XM_69 645 NM_001144043 // gabbr2 // gamma-aminobutyric acid (GABA) B gabbr2 9.43E-05 0.5 receptor, 2 // --- // 560267 AB119258 // acana // aggrecan a // --- // 497505 acana 4.60E-05 0.5 BC155323 // zgc:175108 // zgc:175108 // --- // 569746 /// zgc:175108 9.35E-06 0.5 ENSDART00000111642 // zgc:175 XM_684566 // vsnl1b // visinin-like 1b // --- // 561162 vsnl1b 0.000638 0.5 4 XM_691254 // crhr1 // corticotropin releasing hormone receptor 1 // crhr1 3.07E-05 0.5 --- // 567940 /// E ENSDART00000073815 // PAQR9 // progestin and adipoQ receptor PAQR9 5.06E-05 0.5 family member IX // --- // NM_001111164 // opn3 // opsin 3 (encephalopsin, panopsin) // --- // opn3 4.42E-05 0.5 561815 /// ENSDART0

189

NM_001002537 // parp6a // poly (ADP-ribose) polymerase family, parp6a 2.22E-06 0.5 member 6a // --- // 4368 ENSDART00000144069 // paqr7b // progestin and adipoQ receptor paqr7b 8.53E-05 0.5 family member VII, b // - XM_003199539 // LOC100538000 // bifunctional protein NCOAT- LOC1005380 0.000289 0.5 like // --- // 100538000 /// 00 339 NM_001166331 // nlgn4a // neuroligin 4a // --- // 561122 /// nlgn4a 0.000168 0.4 ENSDART00000113818 // nlgn 735 ENSDART00000017292 // stxbp5l // syntaxin binding protein 5-like stxbp5l 6.19E-05 0.4 // --- // 567605 /// N NM_001109769 // wscd2 // WSC domain-containing protein 2 // --- wscd2 0.000946 0.4 // 561091 /// ENSDART00 805 ENSDART00000065372 // LOC557095 // G protein-activated LOC557095 0.000155 0.4 inward rectifier potassium chann 859 --- 0.000118 0.4 919 ENSDART00000057652 // LOC100538221 // collagen alpha-1(IX) LOC1005382 1.88E-05 0.4 chain-like // --- // 1005382 21 ENSDART00000141372 // LOC571713 // 1-phosphatidylinositol- LOC571713 0.000423 0.4 4,5-bisphosphate phosphodiest 374 NM_001002533 // rab11fip4a // RAB11 family interacting protein 4 rab11fip4a 0.000286 0.4 (class II) a // --- // 995 --- 9.08E-05 0.4 --- 2.83E-05 0.4 --- 7.25E-06 0.4 ENSDART00000042430 // DLG4 (1 of 3) // discs, large homolog 4 DLG4 0.000729 0.4 (Drosophila) // --- // -- 98 XM_681138 // LOC557975 // uncharacterized LOC557975 // --- // LOC557975 0.000820 0.4 557975 817 NM_001003508 // cdk5r2b // cyclin-dependent kinase 5, regulatory cdk5r2b 0.000935 0.4 subunit 2 // --- // 44 589 ENSDART00000002633 // frzb // frizzled-related protein // --- // frzb 0.000877 0.4 30119 /// NM_130943 // 18 --- 0.000385 0.4 984 ENSDART00000085523 // LOC100537012 // brain-specific LOC1005370 0.000141 0.4 angiogenesis inhibitor 1-associate 12 026 --- 0.000703 0.4 238 ENSDART00000010148 // spon2b // spondin 2b, extracellular spon2b 0.000400 0.4 matrix protein // --- // 3020 094 NM_212434 // disp2 // dispatched homolog 2 (Drosophila) // --- // disp2 8.05E-07 0.4 405793 /// ENSDART000 --- 1.93E-05 0.4 --- 0.000750 0.4 517 ENSDART00000121579 // LOC100150472 // sodium- and chloride- LOC1001504 0.000333 0.4 dependent GABA transporter 3 72 655 XM_001919004 // LOC562570 // extracellular leucine-rich repeat LOC562570 8.47E-06 0.4 and fibronectin type-III JN837103 // ntrk3b // neurotrophic tyrosine kinase, receptor, type ntrk3b 1.27E-05 0.4 3b // --- // 798577 ENSDART00000130439 // PRKCA (2 of 3) // protein kinase C, PRKCA 0.000906 0.4 alpha // --- // --- 469 --- 0.000220 0.4 592 XM_684484 // LOC561083 // gamma-aminobutyric acid receptor LOC561083 0.000283 0.4 subunit rho-3-like // --- // 547 BC142872 // gabrb2l // gamma-aminobutyric acid (GABA) A gabrb2l 0.000204 0.4 receptor, beta 2 like // --- // 61

190

ENSDART00000074618 // lrrtm1 // leucine rich repeat lrrtm1 2.49E-07 0.4 transmembrane neuronal 1 // --- // NM_131817 // neurod6b // neurogenic differentiation 6b // --- // neurod6b 4.73E-05 0.4 114415 /// ENSDART0000 ENSDART00000110033 // ABHD8 (2 of 2) // abhydrolase domain ABHD8 0.000149 0.4 containing 8 // --- // --- 127 ENSDART00000101161 // impdh1a // inosine 5'-phosphate impdh1a 0.000172 0.4 dehydrogenase 1a // --- // 431724 505 --- 0.000113 0.4 958 --- 5.66E-05 0.4 ENSDART00000003954 // mapkapk2b // mitogen-activated protein mapkapk2b 0.000366 0.4 kinase-activated protein k 153 ENSDART00000054833 // rgs11 // regulator of G-protein signaling rgs11 0.000734 0.4 11 // --- // 559197 /// 695 ENSDART00000130290 // LOC100537227 // uncharacterized LOC1005372 7.45E-05 0.4 serine/threonine-protein kinase S 27 ENSDART00000092725 // atp2b3b // ATPase, Ca++ transporting, atp2b3b 0.000614 0.4 plasma membrane 3b // --- / 145 DQ017632 // thrab // thyroid hormone receptor alpha b // --- // thrab 0.000355 0.4 558427 /// DQ991962 // 651 --- 0.000248 0.4 905 --- 1.31E-06 0.4 XM_002662191 // LOC100334158 // voltage-gated potassium LOC1003341 0.000646 0.4 channel subunit beta-3-like // 58 713 ENSDART00000129052 // FBXO48 // F-box protein 48 // --- // --- /// FBXO48 0.000756 0.4 XM_003197725 // LOC1 796 ENSDART00000123533 // LOC100538024 // fatty acid desaturase LOC1005380 0.000383 0.4 6-like // --- // 100538024 24 107 NM_001024387 // gabrb2 // gamma-aminobutyric acid (GABA) A gabrb2 0.000574 0.4 receptor, beta 2 // --- // 3 675 NM_001045376 // gabrr2a // gamma-aminobutyric acid (GABA) A gabrr2a 0.000385 0.4 receptor, rho 2a // --- // 217 ENSDART00000022768 // LOC100003595 // novel protein similar LOC1000035 0.000197 0.4 to vertebrate adenylate cyc 95 823 NM_001020603 // trim13 // tripartite motif-containing 13 // --- // trim13 4.90E-07 0.4 553630 /// ENSDART00 --- 0.000868 0.4 363 ENSDART00000076749 // samsn1a // SAM domain, SH3 domain samsn1a 0.000372 0.4 and nuclear localisation signal 676 --- 0.000584 0.4 749 ENSDART00000079840 // rorca // RAR-related orphan receptor C rorca 0.000227 0.4 a // --- // 559245 /// NM_ 635 ENSDART00000136608 // si:dkey-30c15.3 // si:dkey-30c15.3 // --- // si:dkey- 0.000155 0.4 100034584 30c15.3 781 ENSDART00000040086 // pacsin1a // protein kinase C and casein pacsin1a 8.07E-05 0.4 kinase substrate in neuro ENSDART00000090397 // C25H11orf41 (3 of 3) // chromosome 11 C25H11orf41 0.000583 0.4 open reading frame 41 // -- 695 BC081499 // zgc:103625 // zgc:103625 // --- // 447931 /// zgc:103625 0.000251 0.4 ENSDART00000056619 // zgc:103 085 --- 0.000281 0.4 469 NM_001020731 // pcbp3 // poly(rC) binding protein 3 // --- // 553765 pcbp3 0.000147 0.4 /// ENSDART0000001 481 XM_001919172 // lingo3 // leucine rich repeat and Ig domain lingo3 0.000594 0.4 containing 3 // --- // 1001 372

191

XM_002664615 // ulk2 // unc-51-like kinase 2 (C. elegans) // --- // ulk2 3.69E-05 0.4 100329702 ENSDART00000131867 // cnih3 // cornichon homolog 3 cnih3 0.000344 0.4 (Drosophila) // --- // 564015 256 NM_001098755 // slc17a7 // solute carrier family 17 (sodium- slc17a7 2.00E-05 0.4 dependent inorganic phospha ENSDART00000112468 // vip2 // vasoactive intestinal polypeptide vip2 0.000282 0.4 type II // --- // 10000 691 ENSDART00000111841 // LOC559131 // serine/threonine-protein LOC559131 0.000334 0.4 kinase NIM1-like // --- // 292 XM_686547 // LOC563178 // mpv17-like protein-like // --- // 563178 LOC563178 0.000939 0.4 218 ENSDART00000135203 // LOC100536431 // uncharacterized LOC1005364 0.000236 0.4 LOC100536431 // --- // 100536431 31 174 BC153925 // zgc:171453 // zgc:171453 // --- // 565014 /// zgc:171453 7.35E-05 0.4 ENSDART00000086117 // zgc:171 BC134108 // zgc:162825 // zgc:162825 // --- // 100037374 /// zgc:162825 1.06E-05 0.4 ENSDART00000076766 // zgc: --- 2.86E-05 0.4 ENSDART00000098815 // LOC570112 // serine incorporator 4-like LOC570112 0.000691 0.4 // --- // 570112 /// XM_6 926 ENSDART00000067166 // cntn1b // contactin 1b // --- // 541474 /// cntn1b 5.95E-05 0.4 NM_001014814 // cntn1 --- 0.000219 0.4 973 XM_695908 // LOC572215 // solute carrier family 12 member 5-like LOC572215 0.000539 0.4 // --- // 572215 /// E 803 --- 7.97E-05 0.4 --- 0.000785 0.4 392 ENSDART00000054989 // LOC570314 // novel protein similar to LOC570314 9.51E-05 0.4 vertebrate fascin homolog 1 XM_001343070 // LOC100003594 // protein FAM135B-like // --- // LOC1000035 0.000526 0.4 100003594 /// ENSDART000 94 816 --- 0.000967 0.4 068 NM_001111194 // prph2l // peripherin 2, like // --- // 567873 /// prph2l 0.000151 0.4 ENSDART00000010584 // 924 BC095146 // camkvl // CaM kinase-like vesicle-associated, like // --- camkvl 0.000117 0.4 // 553431 284 ENSDART00000147583 // si:ch211-242e8.1 // si:ch211-242e8.1 // --- si:ch211- 0.000263 0.4 // 555427 242e8.1 045 NM_001110392 // ano2 // anoctamin 2 // --- // 566373 /// ano2 0.000143 0.4 ENSDART00000150843 // ano2 // 349 --- 0.000890 0.4 384 ENSDART00000047416 // slc4a8 // solute carrier family 4, sodium slc4a8 0.000181 0.4 bicarbonate cotransport 485 BC124343 // zgc:153394 // zgc:153394 // --- // 767653 /// zgc:153394 0.000730 0.4 ENSDART00000099180 // zgc:153 484 --- 1.21E-05 0.4 ENSDART00000112328 // LOC100001887 // trafficking kinesin- LOC1000018 5.50E-06 0.4 binding protein 1-like // --- 87 --- 0.000604 0.4 022 --- 0.000603 0.4 056 XM_694777 // LOC571208 // potassium voltage-gated channel LOC571208 5.24E-05 0.4 subfamily A member 1-like // ENSDART00000098840 // ralgps1 // Ral GEF with PH domain and ralgps1 1.71E-06 0.4

192

SH3 binding motif 1 // --- ENSDART00000031546 // chrna6 // cholinergic receptor, nicotinic, chrna6 0.000298 0.4 alpha 6 // --- // 5557 579 --- 1.24E-05 0.4 ENSDART00000082000 // EVPL (2 of 2) // envoplakin // --- // --- EVPL 0.000581 0.4 399 NM_203478 // rtn4r // reticulon 4 receptor // --- // 403306 /// rtn4r 6.82E-05 0.4 AY257178 // rtn4r // re ENSDART00000142781 // cald1 // caldesmon 1 // --- // 563892 /// cald1 0.000599 0.4 ENSDART00000121989 // c 083 ENSDART00000041503 // slc4a4a // solute carrier family 4, slc4a4a 0.000349 0.4 member 4a // --- // 568631 // 135 ENSDART00000078460 // LOC100330303 // visceral mesodermal LOC1003303 2.26E-05 0.4 armadillo-repeats-like // --- 03 --- 0.000696 0.4 347 XM_001923224 // LOC100148888 // leucine-rich repeat-containing LOC1001488 0.000348 0.4 protein C22orf36-like // 88 762 NM_001002542 // camk2d2 // calcium/calmodulin-dependent camk2d2 8.29E-05 0.4 protein kinase (CaM kinase) II BC155345 // wu:fb25h12 // wu:fb25h12 // --- // 571418 /// wu:fb25h12 4.94E-06 0.4 ENSDART00000113851 // wu:fb25 XM_002666792 // sv2b // synaptic vesicle glycoprotein 2 b // --- // sv2b 9.41E-06 0.4 100006427 /// XM_00 --- 0.000480 0.4 646 --- 0.000234 0.4 574 --- 0.000775 0.4 416 ENSDART00000148013 // LOC564916 // GDNF family receptor LOC564916 2.59E-06 0.4 alpha-2-like // --- // 564916 / HQ113097 // tcf4 // transcription factor 4 // --- // 100526651 tcf4 3.16E-05 0.4 ENSDART00000067455 // dpysl5b // dihydropyrimidinase-like 5b // dpysl5b 7.56E-05 0.4 --- // 553410 /// NM_00 XM_677888 // adcyap1r1 // adenylate cyclase activating polypeptide adcyap1r1 0.000138 0.4 1 (pituitary) recept 223 ENSDART00000028787 // ahr1b // aryl hydrocarbon receptor 1b // ahr1b 0.000210 0.4 --- // 554265 /// NM_001 359 ENSDART00000123245 // igsf21a // immunoglobin superfamily, igsf21a 1.72E-05 0.4 member 21a // --- // 569872 ENSDART00000003008 // gad1b // glutamate decarboxylase 1b // -- gad1b 3.76E-05 0.4 - // 378441 /// NM_19441 BC122296 // zgc:153443 // zgc:153443 // --- // 751686 /// zgc:153443 0.000146 0.4 ENSDART00000111805 // zgc:153 347 --- 2.42E-05 0.4 NM_001190760 // slc1a7b // solute carrier family 1 (glutamate slc1a7b 0.000187 0.4 transporter), member 7b / 422 ENSDART00000081960 // LOC559157 // contactin-associated LOC559157 5.79E-05 0.4 protein-like 2-like // --- // 5 NM_001166231 // wisp3 // WNT1 inducible signaling pathway wisp3 0.000344 0.4 protein 3 // --- // 794092 // 17 --- 0.000200 0.4 632 --- 2.30E-05 0.4 --- 4.66E-05 0.4 ENSDART00000108959 // LOC796392 // RING finger protein 208- LOC796392 4.38E-09 0.4 like // --- // 796392 /// XM

193

--- 0.000979 0.4 482 ENSDART00000041714 // atp6v0a1b // ATPase, H+ transporting, atp6v0a1b 0.000559 0.4 lysosomal V0 subunit a isof 481 ENSDART00000011078 // CACNA1F (2 of 2) // calcium channel, CACNA1F 0.000403 0.4 voltage-dependent, L type, a 064 --- 0.000159 0.4 289 XM_678907 // LOC556170 // uncharacterized LOC556170 // --- // LOC556170 0.000437 0.4 556170 748 ENSDART00000142653 // grm1a // glutamate receptor, grm1a 1.65E-05 0.4 metabotropic 1a // --- // 555576 /// --- 0.000204 0.3 07 ENSDART00000114936 // LOC562934 // endothelial cell-selective LOC562934 5.09E-06 0.3 adhesion molecule-like // ENSDART00000082688 // si:ch211-271b14.7 // si:ch211-271b14.7 // si:ch211- 0.000396 0.3 --- // 562750 271b14.7 404 ENSDART00000057622 // LOC100006044 // fas apoptotic LOC1000060 0.000336 0.3 inhibitory molecule 2-like // --- / 44 944 --- 0.000908 0.3 455 ENSDART00000122432 // FNDC7 (3 of 3) // fibronectin type III FNDC7 4.07E-05 0.3 domain containing 7 // --- ENSDART00000091707 // dbpa // D site of albumin promoter dbpa 3.02E-05 0.3 (albumin D-box) binding protei --- 0.000429 0.3 113 --- 0.000826 0.3 424 BC129335 // kcnb2 // potassium voltage-gated channel, Shab- kcnb2 0.000249 0.3 related subfamily, member 2 729 --- 3.70E-05 0.3 ENSDART00000064403 // nptnb // neuroplastin b // --- // 403006 /// nptnb 1.90E-05 0.3 NM_205705 // nptnb / NM_201028 // cpne5 // copine V // --- // 394003 /// cpne5 0.000479 0.3 ENSDART00000126071 // cpne5 // copi 046 ENSDART00000055264 // LOC568543 // carbonic anhydrase- LOC568543 1.90E-07 0.3 related protein 10-like // --- // --- 0.000522 0.3 792 NM_001114714 // si:dkey-1h24.2 // si:dkey-1h24.2 // --- // 100137117 si:dkey-1h24.2 0.000310 0.3 /// ENSDART0000010 77 ENSDART00000097822 // atp1b2b // ATPase, Na+/K+ atp1b2b 0.000161 0.3 transporting, beta 2b polypeptide // -. 682 ENSDART00000140365 // LOC797331 // novel protein similar to LOC797331 6.51E-05 0.3 vertebrate solute carrier f ENSDART00000082697 // gabra6a // gamma-aminobutyric acid gabra6a 2.63E-05 0.3 (GABA) A receptor, alpha 6a // XM_003201409 // LOC100536841 // cytosolic carboxypeptidase 1- LOC1005368 6.88E-06 0.3 like // --- // 100536841 41 NM_001030242 // sypb // synaptophysin b // --- // 436774 /// sypb 0.000744 0.3 ENSDART00000088240 // sypb 953 --- 0.000776 0.3 956 --- 0.000325 0.3 004 ENSDART00000128676 // LOC100150385 // uncharacterized LOC1001503 8.34E-06 0.3 LOC100150385 // --- // 100150385 85 --- 0.000690 0.3 029

194

XM_001923562 // LOC100000156 // cytochrome P450 2G1-like // --- LOC1000001 0.000972 0.3 // 100000156 /// ENSDAR 56 568 ENSDART00000127627 // LOC100536392 // cadherin-related LOC1005363 1.47E-05 0.3 family member 5-like // --- // 1 92 --- 2.55E-05 0.3 ENSDART00000114146 // NECAB3 // N-terminal EF-hand calcium NECAB3 0.000415 0.3 binding protein 3 // --- // 707 NM_001018164 // lrit1a // leucine-rich repeat, immunoglobulin-like lrit1a 1.77E-05 0.3 and transmembrane do NM_213523 // krt15 // keratin 15 // --- // 406844 /// krt15 0.000156 0.3 ENSDART00000025016 // krt15 // ke 136 --- 9.37E-06 0.3 --- 0.000337 0.3 675 --- 0.000651 0.3 853 NM_001037574 // zgc:122979 // zgc:122979 // --- // 641576 /// zgc:122979 2.62E-07 0.3 ENSDART00000102559 // zgc --- 5.00E-05 0.3 XM_680598 // LOC557518 // transketolase-like // --- // 557518 /// LOC557518 0.000285 0.3 ENSDART00000045888 // 89 BC115305 // nfixb // nuclear factor I/Xb // --- // 678521 /// nfixb 8.03E-05 0.3 ENSDART00000090235 // nfi ENSDART00000110005 // cdhr1a // cadherin-related family cdhr1a 2.50E-05 0.3 member 1a // --- // 449008 /// XM_001346021 // LOC100007655 // protein Tob2-like // --- // LOC1000076 0.000686 0.3 100007655 /// ENSDART000001 55 415 NM_001004656 // reep6 // receptor accessory protein 6 // --- // reep6 2.34E-05 0.3 447918 /// BC081377 // BC128792 // atp8b5b // ATPase, class I, type 8B, member 5b // --- // atp8b5b 0.000345 0.3 563450 156 --- 0.000621 0.3 578 ENSDART00000131206 // LOC797069 // e3 ubiquitin-protein ligase LOC797069 0.000231 0.3 MARCH1-like // --- // 79 048 ENSDART00000147001 // si:dkeyp-72h1.1 // si:dkeyp-72h1.1 // --- // si:dkeyp- 3.04E-05 0.3 799956 /// BC124643 72h1.1 ENSDART00000058796 // fstl5 // follistatin-like 5 // --- // 566192 /// fstl5 0.000155 0.3 NM_001031842 // 948 --- 0.000211 0.3 288 --- 0.000472 0.3 165 ENSDART00000137900 // grin2ab // glutamate receptor, grin2ab 0.000266 0.3 ionotropic, N-methyl D-aspartate 2 196 ENSDART00000049219 // cacnb2b // calcium channel, voltage- cacnb2b 4.10E-05 0.3 dependent, beta 2b // --- // ENSDART00000100948 // LOC100330754 // tubby-related protein LOC1003307 0.000103 0.3 1-like // --- // 100330754 54 427 NM_001128694 // zgc:195063 // zgc:195063 // --- // 558559 /// zgc:195063 2.50E-05 0.3 ENSDART00000077823 // zgc XM_001919091 // LOC792447 // germ cell-specific gene 1-like LOC792447 4.68E-05 0.3 protein-like // --- // 7924 NM_001045321 // npas4a // neuronal PAS domain protein 4a // --- // npas4a 0.000776 0.3 724016 /// ENSDART00 735 XM_683811 // LOC560410 // adenylate cyclase type 8-like // --- // LOC560410 0.000587 0.3 560410 /// ENSDART000 385 ENSDART00000145155 // LOC571422 // similar to Gamma- LOC571422 1.07E-05 0.3 aminobutyric acid receptor subunit --- 0.000204 0.3

195

002 --- 0.000198 0.3 277 FJ392628 // nrgna // neurogranin (protein kinase C substrate, RC3) nrgna 0.000141 0.3 a // --- // 567608 / 301 ENSDART00000052346 // gnao1b // guanine nucleotide binding gnao1b 3.30E-05 0.3 protein (G protein), alpha a --- 0.000752 0.3 329 ENSDART00000018501 // opn4.1 // opsin 4.1 // --- // 352918 /// opn4.1 0.000195 0.3 NM_178289 // opn4.1 // o 365 BC096789 // zgc:109965 // zgc:109965 // --- // 619266 /// zgc:109965 0.000670 0.3 ENSDART00000122705 // zgc:109 184 ENSDART00000049291 // gria3a // glutamate receptor, ionotropic, gria3a 5.84E-05 0.3 AMPA 3a // --- // 17045 NM_001123277 // si:dkey-91i10.3 // si:dkey-91i10.3 // --- // 565876 /// si:dkey- 0.000209 0.3 ENSDART00000079 91i10.3 448 ENSDART00000140850 // fxyd6 // FXYD domain containing ion fxyd6 0.000893 0.3 transport regulator 6 // --- 611 NM_131866 // gc3 // guanylyl cyclase 3 // --- // 140426 /// gc3 9.76E-05 0.3 ENSDART00000097770 // gc3 / NM_001161488 // pcp4l1 // Purkinje cell protein 4 like 1 // --- // pcp4l1 0.000218 0.3 100008021 /// ENSDAR 687 --- 0.000409 0.3 499 --- 6.54E-05 0.3 --- 6.54E-05 0.3 --- 0.000132 0.3 364 XM_002660888 // si:dkeyp-51f11.8 // si:dkeyp-51f11.8 // --- // si:dkeyp- 0.000341 0.3 100319971 /// ENSDART000 51f11.8 727 BC142843 // lppr5b // lipid phosphate phosphatase-related protein lppr5b 3.03E-06 0.3 type 5b // --- // 100 ENSDART00000128032 // LOC100002104 // complement C1q-like LOC1000021 0.000640 0.3 protein 4-like // --- // 1000 04 004 ENSDART00000141824 // LOC794185 // uncharacterized LOC794185 0.000630 0.3 LOC794185 // --- // 794185 /// XM_00 449 XM_001920557 // cacna2d4 // calcium channel, voltage-dependent, cacna2d4 1.24E-05 0.3 alpha 2/delta subunit 4 NM_001080165 // colm // collomin // --- // 572018 /// colm 0.000851 0.3 ENSDART00000087960 // colm // col 037 --- 0.000492 0.3 535 --- 0.000491 0.3 744 ENSDART00000136525 // LOC563546 // ubiquitin carboxyl- LOC563546 0.000908 0.3 terminal hydrolase 21-like // --- 304 --- 0.000518 0.3 627 HM138700 // slc1a8a // solute carrier family 1 (glutamate slc1a8a 1.78E-05 0.3 transporter), member 8a // -- ENSDART00000136378 // mb // myoglobin // --- // 393558 /// mb 9.44E-05 0.3 NM_200586 // mb // myoglobin ENSDART00000055269 // gng13b // guanine nucleotide binding gng13b 1.04E-06 0.3 protein (G protein), gamma 1 XR_117663 // LOC100537033 // uncharacterized LOC100537033 // LOC1005370 8.44E-05 0.3 --- // 100537033 33 --- 8.71E-05 0.3 ENSDART00000138658 // LOC566599 // uncharacterized LOC566599 0.000410 0.3 LOC566599 // --- // 566599 222

196

--- 6.73E-05 0.3 --- 0.000247 0.3 427 --- 0.000241 0.3 849 ENSDART00000003514 // plp1b // proteolipid protein 1b // --- // plp1b 9.04E-05 0.3 368234 /// NM_001005586 --- 0.000788 0.3 615 --- 0.000358 0.3 974 XM_682733 // LOC559395 // asc-type amino acid transporter 1-like LOC559395 0.000334 0.3 // --- // 559395 /// E 416 NM_001128745 // nrn1lb // neuritin 1-like b // --- // 568997 /// nrn1lb 1.27E-05 0.3 ENSDART00000086905 // ENSDART00000151372 // ncaldb // neurocalcin delta b // --- // ncaldb 0.000284 0.3 445319 /// ENSDART0000001 529 --- 0.000110 0.3 644 --- 0.000987 0.3 269 ENSDART00000074997 // ccdc136b // coiled-coil domain containing ccdc136b 0.000107 0.3 136b // --- // 10032171 442 XM_002667333 // wu:fd14a01 // wu:fd14a01 // --- // 100008376 /// wu:fd14a01 0.000388 0.3 ENSDART00000051360 // 673 NM_001082901 // col8a2 // collagen, type VIII, alpha 2 // --- // col8a2 0.000255 0.3 794479 /// ENSDART0000 619 XM_002665219 // LOC100331800 // photoreceptor outer segment LOC1003318 0.000138 0.3 membrane glycoprotein 2-lik 00 904 ENSDART00000141493 // LOC563500 // anoctamin-1-like // --- // LOC563500 1.49E-06 0.3 563500 /// XM_686862 // L ENSDART00000077724 // gnb5b // guanine nucleotide binding gnb5b 2.11E-05 0.3 protein (G protein), beta 5b --- 0.000372 0.2 866 NM_001007322 // mybpc1 // myosin binding protein C, slow type // mybpc1 9.12E-05 0.2 --- // 492356 /// ENSD --- 5.82E-05 0.2 --- 0.000280 0.2 387 --- 0.000284 0.2 197 BC162874 // si:ch211-81a5.8 // si:ch211-81a5.8 // --- // 560648 /// si:ch211- 0.000187 0.2 ENSDART00000104637 81a5.8 251 ENSDART00000022959 // guk1b // guanylate kinase 1b // --- // guk1b 0.000795 0.2 393697 /// NM_200724 // gu 768 ENSDART00000076322 // zgc:171544 // zgc:171544 // --- // 561738 zgc:171544 0.000308 0.2 /// NM_001114742 // zgc 199 --- 0.000110 0.2 288 NM_001017822 // zgc:110204 // zgc:110204 // --- // 550520 /// zgc:110204 0.000531 0.2 ENSDART00000014454 // zgc 276 ENSDART00000014661 // glmnb // glomulin, FKBP associated glmnb 0.000186 0.2 protein b // --- // 100170791 615 ENSDART00000150894 // LOC100331226 // MAGUK p55 LOC1003312 9.10E-07 0.2 subfamily member 4-like // --- // 10033 26 --- 0.000556 0.2 659 --- 0.000215 0.2 043

197

ENSDART00000145775 // LOC100535324 // MAGUK p55 LOC1005353 6.61E-06 0.2 subfamily member 4-like // --- // 10053 24 --- 8.10E-05 0.2 BC127586 // zgc:158340 // zgc:158340 // --- // 780836 /// zgc:158340 8.70E-05 0.2 ENSDART00000102312 // zgc:158 ENSDART00000125743 // slc25a3a // solute carrier family 25 slc25a3a 0.000240 0.2 (mitochondrial carrier; phos 335 ENSDART00000124154 // LOC100331665 // phenylserine LOC1003316 7.25E-06 0.2 dehydratase-like // --- // 100331665 65 ENSDART00000066501 // zgc:163073 // zgc:163073 // --- // zgc:163073 0.000870 0.2 100037358 /// NM_001089511 // 656 NM_200693 // arl3l1 // ADP-ribosylation factor-like 3, like 1 // --- // arl3l1 0.000376 0.2 393666 /// BC07 916 XM_001345079 // LOC100006333 // teneurin-2-like // --- // LOC1000063 0.000297 0.2 100006333 33 22 ENSDART00000127706 // lrit1b // leucine-rich repeat, lrit1b 8.55E-06 0.2 immunoglobulin-like and transmembr --- 7.74E-05 0.2 ENSDART00000004619 // IMPG2 (3 of 3) // interphotoreceptor IMPG2 4.06E-07 0.2 matrix proteoglycan 2 // --- NR_030507 // mir726 // microRNA 726 // --- // 100033737 mir726 2.58E-05 0.2 ENSDART00000104353 // atp2b1b // ATPase, Ca++ transporting, atp2b1b 1.14E-05 0.2 plasma membrane 1b // --- / NM_001144131 // grin1b // glutamate receptor, ionotropic, N- grin1b 2.19E-05 0.2 methyl D-aspartate 1b // -- --- 7.36E-06 0.2 ENSDART00000086936 // IMPG2 (2 of 3) // interphotoreceptor IMPG2 2.32E-05 0.2 matrix proteoglycan 2 // --- ENSDART00000100287 // grk7a // G-protein-coupled receptor grk7a 7.06E-05 0.2 kinase 7a // --- // 566120 // ENSDART00000105952 // aqp8a.2 // aquaporin 8a, tandem aqp8a.2 0.000334 0.2 duplicate 2 // --- // 563130 /// 548 ENSDART00000145835 // tmx3 // thioredoxin-related tmx3 6.44E-06 0.2 transmembrane protein 3 // --- // 553 NM_001020546 // syt5b // synaptotagmin Vb // --- // 553567 /// syt5b 0.000439 0.2 ENSDART00000013117 // sy 694 ENSDART00000058773 // rgs16 // regulator of G-protein signaling rgs16 3.19E-05 0.2 16 // --- // 569828 /// ENSDART00000016753 // mag // myelin associated glycoprotein // - mag 6.39E-05 0.2 -- // 474346 /// NM_001 XM_003199754 // LOC100535278 // uncharacterized LOC1005352 0.000553 0.2 LOC100535278 // --- // 100535278 78 617 --- 0.000259 0.2 738 --- 6.38E-07 0.2 ENSDART00000145035 // saga // S-antigen; retina and pineal gland saga 0.000103 0.2 (arrestin) a // --- // 284 XM_001335844 // LOC100000241 // uncharacterized LOC1000002 0.000334 0.2 LOC100000241 // --- // 100000241 41 788 XM_001334934 // LOC794903 // collagen alpha-4(VI) chain-like // -- LOC794903 4.88E-05 0.2 - // 794903 --- 0.000123 0.2 745 --- 1.06E-05 0.2 ENSDART00000133035 // syt5a // synaptotagmin Va // --- // 436686 syt5a 0.000350 0.2 /// ENSDART00000059197 739 --- 0.000406 0.2 55

198

ENSDART00000054322 // cnrip1b // cannabinoid receptor cnrip1b 2.62E-05 0.2 interacting protein 1b // --- // --- 6.91E-05 0.2 ENSDART00000054735 // LOC558290 // synaptoporin-like // --- // LOC558290 0.000834 0.2 558290 /// XM_681491 // 405 ENSDART00000006897 // rlbp1a // retinaldehyde binding protein rlbp1a 0.000224 0.2 1a // --- // 393678 /// N 172 ENSDART00000134719 // prom1b // prominin 1 b // --- // 378834 /// prom1b 4.89E-06 0.2 ENSDART00000102768 // NM_205729 // nr1d1 // nuclear receptor subfamily 1, group d, nr1d1 4.52E-05 0.2 member 1 // --- // 494487 --- 0.000158 0.2 573 --- 0.000382 0.2 899 NM_001089376 // stxbp1b // syntaxin binding protein 1b // --- // stxbp1b 0.000202 0.2 557717 /// BC171526 // 175 ENSDART00000058936 // LOC100004357 // secretory carrier- LOC1000043 9.88E-05 0.2 associated membrane protein 5A- 57 NM_001110473 // igsf21b // immunoglobin superfamily, member igsf21b 2.84E-07 0.2 21b // --- // 567714 /// EN --- 2.27E-05 0.2 BC049482 // rom1b // retinal outer segment membrane protein 1b // rom1b 0.000410 0.1 --- // 393989 /// ENS 03 NM_001190305 // slc1a2a // solute carrier family 1 (glial high slc1a2a 6.22E-05 0.1 affinity glutamate trans ENSDART00000084011 // cplx4a // complexin 4a // --- // 768157 /// cplx4a 6.14E-05 0.1 NM_001077300 // cplx4 --- 0.000222 0.1 718 XM_002662494 // LOC566922 // gamma-aminobutyric acid LOC566922 3.20E-06 0.1 receptor subunit beta-3-like // -- ENSDART00000055995 // sagb // S-antigen; retina and pineal gland sagb 9.98E-07 0.1 (arrestin) b // --- // NM_200751 // rpe65a // retinal pigment epithelium-specific protein rpe65a 0.000401 0.1 65a // --- // 393724 295 --- 0.000203 0.1 242 ENSDART00000126830 // opn1mw1 // opsin 1 (cone pigments), opn1mw1 0.000184 0.1 medium-wave-sensitive, 1 // - 83 XM_682552 // LOC559232 // regulator of G-protein signaling 9- LOC559232 3.19E-06 0.1 binding protein-like // -- BC151864 // wu:fb15e04 // wu:fb15e04 // --- // 566445 wu:fb15e04 3.87E-05 0.1 XM_693199 // LOC569792 // uncharacterized LOC569792 // --- // LOC569792 0.000286 0.1 569792 511 XM_001336435 // LOC100000094 // uncharacterized LOC1000000 2.12E-05 0.1 LOC100000094 // --- // 100000094 94 NM_200794 // rom1a // retinal outer segment membrane protein 1a rom1a 6.74E-05 0.1 // --- // 393767 /// EN --- 0.000485 0.1 139 --- 0.000425 0.1 655 ENSDART00000012673 // gnb3a // guanine nucleotide binding gnb3a 3.08E-06 0.1 protein (G protein), beta pol XM_003199054 // LOC799480 // uncharacterized LOC799480 // --- LOC799480 0.000204 0.1 // 799480 /// ENSDART0000 272 NM_001017711 // grk1b // G protein-coupled receptor kinase 1 b // - grk1b 8.90E-05 0.1 -- // 550406 /// ENS --- 9.03E-05 0.1

199

ENSDART00000105741 // AGL (3 of 3) // amylo-alpha-1, 6- AGL 0.000124 0.1 glucosidase, 4-alpha-glucanotran 883 BC076192 // faimb // Fas apoptotic inhibitory molecule b // --- // faimb 0.000111 0.1 436668 /// ENSDART00 959 ENSDART00000130128 // rgs9a // regulator of G-protein signaling rgs9a 3.09E-05 0.1 9a // --- // 767636 /// --- 7.04E-06 0.1 NM_131451 // irbp // interphotoreceptor retinoid-binding protein // irbp 0.000105 0.1 --- // 30735 /// EN 774 --- 3.53E-05 0.1 --- 5.43E-05 0.1 NM_001030248 // zgc:114180 // zgc:114180 // --- // 570333 /// zgc:114180 0.000178 0.1 ENSDART00000074036 // zgc 495 ENSDART00000111499 // IMPG1 // interphotoreceptor matrix IMPG1 1.58E-06 0.1 proteoglycan 1 // --- // --- BC091979 // aglb // amylo-1, 6-glucosidase, 4-alpha- aglb 7.44E-05 0.1 glucanotransferase b // --- // 5533 --- 6.73E-06 0.1 --- 7.14E-06 0.1 ENSDART00000080106 // zgc:158677 // zgc:158677 // --- // zgc:158677 0.000114 0.1 100009626 /// NM_001082995 // 077 --- 2.72E-05 0.1 --- 3.31E-06 0.1 ENSDART00000055415 // prph2a // peripherin 2a (retinal prph2a 8.11E-06 0.1 degeneration, slow) // --- // 58 ENSDART00000036050 // rs1 // retinoschisis (X-linked, juvenile) 1 rs1 0.000342 0.1 // --- // 445044 /// 256 NM_131869 // gnat2 // guanine nucleotide binding protein (G gnat2 1.35E-06 0.1 protein), alpha transducing XM_001339170 // myhb // myosin, heavy chain b // --- // 100002040 myhb 0.000392 0.1 /// ENSDART0000002625 725 NM_200825 // zgc:73075 // zgc:73075 // --- // 572207 /// zgc:73075 2.48E-05 0.1 ENSDART00000128721 // zgc:7307 --- 2.21E-05 0.1 --- 2.21E-05 0.1 --- 0.000151 0.1 521 --- 2.89E-05 0.1 ENSDART00000114673 // LOC100535672 // trypsin-1-like // --- // LOC1005356 0.000199 0.1 100535672 72 248 ENSDART00000078996 // arr3a // arrestin 3a, retinal (X-arrestin) // arr3a 3.74E-06 0.1 --- // 436678 /// N ENSDART00000023543 // rcv1 // recoverin // --- // 335650 /// rcv1 1.33E-05 0.1 NM_199964 // rcv1 // recov ENSDART00000106501 // pde6c // phosphodiesterase 6C, cGMP- pde6c 9.37E-06 0.1 specific, cone, alpha prime / NM_131192 // opn1sw2 // opsin 1 (cone pigments), short-wave- opn1sw2 1.12E-05 0.1 sensitive 2 // --- // 30435 BC060940 // zgc:73359 // zgc:73359 // --- // 393810 zgc:73359 1.09E-05 0.1 --- 9.93E-08 0.1 --- 0.000369 0.1 116 ENSDART00000064896 // gnat1 // guanine nucleotide binding gnat1 0.000455 0.1 protein (G protein), alpha tr 101 ENSDART00000075513 // aqp9b // aquaporin 9b // --- // 570191 /// aqp9b 0.000203 0.1 NM_001177744 // aqp9b 846

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--- 0.000210 0.1 744 NM_200785 // pde6h // phosphodiesterase 6H, cGMP-specific, cone, pde6h 2.37E-05 0.1 gamma // --- // 393758 BC047826 // zgc:56085 // zgc:56085 // --- // 327506 /// zgc:56085 2.53E-05 0.0 ENSDART00000101449 // zgc:56085 ENSDART00000005547 // gnb3b // guanine nucleotide binding gnb3b 2.82E-06 0.0 protein (G protein), beta pol ENSDART00000065940 // opn1lw2 // opsin 1 (cone pigments), long- opn1lw2 7.87E-07 0.0 wave-sensitive, 2 // ------0.000338 0.0 57 --- 0.000296 0.0 576 ENSDART00000020671 // prph2b // peripherin 2b (retinal prph2b 3.18E-06 0.0 degeneration, slow) // --- // 55 ENSDART00000027000 // rho // rhodopsin // --- // 30295 /// rho 2.33E-05 0.0 NM_131084 // rho // rhodopsi NM_001204332 // gngt2b // guanine nucleotide binding protein (G gngt2b 9.53E-07 0.0 protein), gamma transdu --- 7.70E-05 0.0 ENSDART00000067160 // opn1sw1 // opsin 1 (cone pigments), opn1sw1 2.81E-05 0.0 short-wave-sensitive 1 // ------3.69E-06 0.0

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Table 2A. List of selected significantly up and down regulated genes in leptin morphants.

Gene Adj P Fold Gene Assignment Symbol value Change ENSDART00000062845 // mmp9 // matrix metalloproteinase 9 // --- // mmp9 2.5E-04 9.9 406397 /// NM_213123 + NM_001161552 // fosl1a // FOS-like antigen 1a // --- // 564241 /// fosl1a 4.8E-05 8.5 ENSDART00000008373 / + NM_001113589 // hsp70l // heat shock cognate 70-kd protein, like // --- // hsp70l 6.5E-05 7.7 560210 /// N + NM_199896 // iars // isoleucyl-tRNA synthetase // --- // 334393 /// iars 6.1E-07 6.3 ENSDART00000004423 + ENSDART00000006180 // dla // deltaA // --- // 30131 /// NM_130954 // dla 1.3E-06 6.3 dla // deltaA // - + NM_131441 // notch1a // notch homolog 1a // --- // 30718 notch1a 1.7E-06 5.6 + ENSDART00000019259 // dlb // deltaB // --- // 30141 /// NM_130958 // dlb 1.0E-08 4.8 dlb // deltaB // - + ENSDART00000099224 // dld // deltaD // --- // 30138 /// NM_130955 // dld 2.7E-07 3.9 dld // deltaD // - + NM_001044310 // aars // alanyl-tRNA synthetase // --- // 324940 /// aars 6.4E-06 3.9 NM_001040035 // aar + ENSDART00000050855 // notch1b // notch homolog 1b // --- // 794892 /// notch1b 1.6E-05 3.0 NM_131302 // not + ENSDART00000130626 // rars // arginyl-tRNA synthetase // --- // rars 8.2E-05 2.9 337070 /// ENSDART00000 + ENSDART00000102888 // cdk6 // cyclin-dependent kinase 6 // --- // cdk6 3.3E-04 2.8 100034507 /// ENSDART + ENSDART00000047851 // jag1a // jagged 1a // --- // 140421 /// jag1a 7.7E-06 2.5 NM_131861 // jag1a // jag + NM_001204248 // eprs // glutamyl-prolyl-tRNA synthetase // --- // eprs 1.4E-04 2.5 562037 + ENSDART00000073930 // notch3 // notch homolog 3 // --- // 58066 /// notch3 1.7E-05 2.3 NM_131549 // notch3 + ENSDART00000018514 // dlc // deltaC // --- // 30120 /// NM_130944 // dlc 1.5E-04 2.3 dlc // deltaC // - + NM_131025 // ccnd1 // cyclin D1 // --- // 30222 /// ccnd1 4.1E-04 2.3 ENSDART00000051868 // ccnd1 // cycl + ENSDART00000104616 // lepr // leptin receptor // --- // 567241 /// lepr 3.9E-04 2.1 NM_001113376 // lepr + ENSDART00000128602 // tfdp2 // transcription factor Dp-2 // --- // tfdp2 1.0E-04 2.1 338204 /// NM_198208 + BC090426 // vars // valyl-tRNA synthetase // --- // 114427 /// vars 7.8E-04 1.9 ENSDART00000004832 // va + NM_131191 // smn1 // survival motor neuron 1 // --- // 30432 /// smn1 5.7E-05 1.7 ENSDART00000028099 // + NM_200866 // ctnnbl1 // catenin, beta like 1 // --- // 393840 /// ctnnbl1 7.5E-04 1.6 ENSDART00000015535 // + ENSDART00000067160 // opn1sw1 // opsin 1 (cone pigments), short- opn1sw1 2.8E-05 -56.45 - wave-sensitive 1 // --- ENSDART00000027000 // rho // rhodopsin // --- // 30295 /// NM_131084 rho 2.3E-05 -31.72 - // rho // rhodopsi ENSDART00000027000 // rho // rhodopsin // --- // 30295 /// NM_131084 rho 2.3E-05 -31.72 - // rho // rhodopsi ENSDART00000020671 // prph2b // peripherin 2b (retinal prph2b 3.2E-06 -27.83 - degeneration, slow) // --- // 55 ENSDART00000065940 // opn1lw2 // opsin 1 (cone pigments), long- opn1lw2 7.9E-07 -24.87 - wave-sensitive, 2 // --- NM_131192 // opn1sw2 // opsin 1 (cone pigments), short-wave-sensitive opn1sw2 1.1E-05 -15.82 - 2 // --- // 30435 ENSDART00000126830 // opn1mw1 // opsin 1 (cone pigments), opn1mw1 1.8E-04 -7.22 -

202 medium-wave-sensitive, 1 // - ENSDART00000133035 // syt5a // synaptotagmin Va // --- // 436686 /// syt5a 3.5E-04 -5.46 - ENSDART00000059197 NM_001020546 // syt5b // synaptotagmin Vb // --- // 553567 /// syt5b 4.4E-04 -5.04 - ENSDART00000013117 // sy NM_001144131 // grin1b // glutamate receptor, ionotropic, N-methyl D- grin1b 2.2E-05 -4.90 - aspartate 1b // -- ENSDART00000137900 // grin2ab // glutamate receptor, ionotropic, N- grin2ab 2.7E-04 -3.26 - methyl D-aspartate 2 DQ017632 // thrab // thyroid hormone receptor alpha b // --- // 558427 /// thrab 3.6E-04 -2.33 - DQ991962 // NM_001002542 // camk2d2 // calcium/calmodulin-dependent protein camk2d2 8.3E-05 -2.64 - kinase (CaM kinase) II ENSDART00000092725 // atp2b3b // ATPase, Ca++ transporting, atp2b3b 6.1E-04 -2.33 - plasma membrane 3b // --- / NM_001166331 // nlgn4a // neuroligin 4a // --- // 561122 /// nlgn4a 1.7E-04 -2.22 - ENSDART00000113818 // nlgn NM_001076714 // grin1a // glutamate receptor, ionotropic, N-methyl D- grin1a 2.8E-04 -2.17 - aspartate 1a // -- XM_691254 // crhr1 // corticotropin releasing hormone receptor 1 // --- crhr1 3.1E-05 -2.20 - // 567940 /// E NM_200978 // prkcbb // protein kinase C, beta b // --- // 393953 /// prkcbb 8.7E-06 -2.16 - ENSDART00000029451 NM_001003851 // negr1 // neuronal growth regulator 1 // --- // 445374 /// negr1 4.4E-06 -1.93 - ENSDART000000 ENSDART00000112484 // nlgn2a // neuroligin 2a // --- // 100002407 /// nlgn2a 7.1E-04 -1.93 - NM_001166336 // n ENSDART00000081604 // adipor2 // adiponectin receptor 2 // --- // adipor2 8.0E-04 -1.87 - 560140 /// NM_0010255 ENSDART00000148650 // stat2 // signal transducer and activator of stat2 7.8E-04 -1.85 - transcription 2 // -- NM_001002463 // syt1a // synaptotagmin Ia // --- // 436736 /// syt1a 3.9E-04 -1.84 - ENSDART00000066896 // sy NM_213351 // calm1a // calmodulin 1a // --- // 406660 /// calm1a 2.3E-05 -1.78 - ENSDART00000034580 // calm1a XM_687795 // grm7 // glutamate receptor, metabotropic 7 // --- // 564461 grm7 5.3E-04 -1.61 - /// ENSDART000 NM_001199370 // oxtr // oxytocin receptor // --- // 100001530 /// oxtr 9.0E-04 -1.76 - ENSDART00000050114 // ENSDART00000057611 // oprm1 // opioid receptor, mu 1 // --- // 65088 oprm1 9.0E-04 -1.73 - /// NM_131707 // o ENSDART00000100681 // ncam2 // neural cell adhesion molecule 2 // --- ncam2 7.2E-04 -1.65 - // 114441 /// NM_ ENSDART00000145354 // tcirg1 // T-cell, immune regulator 1, ATPase, tcirg1 3.8E-05 -1.68 - H+ transporting, ly

203

Optical Density 570nm @ Density Optical

Time (seconds)

Figure A1. Zebrafish embryo carbonic acid production microplate assay at 72 hpf.

Each line is a representative data course for each individual treatment. Blank= no embryo, LepMO= Zebrafish microinjected with leptin morpholino, Control= wildtype embryo, and Rescue= LepMO + recombinant zebrafish leptin. Y axis is measured optical density at 570nm and X axis is time (in seconds). Regression of microplate data is linear: Control Embryo R2= 0.943, Rescue embryo R2= 0.9628, and LepMO morphant embryo R2= 0.901. For example, control embryo at x= 2000s, y is OD of 0.36. For Rescue, x=1400s, y is OD of -0.20. Leptin Morphant at 1400s, y is a OD of -0.05.

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