PH73CH09-Hammerschmidt ARI 7 January 2011 12:19

Zebrafish in Endocrine Systems: Recent Advances and Implications for Human Disease

Heiko Lohr¨ 1 and Matthias Hammerschmidt1,2,3

1Institute for Developmental Biology, 2Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CEDAD), 3Center for Molecular Medicine Cologne (CMMC), University of Cologne, D-50923 Cologne, Germany; email: [email protected]

Annu. Rev. Physiol. 2011. 73:183–211 Keywords The Annual Review of Physiology is online at hypothalamo-pituitary axis, energy homeostasis, sleep, stress response, physiol.annualreviews.org reproduction, osmoregulation This article’s doi: 10.1146/annurev-physiol-012110-142320 Abstract Copyright c 2011 by Annual Reviews. Since its introduction as a genetic vertebrate model system approxi- All rights reserved mately 30 years ago, the focus of zebrafish research has increasingly

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. 0066-4278/11/0315-0183$20.00 shifted to questions that are also relevant for human development and disease. Here, we review the potential of the zebrafish as a model Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org for human endocrine systems. A recent review compared the func- tions of the different endocrine systems and glands in zebrafish with those in other vertebrates, including humans, coming to the conclu- sion that major aspects are conserved. Here, we present an updated overview of this rapidly growing field of zebrafish research, focusing on the hypothalamo-pituitary axis, which links the central nervous sys- tem with the endocrine systems, and on major processes that are under (neuro)endocrine control and are the subject of intensive current re- search in other endocrine model organisms, such as feeding circuits and energy homeostasis, sleep, stress, reproduction, osmoregulation, and calcium homeostasis. Finally, we summarize the strengths and weak- nesses of zebrafish as a model for studying endocrine systems.

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INTRODUCTION: THE established in zebrafish: TILLING (targeting ZEBRAFISH MODEL induced local lesions in genomes) and, more recently, zinc finger nuclease technology. The prime animal models for studies of Alternatively, specific gene products can be biomedically relevant questions of endocrine inactivated via injection of chemically modified systems are the rat and the mouse, mammals antisense morpholino oligonucleotides (MOs). like humans. Furthermore, the mouse is highly For endocrine systems, this approach is usually suitable for reverse genetics, such as condi- restricted to studying their early development, tional recombinant gene targeting. However, rather than their function, as MOs injected the zebrafish (Danio rerio), a member of the into fertilized eggs are effective only during Cyprinidae group of teleost, native to India and the first 3 to 5 days of development. Burma, and a popular pet fish, may offer crucial Transgenic approaches are used for spa- complementary strengths (for a recent review, tially and/or temporally controlled overexpres- see Reference 1). A major genetic advantage of sion studies, for fluorochrome labeling of spe- the zebrafish compared with nonfish vertebrate cific cell types and live in vivo imaging, and for model systems is its high suitability to forward toxin-driven specific cell ablations. In addition, genetics. Compared with the mouse, zebrafish again taking advantage of the transparency of are small (up to 5 cm in length), can be kept at fish, one can ablate cells with a laser. To al- much higher density, and are easy to maintain low targeted ablation and for control, such ab- under laboratory conditions. In addition, fecun- lation is usually done with transgenic lines in dity is much higher, with single females giving which particular cell lineages are labeled with a weekly clutches of 100 to 500 synchronously fluorochrome (2). developing eggs. Like mice, zebrafish reach Finally, the zebrafish has become a power- sexual maturity at an age of approximately ful system for small-compound screening. This 3 months. However, zebrafish embryonic application again takes advantage of the rela- development is much faster, and most organs, tively small body size of the fish and the possi- including glands, are formed within the first bility of keeping larvae in 96 or 384 well dishes 5 days of development, when fish are only a for large-scale drug testing. This makes the ze- few millimeters long. Together, this makes the brafish a prime organism for pharmacological zebrafish an ideal system for large-scale and toxicity testing (3). As more and more mutant high-throughput mutagenesis screens, allow- zebrafish models for human diseases become ing the identification of novel essential genes on available, the use of zebrafish for therapeutic the basis of the phenotypes caused by randomly screening and de novo drug discovery is likely induced mutations. Mutations are usually intro- by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. to increase (4–6). duced via the chemical N-ethyl-N-nitrosourea More detailed descriptions of all these tech- (ENU) or via different insertional approaches, Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org niques and the respective references can be and techniques for subsequent cloning of the found in the Supplemental Text (follow Supplemental Material affected genes are well established. For many the Supplemental Material link from the purposes, phenotype screening can be done via Annual Reviews home page at http://www. visual inspection, taking advantage of the trans- annualreviews.org). We refer to several of parency of zebrafish larvae. Alternatively, spe- these technologies as we describe the differ- cific cell types can be labeled, for instance, via ent endocrine systems and hormone-controlled whole mount in situ hybridizations, immuno- processes in the following sections, and we re- stainings, or transgene-encoded fluoro- turn to these technologies at the end of this chromes. Furthermore, behavioral assays have review when we summarize the strengths and been established. weaknesses of the zebrafish as a model for en- In addition to forward genetics, differ- docrine systems. ent reverse-genetics approaches have been

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THE HYPOTHALAMO- circadian rhythms, and/or energy homeostasis PITUITARY AXIS (7–10). As described in more detail below, all hypothalamo-pituitary axes also appear to exist The hypothalamo-pituitary axis constitutes the AH: adenohypophysis in zebrafish, and all seem to act in tight coop- physiological and anatomical link between the eration with other neuroendocrine systems in NH: neurohypophysis central nervous system (CNS), represented by control of body physiology (Figure 1). ARC: arcuate nucleus the hypothalamus, and the endocrine system, TRH: thyrotropin- represented by its master gland, the pituitary, releasing hormone also termed the hypophysis. The pituitary CRH: corticotropin- regulates a whole array of basic physiological The Neuroendocrine Hypothalamus releasing hormone processes involved in body homeostasis and The concept of neurosecretion dates back to GHRH: growth reproduction and consists of two parts, the 1928, when a young doctoral student at the hormone–releasing adenohypophysis (AH) and the neurohypoph- University of Munich, Ernst Scharrer, ob- hormone ysis (NH) (see below for details). In this axis, served specialized neurons in the CNS of the GnRH: particular neuroendocrine cells of the hy- teleost Phoxinus laevis (minnow) that secreted gonadotropin- pothalamus control the activity of the different hormones into the bloodstream. In hindsight, releasing hormone adenohypophyseal cell types via specific releas- this was a revolutionary discovery that demon- DA: dopamine ing or release-inhibiting hormones, to which strated the presence of neuroendocrine cells. adenohypophyseal cells respond by secretion Today, it is known that specialized neuronal of their own hormones to evoke peripheral cells in the mammalian hypothalamus inner- gland or end-organ responses, including vate the pituitary and release neuroactive sub- negative feedback loops to the hypothalamus stances, mostly peptides, into the bloodstream and pituitary. In addition, the hypothalamus to act in a hormonal fashion. generates direct effector hormones, which are In the mammalian hypothalamus, two types secreted via the NH (Figure 1). Prominent of neuroendocrine systems that project into glands under adenohypophyseal control are the or toward the pituitary—the parvocellular and cortex of the adrenal gland, which generates the magnocellular systems—can be distin- glucocorticoid hormones like cortisol; the guished. Parvocellular neuroendocrine cells thyroid, which generates thyroid hormones are located in several hypothalamic nuclei (T3 and T4); and the gonads, which generate lining the third ventricle and include the sex steroid hormones (testosterone, estrogen, arcuate nuclei (ARC), tuberal nuclei, anterior and progesterone). Therefore, many authors periventricular nuclei, and preoptic nuclei as prefer to further subdivide the axis into, for well as paraventricular nuclei (PVN). The par- instance, the hypothalamo-pituitary-adrenal vocellular neuroendocrine system comprises by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. (HPA) axis, also termed the hypothalamo- six different neurohormones, all of which are pituitary-interrenal (HPI) axis in fish; the

Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org brought via a small portal blood vessel system hypothalamo-pituitary-thyroid (HPT) axis; to the AH (the anterior lobe of pituitary; see and the hypothalamo-pituitary-gonadal (HPG) below), where they in turn provoke or inhibit axis. Although the HPA axis is concerned pri- release of adenohypophyseal hormones into marily with stress and immune responses, the the bloodstream. These six neurohormones HPT axis with metabolism, and the HPG are thyrotropin-releasing hormone (TRH), axis with reproduction, their total effects are corticotropin-releasing hormone (CRH), much more complex, and they are tightly growth hormone–releasing hormone (GHRH), interconnected. Furthermore, they cooperate somatostatin (SST), gonadotropin-releasing with other neuroendocrine systems of the hormone (GnRH), and dopamine (DA) (the last hypothalamus that are of high biomedical of which is the sole nonpeptidergic hormone relevance and the subject of intensive current in this system). Magnocellular neurons, in con- research, such as the systems regulating sleep, trast, reside in the PVN and supraoptic nucleus

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Hypothalamus Classical neuronal Neuroendocrine systems systems

NPY/AgRP Effectors Regulators CRH POMC/CART TRH HCRT SST GnRH3 MCH GHRH SF1 DA

AVPL Pituitary OXTL NH

Sl C T C Sl L M G S AH

TSH PRL α-MSH FSH SL GH SL β-Endorphin LH GH ACTH SL

Sleep Energy Osmo- Stress Reproduction homeostasis regulation Interrenal Pineal Thyroid Kidney/gills gland Gonads

Melatonin T3, T4 Angiotensin Cortisol Sex steroids by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org

Liver Stomach Pancreas Fat Various organs/tissues ?

Adiponectin Ghrelin Insulin Leptin Calcitonin Stannicalcin PTH Leptin

Energy homeostasis Calcium homeostasis Somatic growth

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(SON) and synthesize oxytocin (OT) or argi- has been isolated, and the hormone’s spatial nine vasopressin (AVP). Both of these peptides distribution has been studied in the adult brain are directly exported via axonal projections to with cross-reacting antibodies (23, 24). OT: oxytocin the NH (the posterior lobe of the pituitary) Transgenic labeling experiments of the and are secreted into the blood. A specific soma and axons with fluorochromes (see Intro- AVP: arginine vasopressin neuroendocrine cell lineage may express more duction and the Supplemental Text) have al- than one of the listed hormones. Moreover, lowed our lab and others to begin analyzing AVPL: arginine vasopressin like most neuropeptides are also produced by other the neuroendocrine network of the zebrafish neuronal cell types within the CNS, where hypothalamus (Figure 2e). Indeed, hypotha- they function as neurotransmitters rather than lamic avpl-expressing cells present in the pre- Supplemental Material as endocrine or paracrine signaling factors. optic area (Figure 2f ) directly project to the Orthologs for all mammalian neuroen- NH, and AVPL can be detected in the NH with docrine peptides have been identified and specific antibodies (H. Lohr,¨ H.-M. Pogoda & localized in zebrafish. However, because most M. Hammerschmidt, unpublished data). Simi- of these hormones are expressed in more than lar results have been obtained for hypothalamic one hypothalamic domain, and because the GnRH cells and the AH. Similar to their mam- anatomical correlation between zebrafish and malian counterparts, zebrafish GnRH neurons mammalian hypothalamic nuclei is not always derive from anterior preplacodal ectoderm and clear, the exact locations of the neuroendocrine only secondarily migrate into the hypothala- cell groups are sometimes difficult to pinpoint. mus (25). Through use of a transgenic zebrafish In the case of trh (11), crh (12, 13), sst (sst1, line recapitulating endogenous gnrh3 expres- sst3) (14, 15), gnrh3 (Figure 2g) (16), oxytocin sion, direct axonal projections from GnRH3 like [oxtl; formerly termed isotocin (17, 18)], cells in the preoptic area of adult zebrafish to and arginin vasopressin like [avpl; formerly the pituitary were revealed (16). termed vasotocin (19)] (Figure 2f ), a common As in mammals, the roles of zebrafish TRH, expression domain in the preoptic area has CRH, SST, GnRH, GHRH, and DA are been identified (20). The localization of DA most likely concerned with the regulation of neurons and projections has been intensively anterior pituitary activity, although direct func- studied in larvae and adult fish using the rate- tional data via in vivo neuropeptide adminis- limiting enzyme of DA biosynthesis, tyrosine tration have been obtained for only some of hydroxylase, as a marker (21, 22). However, the hormones and mainly in other (larger) fish. because the zebrafish hypothalamus harbors For similar treatments in zebrafish, primary pi- several distinct DA cell groups with over- tuitary cell culture systems have been estab-

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. lapping endohypothalamic projection tracts, lished, revealing stimulation of gonadotrope ex- identification of the parvocellular DA system pression upon treatment with salmon GnRH Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org with its projections to the AH has not been peptide (26). Furthermore, zebrafish laser abla- possible. For GHRH, the zebrafish ortholog tion of GnRH3 cells resulted in arrested oocyte

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 1 Schematic of the different hypothalamic regulators ( pink), hormones (blue), sites of their expression ( pink), and regulated processes ( green). Whether leptin is generated in fat tissue has not been investigated (indicated by question mark). Abbreviations: ACTH, adrenocorticotropic hormone; AgRP, agouti-related peptide; AH, adenohypophysis; AVPL, arginine vasopressin like; C, corticotropes; CART, cocaine- and amphetamine-regulated transcript; CRH, corticotropin-releasing hormone; DA, dopamine; FSH, follicle- stimulating hormone; G, gonadotropes; GH, growth hormone; GHRH, growth hormone–releasing hormone; GnRH, gonadotropin- releasing hormone; HRCT, hypocretin; L, lactotropes; LH, luteinizing hormone; M, melanotropes; MCH, melanin-concentrating hormone; MSH, melanocyte-stimulating hormone; NH, neurohypophysis; NPY, neuropeptide Y; OXTL, oxytocin like; POMC, proopiomelanocortin; PRL, prolactin; PTH, parathyroid hormone; S, somatotropes; SF1, steroidogenic factor; Sl, somatolactotropes; SL, somatolactin; SST, somatostatin; T, thyrotropes; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone.

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development and reduced average oocyte diam- section below (27). In sum, both neuroanatomy eter, demonstrating the essential functional role together with data on the development of spe- of these cells within the HPG axis (2). Exper- cific neuroendocrine cell lineages suggest a PRL: prolactin imental evidence for a regulation of prolactin high level of conservation of the hypothala- (PRL)-producing lactotropes of the AH by DA mic control systems between mammals and is described in more detail in the pituitary zebrafish.

abcrabp1a prl

NH AH

oc 7 dpfAH oc NH 2 dpf c pomc::gfp d pomc::gfp prl::rfp prl::rfp

100 μm 100 μm 3 dpf 240 dpf

e otpb::mgfp f avpl prl::rfp m Ce POA

t d by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. OB NH

Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org NLT 5 dpf 30 dpf * ghgnrh3 pomc-a

* 30 dpf* 30 dpf

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Anatomy and Physiology of the likely are subject to regulation by growth factor Zebrafish Pituitary signaling, although no functional analyses have been reported to address this point. Studies focused on the development and OXTL: oxytocin like The AH (anterior pituitary) constitutes the anatomy of the zebrafish pituitary reveal both nonneural part of the pituitary and is embry- similarities with and differences between the ologically derived from placodal ectoderm. It mammalian system (28). Like its mammalian contains distinct endocrine cell lineages, which counterpart, the zebrafish pituitary consists of are characterized by the type of hormone they two different parts (Figure 2a), which differ in secrete (see Table 1 and Figure 1). In the ze- developmental origin and physiology. brafish AH, nine different cell types can be dis- The NH (posterior pituitary) derives tinguished on the basis of anatomical position from a ventral extension of the develop- and hormone expression profile: lactotropes, ing hypothalamus and represents the neu- two distinct groups of corticotropes, thy- ral compartment of the gland. It consists rotropes, somatotropes, two groups of soma- of (a) axonal nerve endings from hypotha- tolactotropes, melanotropes, and gonadotropes lamic magnocellular neurons, which release (see Table 1 for hormones). Expression of the AVP/AVPL or OT/OXTL neurohormones corresponding hormone genes can be readily (see above) into the systemic bloodstream, and detected via in situ hybridization in distinct ade- (b) pituicytes, which do not generate hor- nohypophyseal cells as early as 2–5 days post- mones but most likely have supportive and fertilization (dpf) (31), which is before the fish modulatory functions. In zebrafish, pituicytes swim freely, take up food, and grow in size. Ex- can be readily identified by the expression ceptions may be follicle-stimulating hormone of specific marker genes such as fzd8b (friz- (FSH) and luteinizing hormone (LH) made by zled homolog 8b) (29), encoding a recep- gonadotropes. fshβ and lhβ transcript levels tor for Wnt signals, and crap1b, encod- in the larval zebrafish pituitary are extremely ing cellular retinoic acid–binding protein 1a low and detectable only via reverse tran- (Figure 2b) (28, 30). The expression of these scriptase polymerase chain reaction (RT-PCR) genes suggests that pituicytes receive and most ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 2 Stainings of the zebrafish hypothalamo-pituitary axis and the hypothalamic melanocortin system. Ages of fish are indicated in each panel’s lower left corner [in days postfertilization (dpf)], and used in situ probes or transgenes are indicated in the upper right corners. (a,b) Toluidine blue staining of longitudinal section,

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. revealing the morphology of the larval pituitary gland. oc denotes oral cavity. (b) Double in situ hybridization for prl in the adenohypophysis (AH) and cellular retinoic acid–binding protein crapb1a in the neurohypophysis (NH) anlage; lateral view. (c,d ) Live imaging of PRL- and proopiomelanocortin Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org (POMC)-synthesizing adenohypophyseal cells labeled with transgene-encoded red fluorescent protein (RFP) and green fluorescent protein (GFP), respectively (27, 37, 190); dorsal views. Despite the massive growth of the pituitary, the overall patterning, with lactotropes and corticotropes in the anterior pars distalis and melanotropes in the pars intermedialis, is maintained from the larval to the adult stages. (e)Liveimaging of innervation of the pituitary by axons of hypothalamic neuroendocrine cells, labeled by membrane-tagged GFP generated under the control of the otpb promoter (transgenic line generously supplied by Josh Bonkowsky). The NH is intensively innervated, whereas the anterior pars distalis of the AH is only sparsely innervated. ( f–h) Expression patterns of hypothalamic neuropeptide hormones in juvenile fish via whole- brain in situ hybridization. The pituitaries, whose positions are indicated by asterisks, have been removed during the preparations. ( f ) The bodies of arginine vasopressin–like (Avpl)-generating cells are located in the preoptic area (POA), rather remote from the NH, the site of Avpl secretion. Other abbreviations: Ce, cerebellum; d, diencephalon; m, mesencephalon; NLT, nucleus lateralis tuberis; OB, olfactory bulb; t, telencephalon. ( g) gnrh3 has an additional expression domain in the NLT. (h) The NLT also displays expression of POMC. Therefore, in analogy to mammals, this site is also referred to as the arcuate nucleus (53).

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Table 1 Zebrafish adenohypophyseal hormones, cell types, and a PRL receptor from 24 h postfertilization hypophalamic regulatorsa onward (27). Zebrafish corticotropes may have Hormone Cell type Hypothalamic control References a similar early negative feedback system. Thus, PRL Lactotrope DA 31, 39, 40 treatment of early zebrafish larvae with the GH Somatotrope GHRH, SST 31, 40 glucocorticoid agonist dexamethasone leads to + α-, β-SL Somatolactotrope Unknown (Ca2 , 39–43 downregulation of (37, 38), and knockdown osmolarity?) of the adrenal adrenocorticotropic hormone α-MSH Melanotrope DA, CRH, TRH, AVPL 31, 37, 44, 45 (ACTH) receptor MC2R leads to upregulation ACTH Corticotrope CRH, AVPL 31, 37, 44 of, pomc expression in anterior corticotropes at TSH Thyrotrope TRH 31 2 dpf (38). These findings indicate that negative feedback regulation within the zebrafish HPI FSH, LH Gonadotrope GnRH 32, 33, 36 axis and the PRL system starts within a day aAbbreviations used: ACTH, adrenocorticotropic hormone; AVPL, arginine vasopressin after AH hormone synthesis has initiated. like; CRH, corticotropin-releasing hormone; DA, dopamine; FSH, follicle-stimulating There is one major difference in the hormone; GH, growth hormone; GHRH, growth hormone–releasing hormone; anatomy of the mammalian versus teleostean GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; MSH, melanocyte- hypothalamo-pituitary axis. The median stimulating hormone; PRL, prolactin; SL, somatolactin; SST, somatostatin; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone. eminence, which connects the pituitary with the hypothalamus, is much more prominent in mammals than in teleosts. The mammalian (32), whereas in juvenile and adult pituitaries, median eminence is a stalk-like neurohemal transcripts can be readily stained via in situ hy- structure, which contains axons of magnocellu- bridization (33). This is possibly correlated with lar neuroendocrine cells that project to the NH, the comparably late onset of sex differentiation as well as a portal blood vessel system that trans- in zebrafish, which starts at an age of 3 weeks ports neuropeptides from the parvocellular (34, 35). However, in recent transgenic analy- system to the AH. In zebrafish and all other in- ses with gonadotropin-specific promoters, lhβ vestigated teleosts, this portal system is absent, was detected in the pituitary of early zebrafish the pituitary is positioned directly underneath larvae (36). the hypothalamus, and neuroendocrine systems Activation or inhibition of adenohy- more directly innervate both the NH and AH pophysial hormone release is most likely (Figure 2a,e) (28). These differences may be controlled by preoptic neuroendocrine hor- due to the differential action of axon-guiding mones and peripheral hormones acting through molecules and angiogenic factors during the classical feedback mechanisms. Regulation by development of the hypothalamo-pituitary axis

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. inhibitory dopaminergic inputs that most likely (M. Placzek, personal communication; H.-M. stem from the hypothalamus has been demon- Pogoda & M. Hammerschmidt, unpublished Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org strated for the regulation of PRL-secreting data). Whether these differences in anatomical lactotropes of the AH (27). The AH of ze- organization of the hypothalamo-pituitary brafish larvae displays expression of the DA axis also have functional implications remains receptor Drd2c. Knockdown of this receptor unclear. or treatment with DA antagonists leads to an increase in PRL levels at 2 dpf, whereas treat- ment with DA agonists has the opposite effect FEEDING BEHAVIOR AND and decreases PRL levels (27). These results ENERGY HOMEOSTASIS indicate that endogenous dopaminergic tonic Maintaining the proper energy balance in a inhibition of lactotropes is operative from the multicellular organism requires multiple reg- ACTH: adrenocorticotropic earliest larval stages. In addition, there seems to ulatory steps. This includes control of appetite hormone be a direct feedback mechanism by PRL signal- and food uptake, processes involved in diges- ing to adenohypophyseal cells, which express tion and absorption of nutrients, and control of

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basal cell metabolism, which determines how second-order neurons and further downstream available energy is utilized by the organism for, effectors is far from fully understood. The net- e.g., physical exercise, heat production, storage work appears to include both CNS-internal AgRP: agouti-related in lipid depots, or linear body growth. The de- processing, as well as hormonal output via neu- peptide scribed actions require extensive cross-talk be- roendocrine motor systems and the pituitary. POMC: tween neuronal and hormonal control mecha- Second-order neurons include TRH and CRH proopiomelanocortin nisms, and many aspects of these processes seem cells of the PVN, which in turn are components MSH: melanocyte- to be conserved between mammals and teleosts. of the hypothalamo-pituitary axis (see above), stimulating suggesting a link between the melanocortin sys- hormone tem to the pituitary and its downstream effec- Hypothalamic Control Mechanisms tors and targets (49, 50). Several regions of the brain mediate CNS con- In mouse, loss-of-function mutations in trol of feeding behavior and energy homeosta- many of these anorexigenic or orexigenic sis, with the hypothalamus playing a primary regulators cause obese or lean phenotypes, role. The hypothalamus receives and integrates respectively. Upon starvation or feeding ad inputs from peripheral hormones or metabo- libitum, zebrafish can also become lean or lites that reflect the nutritional state of the or- obese, in addition to effects on linear body ganism and then relay information to down- growth (see below). Furthermore, all crucial stream mediators. regulators known from mammalian systems One prominent hypothalamic circuit asso- have been identified. NPY-positive cell bodies, ciated with mammalian energy homeostasis is neurites, and projections are localized in the the melanocortin system of the ARC and PVN hypothalamus of zebrafish larvae (51, 52). Also, (46, 47). In the center of this system are two dis- hypothalamic expression of the agrp transcript tinct, antagonistic neuronal cell populations in in zebrafish has been detected, and functional the ARC. One cell population coexpresses neu- conservation of agrp neurons for energy ropeptide Y (NPY) and agouti-related peptide homeostasis has been studied. Consistent (AgRP) and has orexigenic effects (promotes with data from the mouse model, the agrp food uptake and inhibits energy expenditure). transcript is upregulated in the hypothalamus The other cell population coexpresses proo- of starved fish, and the creation of a transgenic piomelanocortin (POMC), the precursor of zebrafish line with ubiquitous overexpression α-melanocyte-stimulating hormone (α-MSH), of agrp resulted in fish with increased linear and cocaine- and amphetamine-regulated tran- growth and an obesity phenotype (53, 54). For script (CART) and has anorexigenic effects (in- pomc, researchers have described two potential

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. hibits food uptake and promotes energy expen- zebrafish orthologs, pomca and pomcb,which diture). These cell populations respond to the originate from whole-genome duplication Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org current energy state of the organism, which (31, 44). pomca is expressed in both the hy- they sense via signals from the body deliv- pothalamus (Figure 2h) and the AH, whereas ered to them via the blood, such as the hor- pomcb expression is restricted to the CNS mones leptin, ghrelin, and insulin and glu- (M. Angerer, H. Lohr¨ & M. Hammerschmidt, cose (48). In turn, α-MSH and AgRP neurons unpublished data). Like mammalian POMC, project to various so-called second-order neu- the product of the zebrafish pomca gene con- rons, including neuroendocrine cell lineages of tains sequences encoding the cleavage products the PVN, which express the α-MSH receptor ACTH; α-MSH, which is the supposed MC4R (melanocortin 4 receptor). AgRP also relevant neuropeptide in the mammalian hy- binds to MC4R but fails to activate receptor pothalamic melanocortin system; β-MSH; the signaling and thus blocks second-order neu- lipotropic hormones (lipotropins) β-LPH and ronal activity and the corresponding anorexi- γ-LPH; and the opioid β-endorphin peptide. genic response. The complex network of these In contrast, the zebrafish pomcb gene can give

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rise only to α-MSH and to β-endorphin (44). ing of HCRT neurons, investigators revealed Also for CART, two genes, cart1 and cart2, long, descending projections from the hypotha- are present in the zebrafish genome, and tran- lamus into spinal regions of zebrafish larvae HCRT: hypocretin scripts can be detected in several brain regions, (61). Consistently, the zebrafish hypocretin re- including the hypothalamus (M. Angerer & M. ceptor (hcrtr2) is expressed at several sites in Hammerschmidt, unpublished data). the CNS, including the spinal cord, where the Localization of α-MSH in relation to AgRP HCRT system may be implicated in locomo- has been mapped in zebrafish larvae and adults. tion control (62–64). This possible pathway is Projections derived from both α-MSH and interesting, as it may be a way of regulating AgRP cell populations innervate many of the energy homeostasis via physical activity. Thus same hypothalamic target regions, in line with far, a zebrafish mutant in the sole hypocretin a conservation of the neuroanatomical circuits receptor gene hcrtr2 that was isolated via the of the melanocortin system between zebrafish TILLING approach has been described only and mammals (55). Zebrafish mutants in MC4R in the context of sleep (64) but may be useful (56) were recently generated via the TILLING to investigate possible connections between the approach, but their phenotype has not been HCRT system, locomotion, and energy home- described as yet. ostasis control. Beyond the ARC/PVN melanocortin Another site involved in the regulation of circuit, other hypothalamic nuclei play a energy homeostasis and reproduction in mam- prominent role in the regulation of mammalian mals is the ventromedial hypothalamus, charac- feeding behavior, including the lateral hy- terized by the expression of the nuclear recep- pothalamic area (LHA). The mammalian LHA tor NR5A1 (SF1). Although no functional data contains two distinct neuronal cell groups, exist for the zebrafish ventromedial hypotha- expressing either melanin-concentrating hor- lamus (VMH), researchers have shown that mone (MCH) (57) or hypocretin (HCRT; the genetic program controlling its develop- also termed orexin). Both MCH and HCRT ment is largely conserved between zebrafish and independently stimulate food intake upon mammals (65). central administration. Two promelanin- concentrating hormone ( pmch) genes, pmch1 and pmch2, exist in zebrafish and are expressed Signals from the Periphery by neighboring cells in the hypothalamus An increasing array of peripheral hormones (58). According to the genomic organization and metabolites affect the hypothalamic and the amino acid sequences of the mature melanocortin system and energy homeostasis

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. peptides, zebrafish MCH2 is more closely in mammals (48). Of those, the hormones related to mammalian MCH than is MCH1. leptin, insulin, and ghrelin are most prominent Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org Consistently, pmch2 expression and the number and, among other effects, regulate the expres- of hypothalamic pmch2-expressing cells are sion levels of POMC and AgRP in the ARC. upregulated upon long-term starvation. Leptin is derived mainly from adipose tissue For HCRT, zebrafish research has focused and leads to a decrease in food consumption and mostly on the role of HCRT in regulating an increase in energy expenditure upon bind- the sleep/wake cycle (see section entitled Sleep ing to its receptor on hypothalamic neurons, and Circadian Rhythms, below). However, one including the ARC, LH, and other hypotha- publication reports a significant upregulation of lamic sites. Two leptin genes, termed leptin a hcrt transcript in zebrafish upon long-term fast- and leptin b, may exist in zebrafish (66), and al- ing, accompanied by increased physical activity though amino acid identity shared between ze- (59). In goldfish, intraperitoneal HCRT injec- brafish and mammalian leptins is low, synteny tion caused increased food uptake (60). Using of the genomic loci and conformational pre- transgenic approaches for specific GFP label- dictions indicate that these may be true leptin

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orthologs. Both zebrafish leptin a and leptin b are orexigenic effects of GH secretagogues have expressed in a variety of different tissues, includ- also been observed in trout (73). A zebrafish ing the liver; however, expression in adipose ghrelin ortholog is expressed in various tissues, GH: growth hormone tissue (see below) was not examined. Further- including the brain and digestive organs (74, more, leptin b transcription is downregulated af- 75). As in mammals, ghrelin transcript levels are ter starvation of fish for 1 week. In addition, a upregulated upon starvation (74). Moreover, recently cloned potential zebrafish ortholog of GHSRs are abundantly expressed in the ze- the leptin receptor was expressed during larval brafish brain (76). Knockdown of ghrelin func- stages and in adult fish (67). Functional studies tion in zebrafish using a morpholino approach have not been performed for any of these ze- leads to a decrease in GH expression (78), simi- brafish genes. However, intraperitoneal or in- lar to the situation in mammals, although the ef- tracerebroventricular administration of mam- fect of ghrelin on agrp/npy expression within the malian leptin significantly reduced food intake hypothalamic melanocortin system (77) has not in goldfish (68, 69). been investigated. Nevertheless, in sum, these Insulin, which is secreted by the pancreas in data suggest that the function of ghrelin and response to high blood glucose levels, has an possibly the entire melanocortin system may be anorexigenic effect similar to that of leptin and conserved between fish and mammals. stimulates expression of hypothalamic POMC. Surprisingly little is known about zebrafish insulin in this context. There are two zebrafish Adipocytes, Lipid Metabolism, insulin paralogs, ins and insb, both of which and Somatic Growth are expressed in the pancreas from the early To fully comprehend an endocrine system, one larval stages onward (70). The two zebrafish needs to identify the relevant and important insulin receptor paralogs, insra and insrb,are target tissues of endocrine signaling. With expressed at moderate levels in the developing respect to energy homeostasis, adipose tissue brain. Interestingly, morpholino-mediated is one such target tissue and has only recently insr knockdown caused growth retardation received attention in zebrafish. In mammals, and defects during brain morphogenesis (71). there are two types of adipose tissue: (a)white Genetic mutants of these paralogs should adipose tissue, the body’s main deposit of neu- be isolated to assess their possible metabolic tral lipids that serves to store energy and that, function in zebrafish. as a kind of feedback mechanism, generates Another anorexigenic peripheral hormone, its own hormones like leptin (see above), and made mainly by adipose tissue and the liver, (b) brown adipose tissue, the major site of

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. is adiponectin. Two adiponectin paralogs, to- nonshivering thermogenesis, i.e., the burning gether with three different adiponectin recep- of energy. In the latter tissue type, oxidative en- Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org tors, are expressed in zebrafish. Furthermore, ergy of the respiratory chain is dissipated into similar to the effect of starvation on leptin, heat instead of ATP production through the starvation of adult zebrafish causes reduced action of uncoupling protein 1 (UCP1). In con- adiponectin b expression in the liver (72). trast to mammals, which are homothermic and In contrast to these anorexigenic periph- in which nonshivering thermogenesis is crucial eral hormones, ghrelin stimulates food intake for maintenance of proper body temperature, and decreases energy expenditure in mammals. ectothermic fish lack brown adipose tissue and It binds to so-called growth hormone secre- possess only white adipose tissue. However, fish tagogue receptors (GHSRs), which were ini- do generate heat that can, at least in some cases, tially found on the basis of their ability to be used for temperature adaptation in specific mediate the effect of growth hormone (GH) regions of the body. For example, cranial secretagogues to increase GH secretion from endothermy has been reported in several fish the pituitary. Similar combined endocrine and species, including swordfish and blue marlin.

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In these fish, the brain heater organ warms and total fat and cholesterol content (87), es- the brain and the eye up to 20◦C above water tablishing the zebrafish as a model system for temperature (79). Although in the brain heater development of antiobesity therapeutics. organ, which is a derivative of extraocular An interesting aspect of energy homeostasis muscles, heat is most likely generated via me- in zebrafish is that, despite their capacity chanical forces, fish may also generate heat via to store energy as neutral lipids in adipose uncoupling of the respiratory chain. Intrigu- tissue, these organisms respond to increased ingly, despite the lack of brown adipose tissue, energy supply with linear growth instead of teleosts, including zebrafish, have uncoupling becoming heavily obese (53). This pattern proteins, including UCP1 (80, 81), and ucp ex- applies not only to juvenile stages but also, pression is increased upon food deprivation. In although to a lesser extent, to adult fish. This carp, increased UCP1 expression in the brain phenomenon is even more prominent in other was even observed following cold exposure fish species, such as giant Danio, a close relative (82). Together, these observations are in line of zebrafish, which displays indeterminate and with the notion that even ectothermic fish are GH-inducible growth throughout its entire capable of nonshivering thermogenesis, which life span (89, 90). The same is true for several may be used to eliminate excessive energy. mammalian species. Interestingly, even obese Development of white adipose tissue as well mice display a subtle increase in body length. as adipocyte physiology and metabolism have In this light, it is tempting to speculate that GH recently been studied in zebrafish. Apparently, release, possible via the action of GHRH, may the cellular mechanisms of lipid metabolism are be under the direct control of the hypothalamic conserved between fish and mammals (83–85). melanocortin system, similar to the HPA and Similar to mammals, fish store lipids in the form HPT axes (see above). This general principle of triacylglycerol. Neutral lipid deposits in ze- may apply to all vertebrate species but with brafish can be visualized using the fluorescent different relative strengths of the different vital dye Nile red in living larvae and young branches of the hypothalamic control system. adults (86, 87), and oil red O can be used on In sum, studies on feeding behavior and en- fixed samples (86, 88). Moreover, expression of ergy homeostasis in zebrafish reveal a high level the nuclear receptor pparg and the lipid chaper- of conservation between fish and tetrapods. one fabp11a is a marker for adipocyte lineages Virtually all hypothalamic circuits implicated in zebrafish. During early zebrafish ontogeny, in regulation of the energy homeostat in the yolk is the exclusive provider of body en- mammals are present in zebrafish and are ergy. Only at 5 dpf, when the nutrient supply likely functional. Peripheral hormones, which

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. of the yolk sac has been depleted, does exoge- report the energy state to the brain, have nous feeding start. From 8 dpf onward, lipid been characterized. However, to our knowl- Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org deposits can be detected at multiple sites, such edge, most likely due to technical limitations as in the visceral cavity in close proximity to the (see below), whether serum levels of these pancreas, liver, and intestinal epithelium as well hormones alter in response to the animal’s as at subcutaneous, pericardial, and periorbital metabolic state has not been addressed. Fur- positions (86). These fat deposits respond to thermore, studies on adipocyte development changes in nutrient supply, suggesting that they and metabolism show strong similarities be- operate as flexible energy deposits. Application tween mammals and zebrafish. Comparative re- of known pharmacological modulators of lipid search in different species may be helpful to metabolism to living zebrafish larvae causes elucidate the general principles and variabili- predicted changes in fat mass as measured by ties of the endocrine system regulating energy Nile red fluorescence, transcriptional profiling, homeostasis.

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SLEEP AND CIRCADIAN hypothalamus and other brain regions. In this RHYTHMS context, HCRT systems of the lateral hypotha- As alluded to above, there is a close nexus be- lamus are crucial for maintaining the wake state tween energy homeostasis and the sleep-wake in mammals. Two studies on the potential role cycle (91). Fundamental differences in sleep of such systems for regulating sleep behaviors patterns exist between the different vertebrate in zebrafish were performed (63, 64), with con- species; such differences mainly reflect dif- flicting results. Prober et al. (63) used a trans- ferences in lifestyle. Humans are diurnal (day genic zebrafish line that ubiquitously expressed active); mice and rats, nocturnal (night active). hcrt via an inducible heat-shock promoter and In mammals, sleep state is defined by both found increased locomotor activity during both electrophysiological and behavioral patterns. day and night with a reduced ability to initi- Electroencephalogram (EEG) recordings ate and maintain rest at night. These findings display differences between brain waves that are consistent with the known HCRT function occur during sleep and wakefulness. At a in mammals. In contrast, Yokogawa et al. (64) behavioral level, sleep is characterized as a observed a reduction in locomotor activity to- period of inactivity associated with increased gether with an increase in total sleep time af- arousal thresholds and thereby differs from ter intracerebroventricular injections of human periods of inactivity in the wake state. In or zebrafish hypocretin peptide, suggesting that fish, EEG recordings have been performed, HCRT exerts a mild sedative effect. Consistent and distinct patterns between activity- and with this finding, mutants in the sole zebrafish sleep-like states have been reported. Due to its hypocretin receptor (hcrtr2) displayed short and small size, the zebrafish is not well suited for fragmented night sleep and insensitivity to in- EEG recordings; however, behavioral analyses jected hypocretin peptide. have defined sleep-like states. Inspection of Circadian sleep regulation is controlled larval and adult zebrafish behavior by visual mainly by the hormone melatonin, which is se- observation or automated imaging systems re- creted by the pineal gland (epiphysis). In all ver- vealed increased activity during the day (63, 64, tebrates analyzed, melatonin secretion from the 92–94). Given the short periods of immobility pineal gland peaks at night. High melatonin ex- associated with reduced sensitivity to external pression correlates with phases of sleep in diur- stimuli during both day and night times and the nal species like humans and with phases of ac- prolonged periods of insensitivity at night (63, tivity in nocturnal species like rats and mice. In 64), zebrafish are considered diurnal animals. mammals, the suprachiasmatic nucleus (SCN) Sleep behaviors in several model organisms of the hypothalamus acts as a central pacemaker by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. are thought to be coordinately regulated by two for the circadian clock. SCN neurons receive control mechanisms, homeostatic and circa- electrical input from light-sensing retinal cells Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org dian. Homeostatic regulation reflects the mech- and synchronize downstream peripheral oscil- anism by which sleep deprivation triggers a lators such as the pineal gland for rhythmic compensatory increase in sleep duration and production of the indolamine melatonin. Sim- intensity to restore an organism’s physiologi- ilar to serotonin, melatonin is a derivative of cal performance. Circadian regulation, in con- the amino acid tryptophan and was initially iso- trast, is required to synchronize sleep phases lated as a skin-lightening agent in frogs (96). In with the 24-h day-night cycle. In zebrafish, zebrafish, circadian pacemaker function of the both larvae and adults display a rebound ef- SCN has not been demonstrated. Instead, all fect upon sleep deprivation, demonstrating that body cells undergo autonomous circadian os- sleep is under homeostatic control, as it is in cillations, modulated by the HPI axis (15). The mammals (64, 95). CNS control of sleep home- pineal gland is responsible for adjustment of ostasis includes interaction between wake- this autonomous rhythm, including rhythmic promoting and sleep-promoting neurons in the melatonin secretion of the pineal gland, to the

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light-dark cycle. In contrast to mammals, fish cell axis, which ultimately leads to the release of pineal cells directly sense light via photorecep- catecholamines (adrenaline and noradrenaline), tors, and light pulses reduce melatonin expres- and the HPA axis (the HPI axis in fish), which sion (93, 95, 97, 98). In fact, the impact of light leads to the release of the glucocorticoid steroid on sleep in fish is much more pronounced than hormone cortisol from the adrenal/interrenal in mammals. Unlike mammals, fish hardly sleep gland (102, 103). Both axes are present and when kept under constant light conditions for active in fish. The effects of the catecholamines several days but display normal sleep behavior include glucose mobilization from the liver afterward, pointing to a predominant impact of and muscles, enhanced oxygen uptake from light/melatonin versus the homeostatic/HCRT the gills, and increased oxygen transfer to the system (64). Zebrafish hypocretin may act, at tissues; all these physiological effects provide least in part, via melatonin (99). Thus, axons extra energy to cope with stressors. Prime tar- of hypothalamic HCRT cells project toward gets of cortisol are the gills, intestine, and liver, the pineal gland, which expresses transcript for reflecting its two major actions: adjustment of the hypocretin receptor. Furthermore, appli- energy metabolism (glucocorticoid action) and cation of HCRT peptide to isolated zebrafish regulation of hydromineral balance. Fish lack pineal glands increases melatonin production, the classic mammalian mineralocorticoid hor- whereas hypocretin receptor mutants have re- mone aldosterone. However, they have both duced melatonin production, as judged by the a mineralocorticoid receptor (MR) and a glu- reduced expression of a key enzyme of mela- cocorticoid receptor (GR), and the differential tonin synthesis. However, such a role of HCRT distribution of these receptors on target cells upstream of melatonin cannot explain the dif- determines the physiological effects of cortisol ferent sleep phenotypes displayed by HCRTr2 (104). Generally, the stress response is benefi- mutant zebrafish and mice (64) because mela- cial to an organism in the short term because tonin has—at least at pharmacological levels— energy stores are mobilized and redistributed sleep-promoting effects in both diurnal fish and to cope with the stressor. In the long run, nocturnal rodents (93). Furthermore, zebrafish however, the stress response impairs vital body respond to other known mammalian hypnotic functions, as energy is allocated away from drugs (63, 100, 101). growth, reproduction, and immune systems. Taken together, obtained results indicate In zebrafish, the HPI axis has been well that sleep control mechanisms are conserved studied. Preoptic CRH neurons constitute ho- between fish and mammals, albeit adapted to mologs of mammalian CRH cells in the PVN, their respective lifestyles. That zebrafish carry and ACTH-producing corticotropic cells, the

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. out diurnal behavior makes it an attractive CRH targets, are present in the anterior and model for sleep researchers because the two posterior zebrafish AH (see section entitled Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org favorite mammalian models, mouse and rats, The Hypothalamo-Pituitary Axis, above). The are nocturnal animals. Moreover, the power of mammalian CRH neuropeptide family also in- large-scale compound screens to identify novel cludes the urocortins UCN1–3, and the UCN1 modulators of vertebrate behavior makes the and UCN3 orthologs urotensin and urocortin zebrafish a valuable model for pharmacological 3-like are expressed in the zebrafish brain (105). drug development (101). Furthermore, urotensin can stimulate ACTH expression in goldfish (106). CRH and UCNs bind to two different CRH receptors, CRHR1 STRESS RESPONSES AND THE and CRHR2, which have both been described HYPOTHALAMO-PITUITARY- in zebrafish (12). In carp, two other hormones— ADRENAL AXIS α-MSH and β-endorphin, which like ACTH The stress response is typically characterized by are cleavage products of POMC—play a role in activation of the brain-sympathetic-chromaffin stress response and cortisol release (107). Also

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in zebrafish, α-MSH can be detected in melan- the HPI axis, and stress (118). Researchers also otropes of the AH (37). The corticotropic activ- found an increase in cortisol levels using preda- ity of α-MSH may regulate the chronic stress tor contact as a stressor (119). response in fish, whereas ACTH is a key factor In addition to cortisol level measurements, in mediating the acute stress response (107). other behavioral assays can also monitor re- The fish adrenal gland is termed the in- sponses to stress, including novel tank div- terrenal gland. Unlike the clear separation of ing, dark/light preference, and open field tests. adrenal cortex and medulla, steroidogenic and With such assays, environmental and pharma- chromaffin cells of the interrenal gland are in- cological stressors such as alarm pheromone, termingled and embedded in the head kidney the antidepressant fluoxetine, caffeine, or (108). Despite these differences in anatomical ethanol resulted in robust anxiogenic or anx- positioning and organization of the gland, de- iolytic behavioral patterns similar to those ob- velopment of the steroidogenic cell lineage is served in mammals. These findings illustrate well conserved. For instance, both mammalian the utility of zebrafish as a valuable model to NR5A1 and its zebrafish homolog nr5a1a ( ff1b) study stress and to search for stress-inhibiting are essential for adrenal and interrenal gland substances or genes (120–125). development, respectively (109–111), as well Zebrafish may also serve as a novel in vivo as for activation of the side-chain cleavage model to study glucocorticoid-induced osteo- enzyme cyp11a1, which is the rate-limiting en- porosis, a major clinical problem that stems zyme in steroid biosynthesis (112). Other en- from long-term use of glucocorticoids to treat zymes involved in gland development and cor- human inflammatory symptoms (126). Simi- tisol biosynthesis also seem to be conserved in lar to mammals, treatment of zebrafish lar- fish (112). By 2 dpf, all components of the HPI vae with the glucocorticoid prednisolone leads axis are expressed in zebrafish larvae (see sec- to a quantifiable reduction in bone mass. As tion entitled The Hypothalamo-Pituitary Axis, such, large-scale trials in zebrafish may uncover above). However, the response to stressors—in new, powerful therapeutic strategies for bone this case increased cortisol following a simple recovery in humans afflicted by drug-induced swirling of larvae in a glass vial—is later and osteoporosis. does not occur before 4 dpf (113). Finally, the zebrafish has also been sug- Single zebrafish GR and MR have been de- gested as a novel model to study social stress scribed (38, 114, 115). In humans, two GR tran- and aggression on the basis of dominant- script isoforms, GRα and GRβ, exist due to al- subordinate relationships (127). In several ternative splicing. GRα serves as the canonical vertebrate species, including mammals, the

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. glucocorticoid receptor, whereas GRβ acts as neuropeptides AVP/AVPL affect aggression. a dominant-negative inhibitor of GRα (116). In zebrafish, immunolabeling revealed a signif- Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org Interestingly, the zebrafish is the only organ- icant reduction in the total number and volume ism apart from humans in which an GR isoform of hypothalamic AVPL cells of dominant males similar to GRβ has been identified (117). compared with subordinate males. In addition, Measurement of whole-body plasma corti- the spatial pattern was altered, with AVPL im- sol levels also qualifies as a suitable indicator for munoreactivity in the magnocellular preoptic the stress response in adult zebrafish (118–120). area of dominant males but in the parvocellular Crowded zebrafish displayed a fourfold increase preoptic area of subordinate males. Thus, in cortisol levels compared with control fish. when monitored as a transgenic reporter (see The observed cortisol response to crowding section entitled The Hypothalamo-Pituitary depended on the feeding state of the fish; fed Axis, above), avpl expression may serve as a fish exhibited an only moderate increase in cor- robust molecular reporter of zebrafish aggres- tisol levels, possibly reflecting a physiological sion suitable for genetic and pharmacological link between the energy homeostasis system, screens.

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REPRODUCTION AND THE in the etiology of Kallmann syndrome, a disease HYPOTHALAMO-PITUITARY- characterized by hypogonadism and anosmia. GONADAL AXIS Interestingly, a Kallmann-like phenotype was obtained upon knockdown of a Vertebrates display a large diversity of repro- kallmann gene homolog in zebrafish (132), suggesting ductive strategies that may vary significantly that similar genetic mechanisms underlie from each other. General processes essential migration of hypophysiotropic GnRH neurons for sexual reproduction include (a) sex de- in zebrafish and mammals. The zebrafish termination and differentiation; (b) gamete genome encodes four distinct GnRH receptors development and sexual maturation; and ( gnrhr1–4). All four are differentially expressed (c) behavioral patterns associated with mate in a variety of tissues but share expression in the selection, mating procedure, and parental care. brain and gonads and respond to physiological Remarkably, the general control mechanisms levels of GnRH peptide (133, 134). Zebrafish of all these reproductive events are conserved FSH and LH gonadotropin β subunits ( β throughout vertebrates, with a central role of fsh and β, respectively) as well as their shared α the HPG axis: Hypothalamic neuroendocrine lh subunit ( / ) were described, and pituitary cells secrete GnRH peptides and regulate the gsu cga expression was reported (32, 33, 135). More- release of the gonadotropins LH and FSH over, expression of FSH and LH receptors ( from gonadotrope cells of the anterior pitu- fshr and , respectively) was reported in zebrafish itary. Both gonadotropins stimulate gamete lhr gonads, and functionality was tested (135, maturation and steroid hormone production by 136). Finally, researchers described and char- somatic cells of the gonads: interstitial cells in acterized several zebrafish nuclear sex steroid males and granulosa and theca cells in females. hormone receptors: three estrogen receptors Steroid hormones in turn modulate upstream ( , , ) (137) plus five estrogen- hypothalamic and pituitary components of esr1 esr2a esr2b related receptors ( , , , , the HPG axis via feedback systems to ensure esrra esrrb esrrga esrrgb )(http://zfin.org), a membrane and a coordination of reproductive events. esrrd nuclear progestin receptor ( ) (138–140), Investigators have characterized 15 distinct pgr and one androgen receptor ( ) (141, 142). All vertebrate GnRH variants that are encoded by ar these receptors display strong expression in distinct genes. The hypophysiotrope-specific the gonads and brain. Among these, an GnRH variant expressed in the mammalian hy- esr2a mutant isolated via the TILLING approach pothalamus is GnRH1, whereas it is GnRH3 in has been identified, but the phenotype has yet zebrafish (128, 129). Rodent GnRH1-positive to be reported (http://zfin.org). neurons are scattered throughout the rostral by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. Sex determination in vertebrates can be hypothalamus instead of forming distinct under genetic control, environmental control, expression domains, which reflects mainly the Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org or both. The mechanisms underlying sex de- unusual ontogenic origin of GnRH1 cells. termination in zebrafish are largely unknown. GnRH1 neurons in mammals arise from the Zebrafish lack a sex chromosome; however, forebrain placodal region and migrate tangen- sex-determining genetic factors may exist tially through the brain to ultimately reach (143). In addition, environmental factors in- hypothalamic target regions. In zebrafish, cluding hypoxia and temperature can modulate studies on a transgenic gnrh3::GFP reporter sex ratios in zebrafish populations, although line suggest a similar embryonic origin of only moderately (144). During the first 3 weeks hypophysiotropic GnRH3 neurons (16, 130). of development, zebrafish are hermaphrodites, However, an alternative embryonic origin from with ovary-like gonadal structures, which later neural crest tissue has also been proposed (25, either develop into a mature ovary or transform 131). In mammals, failure of GnRH1 migration into a testis (34, 143). The regulation of oocyte to reach proper target regions and subsequent and ovary differentiation and maturation by loss of GnRH1 neurons have been implicated

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endocrine and paracrine factors has been (149). Thus, estrogen receptors may mediate well studied in zebrafish (145). Maturation of androgen effects on target cells in the brain and oocytes in fish is regulated primarily by FSH gonads according to the presence or absence of and LH and is modulated by paracrine factors aromatase. of the transforming growth factor β family. Researchers recently identified the neu- Moreover, prostaglandins regulate oocyte ropeptide kisspeptin (KISS) and its receptor maturation and ovulation (146, 147). In com- as new regulators of reproduction and activa- parison, molecular cues underlying testis de- tion of GnRH neurons. Neurons expressing velopment in zebrafish are less well understood kisspeptin in mammals coexpress estrogen and (148). However, on the basis of the expression androgen receptors and may therefore be in- of genes like wt1 (Wilms tumor), amh (anti- volved in the mediation of steroid feedback on Mullerian hormone), dax (dosage-dependent GnRH neurons (for a review, see Reference region on X), and others and their known func- 156). Zebrafish kiss1 and kiss2 peptides together tion in mammals and Drosophila, investigators with two receptors, kiss1ra and kiss1rb, have proposed that these genes induce male gonadal been isolated and display activities and expres- differentiation by suppressing the expression sion patterns suggesting that, as in mammals, of cyp19a1a (149), which is expressed only in they may be involved in the initiation of ze- the soma of developing ovaries and encodes brafish puberty (157, 158). a cytochrome P450 aromatase that normally Assays addressing the impact of the HPG converts androgens into estrogens (143). axis on reproductive behavior have not been In line with this notion, steroid hormones reported in zebrafish. However, some data strongly affect zebrafish gonad development are available from other teleost species. For and sex differentiation. Estrogen treatment of example, prostaglandin administration to fe- fish during stages of sexual maturation leads to male goldfish triggers oviposition within sev- a significant shift in sex ratio toward females eral minutes, and castrated males of rainbow within the treated population (150). In con- trout lacking androgen completely lose sex- trast, chemical inhibition of estrogen biosyn- ual drive, which is restored after androgen ad- thesis in female fish results in sex reversal (151, ministration (159). Control by endocrine sys- 152), and treatment with the androgen steroid tems outside the HPG also seems present. trenbolone causes strong masculinization of ze- Thus, neurohypophyseal preparations or syn- brafish (153). Interestingly, most likely by trig- thetic oxytocin can induce reflexive spawning gering female specification of gonadal somatic movements in hypophysectomized or castrated cells and thereby estrogen production (154), killifish (43). Taken together, all these studies

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. primordial germ cells (PGCs) also impact sex indicate that, despite the striking differences in determination. Thus, after early depletion of sex determination and reproduction styles, the Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org PGCs, all zebrafish develop into males, irre- endocrine control systems of reproduction in spective of their genotype (154, 155). In mam- mammals and teleosts are surprisingly similar. mals, depletion of the germ line at later stages leads to a similar transdifferentiation of ovarian granulosa cells into Sertoli-like cells and the dif- OSMOREGULATION AND ferentiation of testis-like cords, suggesting that CALCIUM HOMEOSTASIS germ cells have a common role in female sex Maintenance of a constant ionic composition determination in fish and mammals (154). and content of body fluid is essential for ver- As mentioned above, steroid feedback from tebrate survival, although homeostatic strate- the gonads regulates the release of HPG hor- gies differ according to the lifestyles and ex- mones at the hypothalamus, pituitary, and go- ternal environments of the different species. nads. cyp19a1b, another aromatase gene, is Changes in body osmolarity ultimately lead strongly expressed in zebrafish brain/pituitary to an adaptive response of water and ion

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transport in osmoregulatory organs that in- some of the missing hormones during osmoreg- clude kidneys in land animals and gills in ulation (161). However, no such phenotype was fish. Strikingly, the hormones controlling such observed after antisense MO-mediated knock- TSH: thyroid- stimulating hormone osmoregulatory processes are largely con- down of PRL, GH, the two SLs, or one of the served among vertebrates (160). Hormones two PRL receptors (27, 162), pointing either to with prominent osmoregulatory function are some functional redundancy among the genes, corticoids from the adrenal/interrenal organ; to compensatory mechanisms (loss of prlr leads PRL, GH, and (in fish) somatolactin (SL) from to upregulation of prl expression) (27), or to the AH; AVPL and OXTL from the NH; a functional requirement for osmoregulation atrium natriuretic peptide (ANP) from the later than 3 dpf. Stages later than 3 dpf could heart; and angiotensin from the kidney. Hor- not be addressed in these experiments due to the mones with more specific functions in Ca2+ limited stability of the MOs. However, expres- homeostasis are parathyroid hormone (PTH) sion of prl and gh was strongly enhanced upon and (in fish) SL, which are hypercalcemic (in- incubation of zebrafish 7-dpf larvae in hypo- crease internal Ca2+ levels), and calcitonin (CT) tonic (diluted) medium (27, 163), as was the ex- and stanniocalcin (STC), which are hypo- and pression of the angiotensin-processing enzyme antihypercalcemic (reduce internal Ca2+ lev- gene renin in the pronephric kidney (164) and els). CT is made by C cells of the mammalian the expression of anp in the heart atrium (165), thyroid and made in the ultimobranchial bod- pointing to a role of all four hormones in pre- ies of nonmammalian vertebrates. However, in venting salt loss and water uptake (163). In con- all vertebrates, thyroid follicle cells generat- trast, there is indirect evidence for increased se- ing T3/T4 hormones and CT-generating cells cretion of OXTL from the zebrafish NH (166), initially develop from two distinct primordia, in line with the role of OXTL in promoting the which fuse during later mammalian develop- electrolyte secretion and water conservation de- ment to form the mature thyroid but remain scribed in other teleosts and in mammals. distinct organs in all nonmammalian verte- As to the hormones regulating Ca2+ home- brates. STC was originally identified and char- ostasis, zebrafish CT has been cloned and is acterized in fish, and the exact functions of its expressed in the ultimobranchial bodies during human ortholog are still a matter of debate. the larval, juvenile, and adult stages. However, Supplemental Material The Supplemental Text provides a more de- functional studies have not been reported tailed review of the roles of all these hormones (167). In addition, two stanniocalcin genes (stc1, in mammals and other teleosts. stc2) have been identified (29, 168). They are In zebrafish, functional studies of osmoreg- expressed in multiple tissues of zebrafish larvae,

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. ulatory hormones are still sparse. As mentioned including the corpuscles of Stannius close to above, teleosts lack the capacity to synthesize al- the kidney, distinct nuclei of telencephalon, the Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org dosterone, the mineralocorticoid of mammals. AH, and the pronephric glomeruli. Downregu- Instead, cortisol combines glucocorticoid as lation of stc1 expression either by incubation of well as mineralocorticoid functions but is me- embryos in low-Ca2+ freshwater or by injection diated via distinct MRs and GRs (115). Mu- of stc1 MOs stimulated whole-body Ca2+ influx tants lacking the pituitary-specific transcrip- and zebrafish epithelial Ca2+ channel (trpv6) tion factor pit1 and therefore PRL-, GH-, mRNA expression, suggesting that STC re- SL-, and thyroid-stimulating hormone (TSH)- duces Ca2+ uptake by repressing Ca2+ channel producing cells display linear dwarfism, most gene expression. Finally, four zebrafish genes likely due to the lack of GH. However, in con- encoding parathyroid hormones ( pth1a, pth1b, trast to mouse Pit1 mutant mice, most of the pth2, pthlh) (169, 170) and three parathyroid zebrafish pit1 mutants were not viable but dis- hormone receptors ( pth1ra, pth1rb, pth2r) (171, played edema formation and died at approx- 172) have been identified and characterized. imately 10 dpf, pointing to essential roles of Like all teleosts, zebrafish lack a distinct

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parathyroid gland. Instead, pth expression is understanding of endocrine deficits during later confined to distinct cells of the spinal cord and human life. Furthermore, knowledge of the de- to the neuromasts of the lateral line system of velopment of endocrine cells can be very impor- zebrafish larvae (170). In line with a potential tant for the interpretation of results obtained hypercalcemic function to increase internal in transgenic overexpression or knockout ex- Ca2+ levels, zebrafish pth1 transcript levels are periments. Recent cell-tracing experiments in inversely related to external Ca2+ concentra- mouse have, for instance, revealed that both tions, as observed in mammals (163). Further- AgRP and POMC neurons, central antagonis- more, administration of PTH had profound ef- tic players of the hypothalamic melanocortin fects on the bone mass of zebrafish larvae (173). system in control of energy homeostasis, de- In sum, these data suggest conserved roles of rive from a common pool of POMC-expressing PTH and the other hormones in the regulation progenitor cells (175). This is important be- of Ca2+ homeostasis in fish and mammals cause all floxed alleles crossed with a POMC- and possible implications in Ca2+-dependent Cre driver mouse may affect both AgRP as processes such as bone mineralization and, well as POMC neurons—possibly with seri- under pathological conditions, osteoporosis. ous consequences to the interpretations of sev- eral high-impact studies. Future research into this direction may again be more easily carried CONCLUSIONS: STRENGTHS out in zebrafish, in particular after the estab- AND WEAKNESSES OF lishment of transgenic tools for systematic lin- ZEBRAFISH AS AN eage tracing and stable labeling of particular ENDOCRINE MODEL cells and their descendants, such as lines driv- Thus far, the zebrafish has been used mainly as a ing the expression of the Cre recombinase or, model system to study vertebrate development, for temporally controlled later-lineage analy- and most zebrafish endocrine research has been sis, tamoxifen-inducible Cre-ERt2 (176) under concerned with the development, rather than the control of cell-specific promoters and floxed the function, of the different endocrine systems. fluorescent reporter lines. Many endocrine systems of the zebrafish de- Zebrafish studies directly addressing velop during the first 5 dpf; pituitary hormone later functions of endocrine systems have gene expression starts between 2 and 5 dpf dealt mainly with the activity of endocrine- (31), thyroxin expression of the thyroid gland disrupting chemicals (EDCs), in particular at 4 dpf (167), key steroidogenic gene expres- those that interfere with sex hormone function sion of the interrenal organ at 2 dpf (38), and (177–179). The relatively small size of even

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. pancreatic insulin at 1.5 dpf (174). Research of adult zebrafish, which allows them to be kept these early events can take full advantage of ma- in the laboratory, has facilitated such studies. Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org jor strengths of the zebrafish system: the small Furthermore, aquatic species are particularly size and transparency of the embryos and lar- susceptible to EDCs, and EDCs can be vae and their accessibility for forward genetics, administered by simply adding them to the chemical compound screening, in vivo imaging, water. In addition, there is a vast literature and rapid antisense-mediated gene knockdown devoted to fish endocrinology, motivated by (see Introduction). Of course, such timelines of the wish to improve aquaculturing yields. endocrine development do not mean that the However, for endocrine research in fish to be endocrine systems are already fully functional also relevant to humans, the endocrine systems at these stages. However, human genetics has must be significantly similar in function. revealed that impaired endocrine function in Recent studies, such as those reviewed above, many human patients originates from develop- revealed that crucial principles and players mental defects. Thus, studying endocrine de- of endocrine systems previously identified in velopment can be absolutely crucial for a better mammalian systems are conserved in zebrafish.

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Thus, the zebrafish is a relevant model for endocrine systems are possible, they can be human endocrine systems. However, rather more demanding than was initially assumed. than confirming data from other systems, As an alternative or in addition to genetics, this model should eventually lead to the it appears feasible to take advantage of the identification of thus-far-unknown endocrine enormous natural diversity among teleosts mechanisms, regulators, and therapeutics that to address particular endocrine issues. Thus, are also relevant for human endocrinology. comparative analyses between zebrafish and the There is reasonable hope that zebrafish can aforementioned giant Danio, with its indeter- help achieve this goal mainly on the basis of its minate growth (89), or between zebrafish and aforementioned suitability for large-scale for- the short-lived killifish (182), may be fruitful ward genetics and drug screens. However, the to study endocrine aspects of growth or aging. zebrafish strains used for such screens are not Another strength of the zebrafish is in vivo isogenic. Therefore, when one is screening for imaging, which in the meantime has become complex traits like hormone-regulated behav- a standard technique that is used largely ior, there most likely are multiple polymorphic for time-lapse recordings of developmental modifiers in the genetic background, which processes. Indeed, imaging of lipid uptake and can lead to a high variability of phenotypic storage after feeding of fluorescent phospho- strengths. At the international conference lipids to zebrafish larvae is a robust assay and for zebrafish development and genetics in may be of use in a forward-genetics screen 2000, there was an exciting report on a screen (183). Similarly, synthetic fluorescently labeled for larvae with increased appetite. Taking estradiol was used to track the dynamics of advantage of the transparency of the larvae, estrogen receptor distribution in live zebrafish researchers subsequently fed these larvae with larvae (137). After transparent adult zebrafish (orange) artemia, the organism’s favorite food, mutants became available (184), and after and (green) algae, searching for insatiable the establishment of devices for long-term mutants that continue to feed after the shrimp anesthetization and immobilization of larval meal, indicated by orange-green stomachs and possibly also juvenile and adult fish (185), (180). However, none of the reported three in vivo imaging became applicable to more and putative mutants was ever published. Indeed, more endocrine-related processes. Multiplex in our own screening attempts, using the same fluorescent reporters for the different hypotha- approach or Nile red stainings of neutral lipid lamic cell types could be used to study the stores, we obtained extremely variable and complexity and plasticity of the neuroendocrine irreproducible results and could not confirm network controlling energy homeostasis. In

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. most, if any, of our primary isolates. Thus, the mouse, the system’s synaptic plasticity it is important to develop very robust assays, has been revealed in response to peripheral Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org possibly with transgenic lines providing easily metabolic hormones, using a combination of scorable molecular reporters. immunomicroscopy, electron microcopy, and In addition, the proper developmental electrophysiological analysis on thick brain stage/age of the fish should be chosen. In our sections of treated animals (46). In zebrafish, screens for altered GH production at 5 dpf, through the use of a combination of optical we found only genes required for pituitary imaging and electrophysiology, similar studies formation, rather than those required for are possible in live animals, as was recently function (28, 181). To find the latter, screens done for GnRH3 neurons (186). Furthermore, have to be conducted with older fish after synaptic activity or signal transduction can they have started to feed and grow. This, be visualized via live imaging with biosensors however, requires more space and time, larger on the basis of fluorescence resonance energy facilities, and more personnel. In conclusion, transfer, bimolecular fluorescence comple- although forward-genetic screens of zebrafish mentation, and/or genetically encoded Ca2+

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indicators (187, 188). This way, it should be repeated samplings and does not allow one to possible to follow neuroendocrine signaling distinguish between hormone production and and the dynamics of axonal projections between hormone secretion. The limited availability specifically labeled cell types during real time of suitable antibodies also hinders studies. and in a fully intact in vivo environment. To circumvent the problems of the small Optical (laser-assisted) and transgenic body size and limited accessibility of the approaches can also be used to ablate specific glands, functional zebrafish ex vivo systems endocrine cell types (see Introduction and such as primary pituitary cell lines, pineal Supplemental Text), offering an alternative gland explants, and testis explants have been Supplemental Material to surgical gland removal, a classical exper- established (26, 99, 189). For behavioral studies iment in endocrine research that is difficult linked to energy homeostasis, sleep, and stress, in zebrafish, due to its small body size, and chambers to quantify the physical activity of that is better performed in larger fish species. adult and larval zebrafish have been established Genetics and transgenesis have also been used (see, for instance, References 64 and 120). to eliminate or overexpress specific molecular However, full metabolic chambers to measure endocrine players, such as particular hormone food intake, oxygen consumption, carbon receptors (64) or neuroendocrine substances dioxide production, and thermogenesis are (53, 63). not available and may be difficult to develop. However, classical gain-of-function Altogether, much ground work is still needed approaches like intraperitoneal and intracere- to establish all the molecular and technical broventricular hormone injections, although tools required for zebrafish endocrine research possible (64, 90), are technically much more to reach a level comparable to that attained for challenging than in larger fish species. Blood established mammalian systems. sampling to measure blood hormone levels is In conclusion, the zebrafish has some obvi- also very difficult. Thus, in the few cases in ous limitations that should be considered when which radioimmunoassays (RIAs) or enzyme- one is planning endocrine research. However, linked immunosorbent assays (ELISAs) were the blended use of mammalian models together performed, whole-body hormone levels were with unique features provided by the zebrafish determined from homogenized adult zebrafish should provide a powerful approach to eluci- or pooled (>25) larvae (98, 113, 118). This, date developmental and physiological mecha- of course, hampers long-term studies with nisms of the endocrine system.

by U.S. EPA-AWBERC Library on 04/23/13. For personal use only. DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that Annu. Rev. Physiol. 2011.73:183-211. Downloaded from www.annualreviews.org might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We apologize to all the investigators whose research could not be appropriately cited owing to space constraints. We are very grateful to our colleague Hans-Martin Pogoda for the images shown in Figure 2a–e and to Josh Bonkowsky for sharing the tg(otpb::mGFP) line with us prior to publication. We also thank Rebecca Richardson for critical reading of the manuscript and Anna Nagel for help with figure illustration.

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