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. 183 PH73CH09-Hammerschmidt ARI 7 January 2011 12:19 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 184 Lohr¨ · Hammerschmidt PH73CH09-Hammerschmidt ARI 7 January 2011 12:19 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)