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THE PITUITARY | Development of the Hypothalamus-Pituitary-Interrenal Axis
HORMONAL CONTROLS
The Pituitary
Contents Development of the Hypothalamus-Pituitary-Interrenal Axis Pituitary Gland or Hypophysis
Development of the Hypothalamus-Pituitary-Interrenal Axis D Alsop, McMaster University, Hamilton, ON, Canada N Aluru, Woods Hole Oceanographic Institution, Woods Hole, MA, USA ª 2011 Elsevier Inc. All rights reserved.
Introduction Components of the HPI Axis Ontogeny of the HPI Axis HPI Axis Development in Zebrafish
Glossary Adrenocorticotropic hormone (ACTH) A protein hormone primarily produced by the corticotropic cells of the anterior pituitary that causes release of cortisol from the interregnal. Corticotropin-releasing factor (CRF) A neuropeptide secreted by the paraventricular nucleus of the hypothalamus. CRF stimulates the corticotropic cells of the anterior pituitary to secrete ACTH. Glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) Ligand-activated, nuclear hormone receptors (subfamily 3, group C) that bind steroids such as cortisol. Upon activation, the receptors
Introduction Stress is the disruption of homeostasis from exposure to a biotic or abiotic stressor. In vertebrates, a highly con served response to stress is an elevation of plasma corticosteroid levels, which serve to restore homeostasis. The exact nature of this response is variable and depen dent on a number of factors, including the type, duration, and intensity of the stressor. However, circulating corti costeroid concentrations have long been considered a reliable indicator of the activation of the stress response.
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Stress Axis Development in Other Teleosts Conclusion Further Reading
bind to glucocorticoid response elements in the genome, upstream of target genes. Melanocortin receptor 2 (MC2R) A G-protein-coupled receptor belonging to the melanocortin family of receptors. MC2R specifically binds ACTH and is highly expressed in the cellular membrane of the interrenal cells. Stress The disruption of homeostasis with exposure to a biotic or abiotic factor. Whole genome duplication The duplication of the entire genome. It is thought that a genome duplication event 350 MYA in an ancient fish led to the massive expansion and diversification of the teleost group, and is also responsible for the 2000–3000 duplicate genes that exist in teleosts today.
Cortisol is the primary corticosteroid in teleosts and has been implicated in a wide array of biochemical, phy siological, and behavioral functions, including the release of glucose from stored glycogen to provide energy for the stress recovery process (see also Hormonal Responses to Stress: Hormone Response to Stress). Beyond metabol ism, cortisol action also modulates feeding and growth (see also Hormonal Responses to Stress: Stress Effect on Growth and Metabolism), immune responses, osmo regulation (see also Hormonal Control of Metabolism and Ionic Regulation: The Hormonal Control of
The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
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Figure 1 Overview of the hypothalamus–pituitary–interrenal (HPI)-axis anatomy and signaling pathways. Upon exposure to a stressor, the hypothalamus secrets corticotrophin-releasing factor (CRF), which stimulates the pituitary corticotropic cells to release adernocorticotropic hormone (ACTH) to the circulation. ACTH is one of many signaling molecules derived from the proopiomelanocortin (POMC) gene. Circulating ACTH binds specifically the melanocortin receptor type 2 (MC2R) expressed by the interrenal cells of the headkidney. MC2R induces cAMP signaling pathways that stimulate the cortisol synthesis from cholesterol and subsequent secretion to the bloodstream. In turn, circulating cortisol can enter a cell and bind to the glucocorticoid or mineralocorticoid receptors (GR and MR, respectively). These receptors bind to glucocorticoid response element upstream of target genes, thereby up- or downregulating their expression.
Osmoregulation in Teleost Fish) and reproduction (see also Hormonal Responses to Stress: Impact of Stress in Health and Reproduction). Cortisol signaling is not only critical in the recovery from stress, but also reg ulates many of these processes under unstressed conditions. In teleosts, circulating cortisol levels are regulated by the functioning of the hypothalamus–pituitary– interrenal (HPI) axis (Figure 1), which is homologous to the mammalian hypothalamus–pituitary–adrenal (HPA) axis (see also Hormonal Responses to Stress: Hormone Response to Stress). While the path ways and functions of the stress axes are highly conserved among vertebrates, some differences do exist between fish and mammals. For instance, fish do not possess a discrete adrenal gland, but instead the steroidogenic cells (interrenal cells) are distributed throughout the head kidney region, primarily along the posterior cardinal veins and their branches. Also, the teleost genome underwent a whole genome dupli cation event 350 million years ago (MYA), resulting in two copies of a variety of stress axis genes (which have, in some cases, differentiated) compared to other jawed vertebrates. Another major difference is that only teleosts possess a caudal neurosecretory
system (CNSS), which produces some of the same stress-related neuropeptides as the hypothalamus. The functions of the HPI and HPA axes have been extensively studied in juvenile and adult animals, but little information exist on the ontogeny of the cortisol stress response. There is mounting evidence, primarily in birds and mammals, that stressful events occurring during the early life stages can predispose an organism to different pathological states as an adult. This has led to the paradigm ‘‘developmental origins of adult disease’’. For example, prenatal undernutrition in rats and sheep results in cardiovascular alterations such as hypertension. This is thought to be caused by decreased corticosteroid feedback control of the HPA axis that is programmed during fetal development. Very little is known about the long-term effects of stress axis modulation during embryonic and larval development in fish. This article first reviews the HPI axis and cortisol signaling in teleosts, focusing primarily on molecular pathways. The second section describes the ontogeny of the stress axis, both the molecular pathways and the timing of the functional activation of the cortisol stress response. The subsequent section focuses on zebrafish
1452 The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
(Danio rerio), due to the popularity of this species in deve lopmental and molecular studies. Development of the cortisol stress response in other teleosts, including common carp (Cyprinus carpio) yellow, perch (Perca flavescens) rainbow, trout (Oncorhynchus mykiss), and Atlantic salmon (Salmo salar), are also addressed.
Components of the HPI Axis Hypothalamus The hypothalamus receives inputs from various elements of the central (e.g., cerebral cortex and olfactory system) and peripheral (e.g., viscera) nervous systems. If the hypothalamus interprets an environmental or homeostatic cue as stressful, corticotrophin-releasing factor (CRF; Figure 1) is secreted from the nucleus preopticus (NPO) in the hypophysis and plays a key role in coordi nating the neuroendocrine, autonomic, and behavioral responses to stress. Axons of the NPO CRF cells project directly into the anterior pituitary gland via synapses, or into the circulation via neurosecretion in the pars nervosa. In addition to CRF, CRF-binding protein (CRFBP) and CRF receptors (CRFR1 and R2) are involved in the regulation of the stress response by modulating CRF action. Several CRF-related neuropeptides such as uro tensin I (UI) and urocortin 2 and 3 also control the HPI axis in teleosts through activation of urotensin and CRF receptors, similar to other vertebrates. CRF and UI are not only secreted by the hypothalamus, but are also secreted from the CNSS directly into the circulation in response to stressors to allow direct interaction with interrenal tissue and epithelial tissues. Pituitary CRF released to the anterior pituitary binds to CRF receptors (R1 and R2) in the membrane of the cortico tropic cells. These are G-protein-coupled receptors and activation stimulates cAMP signaling pathways and the release of adrenocorticotropic hormone (ACTH) to the circulation. ACTH is derived from the pro opiomelanocortin (POMC) gene, which is a precursor to a number of smaller signaling peptides (Figure 1). However, ACTH is the main product of POMC in the corticotropic cells. All POMC-derived peptides bind to and activate the G-protein-coupled melanocortin recep tors in target tissues and ACTH specifically binds to the melanocortin 2 receptor (MC2R) located in target tissues. Interrenal ACTH binding to MC2R in the interrenal cells sti mulates adenylate cyclase and cAMP-dependent signaling pathways such as protein kinase A, to
stimulate cortisol synthesis. The increase in cAMP induced by MC2R is dependent upon receptor inte ractions with melanocortin receptor accessory proteins (MRAPs). Three of these proteins are found in zebrafish (MRAP1, 2a, and 2b), whereas two have been identified in mammals. MRAPs are also essential for the initial receptor protein folding, glycosylation, and translocation to the cell membrane. In the interrenal cells, the initial step in steroidogene sis is the transport of cholesterol from the outer to inner mitochondrial membrane. Although the precise mechan ism by which this cholesterol transport is accomplished is still uncertain, the steroidogenic acute regulatory protein (StAR) and the peripheral-type benzodiazepine receptor (PBR) are thought to play a key role in this mitochondrial intermembrane shuttling. The initial, rate-limiting enzymatic step is the conversion of cholesterol to pregnenolone, catalyzed by cyp11a1 (also known as cyto chrome P450 side-chain cleavage (P450scc); Figure 1). Pregnenolone is further metabolized by a series of cytochrome P450 steroid hydroxylases and dehydro genases leading to the production of cortisol. StAR, PBR, and cyp11a1 are particularly responsive to ACTH signaling (see also Hormonal Control of Metabolism and Ionic Regulation: Corticosteroids). Nuclear Receptors and Cortisol Signaling Cortisol signaling in target tissues is mediated by glucocorticoid and mineralocorticoid receptors (GR and MR, respectively), which belong to the nuclear receptor superfamily of ligand-bound transcription factors. GR is ubiquitously expressed, indicating a widespread adaptive role for cortisol. A primary target for cortisol is the GR in the liver, which plays a key metabolic role for providing energy while coping with stressors (see also Hormonal Control of Metabolism and Ionic Regulation: Corticos teroids). The stress-induced disturbance of homeostasis results in the upregulation of energy-demanding pathways, and in fish, cortisol enhances liver gluconeogenic capacity leading to elevated glucose liberation. This response is a key aspect of the stress recovery process, as glucose is the preferred fuel for tissues, in particular the gill and brain. GR activation by cortisol also regulates diverse processes such as osmoregulation, protein metabolism, the immune response, growth, reproduction, and behavior. Cortisol is a very high affinity ligand for MR; however, 11�HSD2 is often co-expressed with MR in tissues such as the gill and kidney. This enzyme deactivates cortisol by metabolizing it to cortisone, which does not bind to MR. In mammals, this enzyme allows hormones such as aldos terone, which is at a much lower concentration than cortisol, access to MR. Aldosterone does not appear to be synthesized in fish, but 11-deoxycorticosterone (DOC) is instead the leading candidate as the teleost MR-specific
The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
Duplicated HPI Axis Genes The whole genome duplication event in fish 350 MYA resulted in a number of duplicated genes in the HPI axis. For example, carp possess two genes for CRF, ACTH (POMC) and GR. However, zebrafish are unique among teleosts in that they have returned to single gene systems for CRF, ACTH, and GR (although zebrafish have two genes that encode POMC, the cleavage site for ACTH is mutated in one copy, resulting in ACTH derived from only one gene). Although this duplicate stress gene loss may have simplified studies on the development of the zebrafish stress axis, it also raises some very interesting questions regarding genome evolution and the func tions of single versus duplicate gene systems.
Ontogeny of the HPI Axis From early embryogenesis, components of the stress axis undergo dynamic changes in expression suggesting that they are functional at this time. However, aside from the well-defined role of cortisol and GR in fetal lung maturation in mammals, the specific stress- or basic developmental-related functions of the HPI/HPA com ponents in the embryo are not well understood. Embryos and larvae of oviparous teleosts have continuous access to cortisol because of a maternal deposit of cortisol in the oocyte. The temporal profile of this steroid during development has been documented in a variety of species (although similar cortisol profiles have yet to be characterized in viviparous and ovoviviparous fish). In all species examined, this deposited cortisol is depleted from the oocyte as embryonic development progresses, reaching a minimum level around hatch, after which time, the larva begins to synthesize cortisol and basal levels increase (Figure 2). However, when is the cortisol stress axis first responsive to stress? This section reviews the molecular ontogeny of the HPI axis with reference to the timing of basal and stress-induced corti sol production. The majority of the work on the temporal expression of signaling molecules, receptors, and enzymes of the HPI axis during teleost development has been performed with zebrafish. The findings from this species are summarized first, followed by information that is available for salmo nids, common carp, and yellow perch (primarily cortisol measurements).
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ligand. However, a physiological role for DOC signaling via MR activation is still lacking in fish. The current thinking is that cortisol may be playing a key role in MR activation, while the specific effect associated with this receptor activation, including expression of MR-responsive genes, is unknown in fish.
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Figure 2 Representative whole-body cortisol levels (arbitrary units) in developing teleost embryos and larvae. Fish oocytes contain a maternal cortisol deposit, and after fertilization basal (open bars) cortisol levels decrease and reach a minimum around the time of hatching (approximately 2–3 dpf for zebrafish, 10 dpf for perch, and 4 wpf for salmonids). The newly hatched larva is able to synthesize cortisol and basal levels begin to rise. In contrast, cortisol levels after a handling stressor (black bars) do not increase above basal levels until after hatch, toward the onset of exogenous feeding. The exception is carp, which display an accelerated HPI-axis development.
HPI Axis Development in Zebrafish The Mature Oocyte Maternal factors are essential during early development as the initial steps of embryogenesis are driven solely by maternal mRNAs or proteins that were produced during oogenesis. In most animals, a substantial portion of embryogenesis occurs prior to the activation of zygotic transcription, which normally begins during the mid-blas tula transition (MBT; approximately 2.5 hours post fertilization (hpf) in zebrafish). In zebrafish, a number of maternal HPI-axis transcripts are expressed in the mature oocyte or zygote. There is an abundance of transcripts for CRF, CRFBP, CRF recep tors (CRFR1 andR2), and GR, while there are very few transcripts for StAR, 11�-hydroxylase and 11�HSD2. In addition, there is a maternal deposit of cortisol (4 pg/egg in zebrafish). Although present, the exact roles of mater nally derived cortisol or HPI-axis transcripts in the early embryo are unknown.
Embryogenesis During embryogenesis, there are dramatic changes in the expression of HPI-axis genes. In addition, the maternal cortisol deposit is being continuously depleted, but
1454 The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
cortisol levels in the embryo are not responsive to a stressor (Figure 2). CRF signaling system. CRF transcripts are detected throughout zebrafish embryogenesis. CRF signal is observed by 25 hpf in the primordial hypothalamus, which has begun to form as a ventral expansion of the diencephalon. By 48 hpf, CRF-positive cells are observed in bilateral pairs of cells in the preoptic region of the hypothalamus, as well as outside the hypothalamus, in the thalamus and rostral medulla oblongata (Figure 3(b)). The distribution of CRF expression outside the hypothalamus suggests that CRF may be involved in processes other than HPI-axis signaling, especially since the
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Figure 3 Summary of regional gene expression for corticotrophin-releasing factor (CRF), urotensin I (UI), and CRF-binding protein (CRFBP) in the zebrafish brain at 2 days post-fertilization (dpf; left panel) and in adult male fish (right panel). Schematics of the brain for 2 dpf (a) and adults (e) in rostro-caudal orientation are shown for reference, and brain regions that are identifiable at 2 dpf are indicated. A comparison between 2 dpf and adult regional expression is shown for CRF ((b) and (f), respectively), UI ((c) and (g), respectively), and CRFBP ((d) and (h), respectively). Each gray circle in (b), (c), and (d) represents the approximate location of a pair of cells that expressed the respective gene of interest in all 2 dpf brains analyzed. The open circle in (c) represents the approximate location of a pair of cells that expressed UI in half of the brains analyzed. Ce, cerebellum; H, hypothalamus; MO, medulla oblongata; Po, pre-optic region; S, subpallium; TeO, optic tectum; Th, thalamus; Ve, ventricle. Courtesy of Sarah Alderman and Nicholas Bernier, University of Guelph, Canada.
cortisol stress response is not activated at this time in response to a stressor. Indeed, CRF has been shown to play other roles in mammals, where it induces astrocyte proliferation in the developing rat brain. CRFBP is also expressed in many brain regions (Figure 3(d)), although the exact role of CRFBP in CRF signaling is unclear. Studies, to date, suggest that CRFBP prevents CRF from binding to its receptors and subsequent signaling. UI was not detected in the embryonic or larval hypothalamus, although low levels were observed in the thalamus and medulla oblongata (Figure 3(c)). In con trast, UI is highly expressed in the adult brain (Figure 3(g)). CNSS. There is no change in CRF or UI transcript abundance in the tail region during embryonic or larval development, although levels are much higher in the adult. The functional significance or stress responsive ness of the CNSS during development is unknown in fish. POMC/ACTH. POMC is first detected in the pituitary anlage at 24 hpf and is expressed in an anterior/posterior organization starting around 48 hpf. At this time, the anterior corticotropic cells synthesize the ACTH peptide. It is unknown whether development of corticotropic cells is regulated by the hypothalamus and CRF secretion, as is the exact timing of corticotropic responsiveness to CRF. Regardless, POMC and ACTH are strongly localized in the anterior pituitary prior to hatch, although the stress response is nonfunctional. Cortisol biosynthesis. One of the earliest molecular markers of the developing interrenal tissue is the localized expression of the fushi tarazu factor 1� (ff1b), a transcription factor, and homolog of the mammalian ste roidogenic factor 1. The initial expression of ff1b occurs at 22 hpf and is required for interrenal cell differentiation. It is also responsible for the initiation of P450scc expression at 24 hpf, which is the rate-limiting enzyme in steroido genesis. StAR is also initially detected in the primordial interrenals at 24 hpf, followed by 3�-hydroxysteroid dehydrogenase (3�-HSD) (28 hpf) and MC2R (32 hpf). The expression of 11�-hydroxylase, which catalyzes the final step of cortisol synthesis, increases by 48 hpf, around the time of hatch. This is immediately followed by a rise in basal whole-body cortisol levels. Interrenal development over the first 48 hpf occurs independently from higher signals such as ACTH. This is demonstrated by MC2R knockdown morphants or mutants that lack corticotropic cells (and therefore ACTH), which maintains both normal interrenal tissue development and the expression of steroidogenic enzymes. However, by 5 days post-fertilization (dpf), a lack of ACTH signaling results in the loss of steroid synthesizing capacity as genes involved in steroidogenesis are undetectable at this time. This indicates that
The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
Transcript abundance
embryogenesis. Whether the cortisol response is delayed simply due to developmental constraints or is intentional to protect against elevated cortisol signaling during sen sitive stages (similar to the stress hypo-responsive period in postnatal mammals) is unknown.
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Figure 4 Glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11�-hydroxysteroid dehydrogenase type 2 (11�HSD2) expression (arbitrary units) in zebrafish embryos. The paucity of 11�HSD2 transcripts suggests that cortisol has access to MR during embryonic development, which is steadily increasing in transcript number.
interrenal function is autonomous over the first 2 dpf, but control is transferred to higher levels by 5 dpf. ACTH and CRF in the pituitary and hypothalamus, respectively, are expressed by 48 hpf, and the interrenals are beginning to produce cortisol. The brain appears to be poised to activate cortisol production in response to a stress. However, salinity and handling stressors do not stimulate a cortisol stress response at this point in time. Glucocorticoid and mineralocorticoid receptors. The initial abundance of maternally derived GR transcripts in the mature oocyte is unstable and is rapidly degraded (Figure 4). This suggests that GR signaling may not be critical during early embryonic development. By hatch, GR transcript numbers have rebounded and remain elevated. GR signaling may be critical at this time for metabolic energy repartitioning during the onset of feeding and growth. In contrast to GR, MR transcripts increase steadily from fertilization to feeding (Figure 4). In addition, there are very few 11�HSD2 transcripts in the embryo, which would suggest that cortisol is not deactivated and may be a ligand for MR. The embryo has continuous access to cortisol, from the maternal deposit during the early stages, or from de novo synthesis beginning around hatch. The lack of a cortisol stress response during embryo genesis suggests that GR and MR signaling may be regulating basic aspects of development such as metabol ism or osmoregulation as in adults. In mammals, GR regulates fetal carbohydrate metabolism, based on obser vations in GR knockout animals that display altered expression of key gluconeogenic enzymes. Summary. The HPI axis appears to be fully expressed and the interrenal cells are producing cortisol by hatch. However, there is no cortisol stress response during early
Hatching begins at 48 hpf in zebrafish and this coincides with further increases in the expression of CRF, StAR, 11�-hydroxylase, 11�HSD2, and the rebound of GR. At hatch, whole-body cortisol levels are beginning to rise as synthesis is initiated. In spite of these developments, cortisol levels in zebrafish are unresponsive to salinity or handling stressors. It appears that development of the sensory inputs to the hypothalamus, critical for stress perception and CRF release, is the limiting step delaying the activation of the cortisol stress response during development. The stress response is first observed at 3 dpf (72 hpf), where cortisol levels in freshwater reared larvae are ele vated after exposure to increased salinity (seawater). Although this is the earliest indication of a functional HPI axis, a handling stress (swirling around in a glass vial) at this same stage does not elicit an increase in cortisol. One hypothesis to explain this difference is that handling and osmotic challenges activate different signaling pathways, which have reached different degrees of functional development by 3 dpf. The HPI axis appears to be fully functional and receptive to different types of stressors at 4 dpf (approaching the onset of exogenous feeding). At this time both an osmotic challenge and handling stress elicit an increase in cortisol levels.
Stress Axis Development in Other Teleosts Common Carp The common carp and zebrafish are both cyprinids and have similar developmental rates and times to hatch (2–3 dpf). However, the cortisol stress response develops at an earlier stage in carp: basal cortisol and ACTH levels increase well before hatch, and also increase in response to stressor exposure before hatching. The evolutionary pressures that would lead to an accelerated development of HPI axis in carp are unknown. Rainbow Trout, Atlantic Salmon, and Yellow Perch Relative to hatching time, the temporal development of basal and stress-induced cortisol levels in rainbow trout, Atlantic salmon, and yellow perch are very similar to zebrafish. Cortisol levels are at a minimum around
1456 The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis (a) CRF
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Catecholamines Figure 5 The HPI-axis signaling pathway: (a) simplified and (b) with some of the known signaling pathways, modulators, and functions of the HPI axis.
hatching (hatching times: zebrafish – 2–3 dpf; perch – 10 dpf; salmonids – 4 wpf), and then begin to increase. However, stress-induced elevations of cortisol are not observed until after hatching (1 week post-hatch in perch and 2 weeks post-hatch in salmonids). Early exposure to stressors can alter the activation of the stress response later on in life. For example, rainbow trout embryos and larvae exposed to a stressor have a depressed cortisol response as juveniles (5 months post fertilization). This dampening of the stress response is also observed in juvenile rainbow trout that were exposed to waterborne cortisol immediately after fertilization for approximately 2 days. This illustrates that the devel opment and programming of the HPI axis is sensitive to stressor exposure, and long-lasting effects can be imprinted early on during development. However, determining the physiological consequences of these changes in the stress response is still required, and is essential in furthering our understanding of the development of the HPI axis.
Conclusion There is clearly a good understanding of the molecular ontogeny of the HPI axis in teleosts, or more specifically, the CRF-ACTH-cortisol axis (Figure 5(a)). However, this is an extremely simplified view of a complex system that possesses multiple paths of regulation and function (Figure 5(b)). To date, the majority of experiments have focused on the regulation of stress axis genes in terms of transcript abundance under various conditions. However,
there are several genetic and epigenetic mechanisms that influence these parameters. Characterizing these molecu lar mechanisms is useful not only to understand gene regulation but also to identify novel pathways, which can be used as bio-indicators of exposure to stressful stimuli. In addition, perhaps one of the more significant issues that currently remains unresolved is determining the basic functions of the stress axis components such as CRF, ACTH, GR, and MR during embryogenesis, given that there is no active stress response during this time. Model species such as zebrafish have occupied a pivo tal position in the study of physiological and molecular pathways of the HPI axis. However, teleosts are a large group with diverse developmental patterns, so it would be presumptuous to believe that the mechanisms are similar between different species, especially in light of the accele rated HPI-axis development in carp and the unique situation of single gene systems for CRF, ACTH, and GR in zebrafish. Hence, studies conducted in nonmodel species that are of importance to aquaculture, environ mental conservation, and management are necessary. See also: Hormonal Control of Metabolism and Ionic Regulation: Corticosteroids; The Hormonal Control of Osmoregulation in Teleost Fish. Hormonal Responses to Stress: Hormone Response to Stress; Stress Effect on Growth and Metabolism; Impact of Stress in Health and Reproduction.
Further Reading Alsop D and Vijayan MM (2009) The zebrafish stress axis: Molecular fallout from the teleost-specific genome duplication event. General and Comparative Endocrinology 161: 62–66. Alsop D and Vijayan MM (2009) Review: Molecular programming of the corticosteroid stress axis during zebrafish development. Comparative Biochemistry and Physiology. Part A: Molecular and Integrative Physiology 153: 49–54. Aluru N and Vijayan MM (2009) Stress transcriptomics in fish: A role for genomic cortisol signaling. General and Comparative Endocrinology 164: 142–150. Bernier NJ (2006) Review: The corticotropin-releasing factor system as a mediator of the appetite-suppressing effects of stress in fish. General and Comparative Endocrinology 146: 45–55. Matthews SG and Phillips DIW (2010) Minireview: Transgenerational inheritance of the stress response: A new frontier in stress research. Endocrinology 151: 7–13. Wintermantel TM, Berger S, Greiner EF, and Schu¨tz G (2005) Evaluation of steroid receptor function by gene targeting in mice. Journal of Steroid Biochemistry and Molecular Biology 93: 107–112.
The Pituitary
Contents Development of the Hypothalamus-Pituitary-Interrenal Axis Pituitary Gland or Hypophysis
Development of the Hypothalamus-Pituitary-Interrenal Axis D Alsop, McMaster University, Hamilton, ON, Canada N Aluru, Woods Hole Oceanographic Institution, Woods Hole, MA, USA ª 2011 Elsevier Inc. All rights reserved.
Introduction Components of the HPI Axis Ontogeny of the HPI Axis HPI Axis Development in Zebrafish
Glossary Adrenocorticotropic hormone (ACTH) A protein hormone primarily produced by the corticotropic cells of the anterior pituitary that causes release of cortisol from the interregnal. Corticotropin-releasing factor (CRF) A neuropeptide secreted by the paraventricular nucleus of the hypothalamus. CRF stimulates the corticotropic cells of the anterior pituitary to secrete ACTH. Glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) Ligand-activated, nuclear hormone receptors (subfamily 3, group C) that bind steroids such as cortisol. Upon activation, the receptors
Introduction Stress is the disruption of homeostasis from exposure to a biotic or abiotic stressor. In vertebrates, a highly con served response to stress is an elevation of plasma corticosteroid levels, which serve to restore homeostasis. The exact nature of this response is variable and depen dent on a number of factors, including the type, duration, and intensity of the stressor. However, circulating corti costeroid concentrations have long been considered a reliable indicator of the activation of the stress response.
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Stress Axis Development in Other Teleosts Conclusion Further Reading
bind to glucocorticoid response elements in the genome, upstream of target genes. Melanocortin receptor 2 (MC2R) A G-protein-coupled receptor belonging to the melanocortin family of receptors. MC2R specifically binds ACTH and is highly expressed in the cellular membrane of the interrenal cells. Stress The disruption of homeostasis with exposure to a biotic or abiotic factor. Whole genome duplication The duplication of the entire genome. It is thought that a genome duplication event 350 MYA in an ancient fish led to the massive expansion and diversification of the teleost group, and is also responsible for the 2000–3000 duplicate genes that exist in teleosts today.
Cortisol is the primary corticosteroid in teleosts and has been implicated in a wide array of biochemical, phy siological, and behavioral functions, including the release of glucose from stored glycogen to provide energy for the stress recovery process (see also Hormonal Responses to Stress: Hormone Response to Stress). Beyond metabol ism, cortisol action also modulates feeding and growth (see also Hormonal Responses to Stress: Stress Effect on Growth and Metabolism), immune responses, osmo regulation (see also Hormonal Control of Metabolism and Ionic Regulation: The Hormonal Control of
The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
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Figure 1 Overview of the hypothalamus–pituitary–interrenal (HPI)-axis anatomy and signaling pathways. Upon exposure to a stressor, the hypothalamus secrets corticotrophin-releasing factor (CRF), which stimulates the pituitary corticotropic cells to release adernocorticotropic hormone (ACTH) to the circulation. ACTH is one of many signaling molecules derived from the proopiomelanocortin (POMC) gene. Circulating ACTH binds specifically the melanocortin receptor type 2 (MC2R) expressed by the interrenal cells of the headkidney. MC2R induces cAMP signaling pathways that stimulate the cortisol synthesis from cholesterol and subsequent secretion to the bloodstream. In turn, circulating cortisol can enter a cell and bind to the glucocorticoid or mineralocorticoid receptors (GR and MR, respectively). These receptors bind to glucocorticoid response element upstream of target genes, thereby up- or downregulating their expression.
Osmoregulation in Teleost Fish) and reproduction (see also Hormonal Responses to Stress: Impact of Stress in Health and Reproduction). Cortisol signaling is not only critical in the recovery from stress, but also reg ulates many of these processes under unstressed conditions. In teleosts, circulating cortisol levels are regulated by the functioning of the hypothalamus–pituitary– interrenal (HPI) axis (Figure 1), which is homologous to the mammalian hypothalamus–pituitary–adrenal (HPA) axis (see also Hormonal Responses to Stress: Hormone Response to Stress). While the path ways and functions of the stress axes are highly conserved among vertebrates, some differences do exist between fish and mammals. For instance, fish do not possess a discrete adrenal gland, but instead the steroidogenic cells (interrenal cells) are distributed throughout the head kidney region, primarily along the posterior cardinal veins and their branches. Also, the teleost genome underwent a whole genome dupli cation event 350 million years ago (MYA), resulting in two copies of a variety of stress axis genes (which have, in some cases, differentiated) compared to other jawed vertebrates. Another major difference is that only teleosts possess a caudal neurosecretory
system (CNSS), which produces some of the same stress-related neuropeptides as the hypothalamus. The functions of the HPI and HPA axes have been extensively studied in juvenile and adult animals, but little information exist on the ontogeny of the cortisol stress response. There is mounting evidence, primarily in birds and mammals, that stressful events occurring during the early life stages can predispose an organism to different pathological states as an adult. This has led to the paradigm ‘‘developmental origins of adult disease’’. For example, prenatal undernutrition in rats and sheep results in cardiovascular alterations such as hypertension. This is thought to be caused by decreased corticosteroid feedback control of the HPA axis that is programmed during fetal development. Very little is known about the long-term effects of stress axis modulation during embryonic and larval development in fish. This article first reviews the HPI axis and cortisol signaling in teleosts, focusing primarily on molecular pathways. The second section describes the ontogeny of the stress axis, both the molecular pathways and the timing of the functional activation of the cortisol stress response. The subsequent section focuses on zebrafish
1452 The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
(Danio rerio), due to the popularity of this species in deve lopmental and molecular studies. Development of the cortisol stress response in other teleosts, including common carp (Cyprinus carpio) yellow, perch (Perca flavescens) rainbow, trout (Oncorhynchus mykiss), and Atlantic salmon (Salmo salar), are also addressed.
Components of the HPI Axis Hypothalamus The hypothalamus receives inputs from various elements of the central (e.g., cerebral cortex and olfactory system) and peripheral (e.g., viscera) nervous systems. If the hypothalamus interprets an environmental or homeostatic cue as stressful, corticotrophin-releasing factor (CRF; Figure 1) is secreted from the nucleus preopticus (NPO) in the hypophysis and plays a key role in coordi nating the neuroendocrine, autonomic, and behavioral responses to stress. Axons of the NPO CRF cells project directly into the anterior pituitary gland via synapses, or into the circulation via neurosecretion in the pars nervosa. In addition to CRF, CRF-binding protein (CRFBP) and CRF receptors (CRFR1 and R2) are involved in the regulation of the stress response by modulating CRF action. Several CRF-related neuropeptides such as uro tensin I (UI) and urocortin 2 and 3 also control the HPI axis in teleosts through activation of urotensin and CRF receptors, similar to other vertebrates. CRF and UI are not only secreted by the hypothalamus, but are also secreted from the CNSS directly into the circulation in response to stressors to allow direct interaction with interrenal tissue and epithelial tissues. Pituitary CRF released to the anterior pituitary binds to CRF receptors (R1 and R2) in the membrane of the cortico tropic cells. These are G-protein-coupled receptors and activation stimulates cAMP signaling pathways and the release of adrenocorticotropic hormone (ACTH) to the circulation. ACTH is derived from the pro opiomelanocortin (POMC) gene, which is a precursor to a number of smaller signaling peptides (Figure 1). However, ACTH is the main product of POMC in the corticotropic cells. All POMC-derived peptides bind to and activate the G-protein-coupled melanocortin recep tors in target tissues and ACTH specifically binds to the melanocortin 2 receptor (MC2R) located in target tissues. Interrenal ACTH binding to MC2R in the interrenal cells sti mulates adenylate cyclase and cAMP-dependent signaling pathways such as protein kinase A, to
stimulate cortisol synthesis. The increase in cAMP induced by MC2R is dependent upon receptor inte ractions with melanocortin receptor accessory proteins (MRAPs). Three of these proteins are found in zebrafish (MRAP1, 2a, and 2b), whereas two have been identified in mammals. MRAPs are also essential for the initial receptor protein folding, glycosylation, and translocation to the cell membrane. In the interrenal cells, the initial step in steroidogene sis is the transport of cholesterol from the outer to inner mitochondrial membrane. Although the precise mechan ism by which this cholesterol transport is accomplished is still uncertain, the steroidogenic acute regulatory protein (StAR) and the peripheral-type benzodiazepine receptor (PBR) are thought to play a key role in this mitochondrial intermembrane shuttling. The initial, rate-limiting enzymatic step is the conversion of cholesterol to pregnenolone, catalyzed by cyp11a1 (also known as cyto chrome P450 side-chain cleavage (P450scc); Figure 1). Pregnenolone is further metabolized by a series of cytochrome P450 steroid hydroxylases and dehydro genases leading to the production of cortisol. StAR, PBR, and cyp11a1 are particularly responsive to ACTH signaling (see also Hormonal Control of Metabolism and Ionic Regulation: Corticosteroids). Nuclear Receptors and Cortisol Signaling Cortisol signaling in target tissues is mediated by glucocorticoid and mineralocorticoid receptors (GR and MR, respectively), which belong to the nuclear receptor superfamily of ligand-bound transcription factors. GR is ubiquitously expressed, indicating a widespread adaptive role for cortisol. A primary target for cortisol is the GR in the liver, which plays a key metabolic role for providing energy while coping with stressors (see also Hormonal Control of Metabolism and Ionic Regulation: Corticos teroids). The stress-induced disturbance of homeostasis results in the upregulation of energy-demanding pathways, and in fish, cortisol enhances liver gluconeogenic capacity leading to elevated glucose liberation. This response is a key aspect of the stress recovery process, as glucose is the preferred fuel for tissues, in particular the gill and brain. GR activation by cortisol also regulates diverse processes such as osmoregulation, protein metabolism, the immune response, growth, reproduction, and behavior. Cortisol is a very high affinity ligand for MR; however, 11�HSD2 is often co-expressed with MR in tissues such as the gill and kidney. This enzyme deactivates cortisol by metabolizing it to cortisone, which does not bind to MR. In mammals, this enzyme allows hormones such as aldos terone, which is at a much lower concentration than cortisol, access to MR. Aldosterone does not appear to be synthesized in fish, but 11-deoxycorticosterone (DOC) is instead the leading candidate as the teleost MR-specific
The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
Duplicated HPI Axis Genes The whole genome duplication event in fish 350 MYA resulted in a number of duplicated genes in the HPI axis. For example, carp possess two genes for CRF, ACTH (POMC) and GR. However, zebrafish are unique among teleosts in that they have returned to single gene systems for CRF, ACTH, and GR (although zebrafish have two genes that encode POMC, the cleavage site for ACTH is mutated in one copy, resulting in ACTH derived from only one gene). Although this duplicate stress gene loss may have simplified studies on the development of the zebrafish stress axis, it also raises some very interesting questions regarding genome evolution and the func tions of single versus duplicate gene systems.
Ontogeny of the HPI Axis From early embryogenesis, components of the stress axis undergo dynamic changes in expression suggesting that they are functional at this time. However, aside from the well-defined role of cortisol and GR in fetal lung maturation in mammals, the specific stress- or basic developmental-related functions of the HPI/HPA com ponents in the embryo are not well understood. Embryos and larvae of oviparous teleosts have continuous access to cortisol because of a maternal deposit of cortisol in the oocyte. The temporal profile of this steroid during development has been documented in a variety of species (although similar cortisol profiles have yet to be characterized in viviparous and ovoviviparous fish). In all species examined, this deposited cortisol is depleted from the oocyte as embryonic development progresses, reaching a minimum level around hatch, after which time, the larva begins to synthesize cortisol and basal levels increase (Figure 2). However, when is the cortisol stress axis first responsive to stress? This section reviews the molecular ontogeny of the HPI axis with reference to the timing of basal and stress-induced corti sol production. The majority of the work on the temporal expression of signaling molecules, receptors, and enzymes of the HPI axis during teleost development has been performed with zebrafish. The findings from this species are summarized first, followed by information that is available for salmo nids, common carp, and yellow perch (primarily cortisol measurements).
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ligand. However, a physiological role for DOC signaling via MR activation is still lacking in fish. The current thinking is that cortisol may be playing a key role in MR activation, while the specific effect associated with this receptor activation, including expression of MR-responsive genes, is unknown in fish.
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Figure 2 Representative whole-body cortisol levels (arbitrary units) in developing teleost embryos and larvae. Fish oocytes contain a maternal cortisol deposit, and after fertilization basal (open bars) cortisol levels decrease and reach a minimum around the time of hatching (approximately 2–3 dpf for zebrafish, 10 dpf for perch, and 4 wpf for salmonids). The newly hatched larva is able to synthesize cortisol and basal levels begin to rise. In contrast, cortisol levels after a handling stressor (black bars) do not increase above basal levels until after hatch, toward the onset of exogenous feeding. The exception is carp, which display an accelerated HPI-axis development.
HPI Axis Development in Zebrafish The Mature Oocyte Maternal factors are essential during early development as the initial steps of embryogenesis are driven solely by maternal mRNAs or proteins that were produced during oogenesis. In most animals, a substantial portion of embryogenesis occurs prior to the activation of zygotic transcription, which normally begins during the mid-blas tula transition (MBT; approximately 2.5 hours post fertilization (hpf) in zebrafish). In zebrafish, a number of maternal HPI-axis transcripts are expressed in the mature oocyte or zygote. There is an abundance of transcripts for CRF, CRFBP, CRF recep tors (CRFR1 andR2), and GR, while there are very few transcripts for StAR, 11�-hydroxylase and 11�HSD2. In addition, there is a maternal deposit of cortisol (4 pg/egg in zebrafish). Although present, the exact roles of mater nally derived cortisol or HPI-axis transcripts in the early embryo are unknown.
Embryogenesis During embryogenesis, there are dramatic changes in the expression of HPI-axis genes. In addition, the maternal cortisol deposit is being continuously depleted, but
1454 The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
cortisol levels in the embryo are not responsive to a stressor (Figure 2). CRF signaling system. CRF transcripts are detected throughout zebrafish embryogenesis. CRF signal is observed by 25 hpf in the primordial hypothalamus, which has begun to form as a ventral expansion of the diencephalon. By 48 hpf, CRF-positive cells are observed in bilateral pairs of cells in the preoptic region of the hypothalamus, as well as outside the hypothalamus, in the thalamus and rostral medulla oblongata (Figure 3(b)). The distribution of CRF expression outside the hypothalamus suggests that CRF may be involved in processes other than HPI-axis signaling, especially since the
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Figure 3 Summary of regional gene expression for corticotrophin-releasing factor (CRF), urotensin I (UI), and CRF-binding protein (CRFBP) in the zebrafish brain at 2 days post-fertilization (dpf; left panel) and in adult male fish (right panel). Schematics of the brain for 2 dpf (a) and adults (e) in rostro-caudal orientation are shown for reference, and brain regions that are identifiable at 2 dpf are indicated. A comparison between 2 dpf and adult regional expression is shown for CRF ((b) and (f), respectively), UI ((c) and (g), respectively), and CRFBP ((d) and (h), respectively). Each gray circle in (b), (c), and (d) represents the approximate location of a pair of cells that expressed the respective gene of interest in all 2 dpf brains analyzed. The open circle in (c) represents the approximate location of a pair of cells that expressed UI in half of the brains analyzed. Ce, cerebellum; H, hypothalamus; MO, medulla oblongata; Po, pre-optic region; S, subpallium; TeO, optic tectum; Th, thalamus; Ve, ventricle. Courtesy of Sarah Alderman and Nicholas Bernier, University of Guelph, Canada.
cortisol stress response is not activated at this time in response to a stressor. Indeed, CRF has been shown to play other roles in mammals, where it induces astrocyte proliferation in the developing rat brain. CRFBP is also expressed in many brain regions (Figure 3(d)), although the exact role of CRFBP in CRF signaling is unclear. Studies, to date, suggest that CRFBP prevents CRF from binding to its receptors and subsequent signaling. UI was not detected in the embryonic or larval hypothalamus, although low levels were observed in the thalamus and medulla oblongata (Figure 3(c)). In con trast, UI is highly expressed in the adult brain (Figure 3(g)). CNSS. There is no change in CRF or UI transcript abundance in the tail region during embryonic or larval development, although levels are much higher in the adult. The functional significance or stress responsive ness of the CNSS during development is unknown in fish. POMC/ACTH. POMC is first detected in the pituitary anlage at 24 hpf and is expressed in an anterior/posterior organization starting around 48 hpf. At this time, the anterior corticotropic cells synthesize the ACTH peptide. It is unknown whether development of corticotropic cells is regulated by the hypothalamus and CRF secretion, as is the exact timing of corticotropic responsiveness to CRF. Regardless, POMC and ACTH are strongly localized in the anterior pituitary prior to hatch, although the stress response is nonfunctional. Cortisol biosynthesis. One of the earliest molecular markers of the developing interrenal tissue is the localized expression of the fushi tarazu factor 1� (ff1b), a transcription factor, and homolog of the mammalian ste roidogenic factor 1. The initial expression of ff1b occurs at 22 hpf and is required for interrenal cell differentiation. It is also responsible for the initiation of P450scc expression at 24 hpf, which is the rate-limiting enzyme in steroido genesis. StAR is also initially detected in the primordial interrenals at 24 hpf, followed by 3�-hydroxysteroid dehydrogenase (3�-HSD) (28 hpf) and MC2R (32 hpf). The expression of 11�-hydroxylase, which catalyzes the final step of cortisol synthesis, increases by 48 hpf, around the time of hatch. This is immediately followed by a rise in basal whole-body cortisol levels. Interrenal development over the first 48 hpf occurs independently from higher signals such as ACTH. This is demonstrated by MC2R knockdown morphants or mutants that lack corticotropic cells (and therefore ACTH), which maintains both normal interrenal tissue development and the expression of steroidogenic enzymes. However, by 5 days post-fertilization (dpf), a lack of ACTH signaling results in the loss of steroid synthesizing capacity as genes involved in steroidogenesis are undetectable at this time. This indicates that
The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis
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embryogenesis. Whether the cortisol response is delayed simply due to developmental constraints or is intentional to protect against elevated cortisol signaling during sen sitive stages (similar to the stress hypo-responsive period in postnatal mammals) is unknown.
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Figure 4 Glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11�-hydroxysteroid dehydrogenase type 2 (11�HSD2) expression (arbitrary units) in zebrafish embryos. The paucity of 11�HSD2 transcripts suggests that cortisol has access to MR during embryonic development, which is steadily increasing in transcript number.
interrenal function is autonomous over the first 2 dpf, but control is transferred to higher levels by 5 dpf. ACTH and CRF in the pituitary and hypothalamus, respectively, are expressed by 48 hpf, and the interrenals are beginning to produce cortisol. The brain appears to be poised to activate cortisol production in response to a stress. However, salinity and handling stressors do not stimulate a cortisol stress response at this point in time. Glucocorticoid and mineralocorticoid receptors. The initial abundance of maternally derived GR transcripts in the mature oocyte is unstable and is rapidly degraded (Figure 4). This suggests that GR signaling may not be critical during early embryonic development. By hatch, GR transcript numbers have rebounded and remain elevated. GR signaling may be critical at this time for metabolic energy repartitioning during the onset of feeding and growth. In contrast to GR, MR transcripts increase steadily from fertilization to feeding (Figure 4). In addition, there are very few 11�HSD2 transcripts in the embryo, which would suggest that cortisol is not deactivated and may be a ligand for MR. The embryo has continuous access to cortisol, from the maternal deposit during the early stages, or from de novo synthesis beginning around hatch. The lack of a cortisol stress response during embryo genesis suggests that GR and MR signaling may be regulating basic aspects of development such as metabol ism or osmoregulation as in adults. In mammals, GR regulates fetal carbohydrate metabolism, based on obser vations in GR knockout animals that display altered expression of key gluconeogenic enzymes. Summary. The HPI axis appears to be fully expressed and the interrenal cells are producing cortisol by hatch. However, there is no cortisol stress response during early
Hatching begins at 48 hpf in zebrafish and this coincides with further increases in the expression of CRF, StAR, 11�-hydroxylase, 11�HSD2, and the rebound of GR. At hatch, whole-body cortisol levels are beginning to rise as synthesis is initiated. In spite of these developments, cortisol levels in zebrafish are unresponsive to salinity or handling stressors. It appears that development of the sensory inputs to the hypothalamus, critical for stress perception and CRF release, is the limiting step delaying the activation of the cortisol stress response during development. The stress response is first observed at 3 dpf (72 hpf), where cortisol levels in freshwater reared larvae are ele vated after exposure to increased salinity (seawater). Although this is the earliest indication of a functional HPI axis, a handling stress (swirling around in a glass vial) at this same stage does not elicit an increase in cortisol. One hypothesis to explain this difference is that handling and osmotic challenges activate different signaling pathways, which have reached different degrees of functional development by 3 dpf. The HPI axis appears to be fully functional and receptive to different types of stressors at 4 dpf (approaching the onset of exogenous feeding). At this time both an osmotic challenge and handling stress elicit an increase in cortisol levels.
Stress Axis Development in Other Teleosts Common Carp The common carp and zebrafish are both cyprinids and have similar developmental rates and times to hatch (2–3 dpf). However, the cortisol stress response develops at an earlier stage in carp: basal cortisol and ACTH levels increase well before hatch, and also increase in response to stressor exposure before hatching. The evolutionary pressures that would lead to an accelerated development of HPI axis in carp are unknown. Rainbow Trout, Atlantic Salmon, and Yellow Perch Relative to hatching time, the temporal development of basal and stress-induced cortisol levels in rainbow trout, Atlantic salmon, and yellow perch are very similar to zebrafish. Cortisol levels are at a minimum around
1456 The Pituitary | Development of the Hypothalamus-Pituitary-Interrenal Axis (a) CRF
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hatching (hatching times: zebrafish – 2–3 dpf; perch – 10 dpf; salmonids – 4 wpf), and then begin to increase. However, stress-induced elevations of cortisol are not observed until after hatching (1 week post-hatch in perch and 2 weeks post-hatch in salmonids). Early exposure to stressors can alter the activation of the stress response later on in life. For example, rainbow trout embryos and larvae exposed to a stressor have a depressed cortisol response as juveniles (5 months post fertilization). This dampening of the stress response is also observed in juvenile rainbow trout that were exposed to waterborne cortisol immediately after fertilization for approximately 2 days. This illustrates that the devel opment and programming of the HPI axis is sensitive to stressor exposure, and long-lasting effects can be imprinted early on during development. However, determining the physiological consequences of these changes in the stress response is still required, and is essential in furthering our understanding of the development of the HPI axis.
Conclusion There is clearly a good understanding of the molecular ontogeny of the HPI axis in teleosts, or more specifically, the CRF-ACTH-cortisol axis (Figure 5(a)). However, this is an extremely simplified view of a complex system that possesses multiple paths of regulation and function (Figure 5(b)). To date, the majority of experiments have focused on the regulation of stress axis genes in terms of transcript abundance under various conditions. However,
there are several genetic and epigenetic mechanisms that influence these parameters. Characterizing these molecu lar mechanisms is useful not only to understand gene regulation but also to identify novel pathways, which can be used as bio-indicators of exposure to stressful stimuli. In addition, perhaps one of the more significant issues that currently remains unresolved is determining the basic functions of the stress axis components such as CRF, ACTH, GR, and MR during embryogenesis, given that there is no active stress response during this time. Model species such as zebrafish have occupied a pivo tal position in the study of physiological and molecular pathways of the HPI axis. However, teleosts are a large group with diverse developmental patterns, so it would be presumptuous to believe that the mechanisms are similar between different species, especially in light of the accele rated HPI-axis development in carp and the unique situation of single gene systems for CRF, ACTH, and GR in zebrafish. Hence, studies conducted in nonmodel species that are of importance to aquaculture, environ mental conservation, and management are necessary. See also: Hormonal Control of Metabolism and Ionic Regulation: Corticosteroids; The Hormonal Control of Osmoregulation in Teleost Fish. Hormonal Responses to Stress: Hormone Response to Stress; Stress Effect on Growth and Metabolism; Impact of Stress in Health and Reproduction.
Further Reading Alsop D and Vijayan MM (2009) The zebrafish stress axis: Molecular fallout from the teleost-specific genome duplication event. General and Comparative Endocrinology 161: 62–66. Alsop D and Vijayan MM (2009) Review: Molecular programming of the corticosteroid stress axis during zebrafish development. Comparative Biochemistry and Physiology. Part A: Molecular and Integrative Physiology 153: 49–54. Aluru N and Vijayan MM (2009) Stress transcriptomics in fish: A role for genomic cortisol signaling. General and Comparative Endocrinology 164: 142–150. Bernier NJ (2006) Review: The corticotropin-releasing factor system as a mediator of the appetite-suppressing effects of stress in fish. General and Comparative Endocrinology 146: 45–55. Matthews SG and Phillips DIW (2010) Minireview: Transgenerational inheritance of the stress response: A new frontier in stress research. Endocrinology 151: 7–13. Wintermantel TM, Berger S, Greiner EF, and Schu¨tz G (2005) Evaluation of steroid receptor function by gene targeting in mice. Journal of Steroid Biochemistry and Molecular Biology 93: 107–112.