Identification of signalling pathways involved in gill regeneration in zebrafish.

The occurrence of regeneration of the organs involved in respiratory gas exchange amongst vertebrates is heterogeneous. In some species of amphibians and fishes, the gills regenerate completely following resection or amputation, whereas in mammals, only partial, facultative regeneration of lung tissue occurs following injury. Given the homology between gills and lungs, the capacity of gill regeneration in aquatic species is of major interest in determining the underlying molecular or signalling pathways involved in respiratory organ regeneration. In the present study, we used adult zebrafish (Danio rerio) to characterize signalling pathways involved in the early stages of gill regeneration. Regeneration of the gills was induced by resection of gill filaments and observed over a period of up to 10 days. We screened for the effects on regeneration of the drugs SU5402, dorsomorphin and LY411575, which inhibit FGF, BMP or Notch signalling pathways, respectively. Exposure to each drug for 5 days significantly reduced regrowth of filament tips in regenerating tissue, compared with unresected controls. In separate experiments under normal conditions of regeneration, we used reverse transcription quantitative PCR and observed an increased expression of genes encoding for the bone morphogenetic factor, Bmp2b, fibroblast growth factor, Fgf8a, a transcriptional regulator (Her6) involved in Notch signalling, and Sonic Hedgehog (Shha), in regenerating gills at 10 day post-resection, compared with unresected controls. In situ hybridization confirmed that all four genes were expressed in regenerating gill tissue. This study implicates BMP, FGF, Notch and Shh signalling in gill regeneration in zebrafish.

Seasonal changes in membrane structure and excitability in retinal neurons of goldfish (Carassius auratus) under constant environmental conditions.

Seasonal modifications in the structure of cellular membranes occur as an adaptive measure to withstand exposure to prolonged environmental change. Little is known about whether such changes occur independently of external cues, such as photoperiod or temperature, or how they may impact the central nervous system. We compared membrane properties of neurons isolated from the retina of goldfish (Carassius auratus), an organism well adapted to extreme environmental change, during the summer and winter months. Goldfish were maintained in a facility under constant environmental conditions throughout the year. Analysis of whole-retina phospholipid composition using mass spectrometry-based lipidomics revealed a twofold increase in phosphatidylethanolamine species during the winter, suggesting an increase in cell membrane fluidity. Atomic force microscopy was used to produce localized, nanoscale-force deformation of neuronal membranes. Measurement of Young's modulus indicated increased membrane-cortical stiffness (or decreased elasticity) in neurons isolated during the winter. Voltage-clamp electrophysiology was used to assess physiological changes in neurons between seasons. Winter neurons displayed a hyperpolarized reversal potential (Vrev) and a significantly lower input resistance (Rin) compared with summer neurons. This was indicative of a decrease in membrane excitability during the winter. Subsequent measurement of intracellular Ca2+ activity using Fura-2 microspectrofluorometry confirmed a reduction in action potential activity, including duration and action potential profile, in neurons isolated during the winter. These studies demonstrate chemical and biophysical changes that occur in retinal neurons of goldfish throughout the year without exposure to seasonal cues, and suggest a novel mechanism of seasonal regulation of retinal activity.

Goldfish and crucian carp are natural models of anoxia tolerance in the retina.

Vertebrates need oxygen to survive. The central nervous system has an especially high energy demand, so brain and retinal neurons quickly die in anoxia. But fish of the genus Carassius are exceptionally anoxia-tolerant: the crucian carp (C. carassius) can survive months without oxygen in ice-covered ponds, and the common goldfish (C. auratus) can withstand hours of anoxia at room temperature. These fish previously offered insights into anoxia tolerance in the brain, heart, and liver. Here, we advance Carassius spp. as models to study anoxia tolerance in the retina. Electroretinogram and evoked potential recordings show that crucian carp reversibly downregulate their visual systems in anoxia, probably to save ATP. Notably, Carassius suppress their visual systems nearly twice as much as anoxia-tolerant turtles, Trachemys and Chrysemys spp., which are often promoted as the champions of anoxia tolerance. We summarize what is known about anoxia tolerance in the goldfish and crucian carp retinas, including cellular pathways which may protect retinal neurons from excitotoxic cell death. We compare the Carassius retina with two relevant models: natural anoxia tolerance in the turtle brain, and ischemic preconditioning in the rat retina. All three models include mitochondria as oxygen sensors: mitochondria depolarize due to mitochondrial ATP-dependent K+ channels, possibly to trigger neuroprotective second messenger cascades. The Carassius retina is an accessible and inexpensive model, with over 70 fruitful years of history in vision research. As a model for anoxia tolerance, it may provide new insights into diseases of the eye (like diabetes, macular degeneration, and eye stroke).

Identification of oxygen-sensitive neuroepithelial cells through an endogenous reporter gene in larval and adult transgenic zebrafish.

In teleost fish, specialized oxygen (O2) chemoreceptors, called neuroepithelial cells (NECs), are found in the gill epithelium in adults. During development, NECs are present in the skin before the formation of functional gills. NECs are known for retaining the monoamine neurotransmitter, serotonin (5-HT) and are conventionally identified through immunoreactivity with antibodies against 5-HT or synaptic vesicle protein (SV2). However, identification of NECs in live tissue and isolated cell preparations has been challenging due to the lack of a specific marker. The present study explored the use of the transgenic zebrafish, ETvmat2:GFP, which expresses green fluorescent protein (GFP) under the control of the vesicular monoamine transporter 2 (vmat2) regulatory element, to identify NECs. Using immunohistochemistry and confocal microscopy, we confirmed that the endogenous GFP in ETvmat2:GFP labelled serotonergic NECs in the skin of larvae and in the gills of adults. NECs of the gill filaments expressed a higher level of endogenous GFP compared with other cells. The endogenous GFP also labelled intrabranchial neurons of the gill filaments. Flow cytometric analysis demonstrated that filamental NECs could be distinguished from other dissociated gill cells based on high GFP expression alone. Acclimation to 2 weeks of severe hypoxia (PO2 = 35 mmHg) induced an increase in filamental NEC frequency, size and GFP gene expression. Here we present for the first time a transgenic tool that labels O2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

Mitochondrial KATP channels stabilize intracellular Ca2+ during hypoxia in retinal horizontal cells of goldfish (Carassius auratus).

Neurons of the retina require oxygen to survive. In hypoxia, neuronal ATP production is impaired, ATP-dependent ion pumping is reduced, transmembrane ion gradients are dysregulated, and intracellular Ca2+ concentration ([Ca2+]i) increases enough to trigger excitotoxic cell death. Central neurons of the common goldfish (Carassius auratus) are hypoxia tolerant, but little is known about how goldfish retinas withstand hypoxia. To study the cellular mechanisms of hypoxia tolerance, we isolated retinal interneurons (horizontal cells; HCs), and measured [Ca2+]i with Fura-2. Goldfish HCs maintained [Ca2+]i throughout 1 h of hypoxia, whereas [Ca2+]i increased irreversibly in HCs of the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss) with just 20 min of hypoxia. Our results suggest mitochondrial ATP-dependent K+ channels (mKATP) are necessary to stabilize [Ca2+]i throughout hypoxia. In goldfish HCs, [Ca2+]i increased when mKATP channels were blocked with glibenclamide or 5-hydroxydecanoic acid, whereas the mKATP channel agonist diazoxide prevented [Ca2+]i from increasing in hypoxia in trout HCs. We found that hypoxia protects against increases in [Ca2+]i in goldfish HCs via mKATP channels. Glycolytic inhibition with 2-deoxyglucose increased [Ca2+]i, which was rescued by hypoxia in a mKATP channel-dependent manner. We found no evidence of plasmalemmal KATP channels in patch-clamp experiments. Instead, we confirmed the involvement of KATP in mitochondria with TMRE imaging, as hypoxia rapidly (<5 min) depolarized mitochondria in a mKATP channel-sensitive manner. We conclude that mKATP channels initiate a neuroprotective pathway in goldfish HCs to maintain [Ca2+]i and avoid excitotoxicity in hypoxia. This model provides novel insight into the cellular mechanisms of hypoxia tolerance in the retina.

A comparative perspective on lung and gill regeneration.

The ability to continuously grow and regenerate the gills throughout life is a remarkable property of fish and amphibians. Considering that gill regeneration was first described over one century ago, it is surprising that the underlying mechanisms of cell and tissue replacement in the gills remain poorly understood. By contrast, the mammalian lung is a largely quiescent organ in adults but is capable of facultative regeneration following injury. In the course of the past decade, it has been recognized that lungs contain a population of stem or progenitor cells with an extensive ability to restore tissue; however, despite recent advances in regenerative biology of the lung, the signaling pathways that underlie regeneration are poorly understood. In this Review, we discuss the common evolutionary and embryological origins shared by gills and mammalian lungs. These are evident in homologies in tissue structure, cell populations, cellular function and genetic pathways. An integration of the literature on gill and lung regeneration in vertebrates is presented using a comparative approach in order to outline the challenges that remain in these areas, and to highlight the importance of using aquatic vertebrates as model organisms. The study of gill regeneration in fish and amphibians, which have a high regenerative potential and for which genetic tools are widely available, represents a unique opportunity to uncover common signaling mechanisms that may be important for regeneration of respiratory organs in all vertebrates. This may lead to new advances in tissue repair following lung disease.

Spontaneous action potentials in retinal horizontal cells of goldfish (Carassius auratus) are dependent upon L-type Ca2+ channels and ryanodine receptors.

Horizontal cells (HCs) are interneurons of the outer retina that undergo graded changes in membrane potential during the light response and provide feedback to photoreceptors. We characterized spontaneous Ca2+-based action potentials (APs) in isolated goldfish (Carassius auratus) HCs with electrophysiological and intracellular imaging techniques. Transient changes in intracellular Ca2+ concentration ([Ca2+]i) were observed with fura-2 and were abolished by removal of extracellular Ca2+ or by inhibition of Ca2+ channels by 50 µM Cd2+ or 100 µM nifedipine. Inhibition of Ca2+ release from stores with 20 µM ryanodine or 50 µM dantrolene abolished Ca2+ transients and increased baseline [Ca2+]i. This increased baseline was prevented by blocking L-type Ca2+ channels with nifedipine, suggesting that Ca2+-induced Ca2+ release from stores may be needed to inactivate membrane Ca2+ channels. Caffeine (3 mM) increased the frequency of Ca2+ transients, and the store-operated channel antagonist 2-aminoethyldiphenylborinate (100 µM) counteracted this effect. APs were detected with voltage-sensitive dye imaging (FluoVolt) and current-clamp electrophysiology. In current-clamp recordings, regenerative APs were abolished by removal of extracellular Ca2+ or in the presence of 5 mM Co2+ or 100 µM nifedipine, and APs were amplified with 15 mM Ba2+. Collectively, our data suggest that during APs Ca2+ enters through L-type Ca2+ channels and that Ca2+ stores (gated by ryanodine receptors) contribute to the rise in [Ca2+]i. This work may lead to further understanding of the possible role APs have in vision, such as transitioning from light to darkness or modulating feedback from HCs to photoreceptors.NEW & NOTEWORTHY Horizontal cells (HCs) are interneurons of the outer retina that provide inhibitory feedback onto photoreceptors. HCs respond to light via graded changes in membrane potential. We characterized spontaneous action potentials in HCs from goldfish and linked action potential generation to a rise in intracellular Ca2+ via plasma membrane channels and ryanodine receptors. Action potentials may play a role in vision, such as transitioning from light to darkness, or in modulating feedback from HCs to photoreceptors.

Calcium dynamics and regulation in horizontal cells of the vertebrate retina: lessons from teleosts.

Horizontal cells (HCs) are inhibitory interneurons of the vertebrate retina. Unlike typical neurons, HCs are chronically depolarized in the dark, leading to a constant influx of Ca2+ Therefore, mechanisms of Ca2+ homeostasis in HCs must differ from neurons elsewhere in the central nervous system, which undergo excitotoxicity when they are chronically depolarized or stressed with Ca2+ HCs are especially well characterized in teleost fish and have been used to unlock mysteries of the vertebrate retina for over one century. More recently, mammalian models of the retina have been increasingly informative for HC physiology. We draw from both teleost and mammalian models in this review, using a comparative approach to examine what is known about Ca2+ pathways in vertebrate HCs. We begin with a survey of Ca2+-permeable ion channels, exchangers, and pumps and summarize Ca2+ influx and efflux pathways, buffering, and intracellular stores. This includes evidence for Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors and for voltage-gated Ca2+ channels. Special attention is given to interactions between ion channels, to differences among species, and in which subtypes of HCs these channels have been found. We then discuss a number of unresolved issues pertaining to Ca2+ dynamics in HCs, including a potential role for Ca2+ in feedback to photoreceptors, the role for Ca2+-induced Ca2+ release, and the properties and functions of Ca2+-based action potentials. This review aims to highlight the unique Ca2+ dynamics in HCs, as these are inextricably tied to retinal function.

Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus).

The neuroepithelial cell (NEC) of the fish gill is an important model for O2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O2 chemoreception in vertebrates that are tolerant to anoxia has not yet been explored. Using whole cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca2+-dependent K+ current (IKCa) peaked at ~20 mV, was potentiated by increased intracellular Ca2+, and was reduced by 100 µM Cd2+ A voltage-dependent inward current in Ba2+ solution, with peak at 0 mV, confirmed the presence of Ca2+ channels. A voltage-dependent K+ current (IKV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K+ current (IKB) with open rectification. Mean resting membrane potential of -45.2 ± 11.6 mV did not change upon administration of hypoxia (Po2 = 11 mmHg), nor were any of the K+ currents sensitive to changes in Po2 during whole cell recording. By contrast, when the membrane and cytosol were left undisturbed during fura-2 or FM 1-43 imaging experiments, hypoxia increased intracellular Ca2+ concentration and initiated synaptic vesicle activity. 100 µM Cd2+ and 50 µM nifedipine eliminated uptake of FM 1-43. We conclude that Ca2+ influx via L-type Ca2+ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of IKCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia.NEW & NOTEWORTHY This study provides the first physiological characterization of oxygen chemoreceptors from an anoxia-tolerant vertebrate. Neuroepithelial cells (NECs) from the gills of goldfish displayed L-type Ca2+ channels and three types of K+ channels, one of which was dependent upon intracellular Ca2+ Although membrane currents were not inhibited by hypoxia during patch-clamp recording, this study is the first to show that NECs with an undisturbed cytosol responded to hypoxia with increased intracellular Ca2+ and synaptic vesicle activity.

Distribution and morphology of cholinergic cells in the branchial epithelium of zebrafish (Danio rerio).

Acetylcholine is an excitatory neurotransmitter important for oxygen sensing in mammals. A cholinergic mechanism in the fish gill has been implicated in the hyperventilatory response to acute hypoxia; however, the identity and distribution of acetylcholine-containing cells in the gills is poorly defined. We test the hypothesis that cholinergic cells are present in the gill filament epithelium in zebrafish (Danio rerio), a model vertebrate for which oxygen chemoreceptors are well characterized, and that these cells would receive nervous innervation. Using immunohistochemistry and confocal microscopy, we observed 10.2 ± 0.6 cells immunoreactive for the vesicular acetylcholine transporter (VAChT) on the efferent aspect of each gill filament, where a high density of serotonergic oxygen-sensitive neuroepithelial cells (NECs) were located. VAChT-positive cells of the efferent epithelium were positioned within 10 µm of NECs. On the afferent aspect of the gill filaments, VAChT-positive cells were greater in number (30.8 ± 3.1 per filament). On the efferent and afferent filament aspects, VAChT-positive cells did not contain serotonin, but did express choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, and were often closely apposed to nerve fibers labeled with the neuronal marker, zn-12. We conclude that cholinergic cells in the zebrafish gills were present in the primary epithelium of gill filaments, and formed contacts with nerve fibers. These studies provide morphological evidence for the presence of a cholinergic system in the zebrafish gill. Such a pathway may contribute to the reflex hyperventilatory response during hypoxia.

Purinergic and adenosine receptors contribute to hypoxic hyperventilation in zebrafish (Danio rerio).

The chemoreceptors involved in oxygen sensing in teleost fish are neuroepithelial cells (NECs) in the gills, and are analogous to glomus cells in the mammalian carotid body. Purinergic signalling mechanisms involving the neurotransmitters, ATP and adenosine, have been identified in mediating hypoxic signalling in the carotid body, but these pathways are not well understood in the fish gill. The present study used a behavioural assay to screen for the effects of drugs, that target purinergic and adenosine receptors, on the hyperventilatory response to hypoxia in larval zebrafish (Danio rerio) in order to determine if the receptors on which these drugs act may be involved in hypoxic signalling. The purinergic receptor antagonist, PPADS, targets purinergic P2X2/3 receptors and inhibited the hyperventilatory response to hypoxia (IC50=18.9µM). The broad-spectrum purinergic agonist, ATPγS, elicited a hyperventilatory response (EC50=168µM). The non-specific adenosine receptor antagonist, caffeine, inhibited the hyperventilatory response to hypoxia, as did the specific A2a receptor antagonist, SCH58261 (IC50=220nM). These results suggest that P2X2/3 and A2a receptors are candidates for mediating hypoxic hyperventilation in zebrafish. This study highlights the potential of applying chemical screening to ventilatory behaviour in zebrafish to further our understanding of the pathways involved in signalling by gill NECs and oxygen sensing in vertebrates.

Extracellular H+ induces Ca2+ signals in respiratory chemoreceptors of zebrafish.

Neuroepithelial cells (NECs) of the fish gill are respiratory chemoreceptors that detect changes in O2 and CO2/H(+) and are homologous to type I cells of the mammalian carotid body. In zebrafish (Danio rerio), stimulation of NECs by hypoxia or hypercapnia initiates inhibition of K(+) channels and subsequent membrane depolarisation. The goal of the present study was to further elucidate, in zebrafish NECs, the signalling pathways that underlie CO2/H(+) sensing and generate intracellular Ca(2+) ([Ca(2+)]i) signals. Breathing frequency was elevated maximally in fish exposed to 5 % CO2 (~37.5 mmHg). Measurement of [Ca(2+)]i in isolated NECs using Fura-2 imaging indicated that [Ca(2+)]i increased in response to acidic hypercapnia (5 % CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5 % CO2, pH 7.6). Measurement of intracellular pH (pHi) using BCECF demonstrated a rapid decrease in pHi in response to acidic and isohydric hypercapnia, while isocapnic acidosis produced a smaller change in pHi. Intracellular acidification was reduced by the carbonic anhydrase inhibitor, acetazolamide, without affecting [Ca(2+)]i responses. Moreover, intracellular acidification using acetate (at constant extracellular pH) was without effect on [Ca(2+)]i. The acid-induced increase in [Ca(2+)]i persisted in the absence of extracellular Ca(2+) and was unaffected by Ca(2+) channel blockers (Cd(2+), Ni(2+) or nifedipine). The results of this study demonstrate that, unlike type I cells, extracellular H(+) is critical to the hypercapnia-induced increase in [Ca(2+)]i in NECs. The increase in [Ca(2+)]i occurs independently of pHi and appears to originate primarily from Ca(2+) derived from intracellular stores.

Aquatic surface respiration and swimming behaviour in adult and developing zebrafish exposed to hypoxia.

Severe hypoxia elicits aquatic surface respiration (ASR) behaviour in many species of fish, where ventilation of the gills at the air-water interface improves O2 uptake and survival. ASR is an important adaptation that may have given rise to air breathing in vertebrates. The neural substrate of this behaviour, however, is not defined. We characterized ASR in developing and adult zebrafish (Danio rerio) to ascertain a potential role for peripheral chemoreceptors in initiation or modulation of this response. Adult zebrafish exposed to acute, progressive hypoxia (PO2 from 158 to 15 mmHg) performed ASR with a threshold of 30 mmHg, and spent more time at the surface as PO2 decreased. Acclimation to hypoxia attenuated ASR responses. In larvae, ASR behaviour was observed between 5 and 21 days postfertilization with a threshold of 16 mmHg. Zebrafish decreased swimming behaviour (i.e. distance, velocity and acceleration) as PO2 was decreased, with a secondary increase in behaviour near or below threshold PO2 . In adults that underwent a 10-day intraperitoneal injection regime of 10 µg g(-1) serotonin (5-HT) or 20 µg g(-1) acetylcholine (ACh), an acute bout of hypoxia (15 mmHg) increased the time engaged in ASR by 5.5 and 4.9 times, respectively, compared with controls. Larvae previously immersed in 10 µmol l(-1) 5-HT or ACh also displayed an increased ASR response. Our results support the notion that ASR is a behavioural response that is reliant upon input from peripheral O2 chemoreceptors. We discuss implications for the role of chemoreceptors in the evolution of air breathing.

Hypercapnia and low pH induce neuroepithelial cell proliferation and emersion behaviour in the amphibious fish Kryptolebias marmoratus.

Aquatic hypercapnia may have helped to drive ancestral vertebrate invasion of land. We tested the hypothesis that amphibious fishes sense and respond to elevated aquatic PCO2 by behavioural avoidance mechanisms, and by morphological changes at the chemoreceptor level. Mangrove rivulus (Kryptolebias marmoratus) were exposed to 1 week of normocapnic control water (pH 8), air, hypercapnia (5% CO2, pH 6.8) or isocapnic acidosis (pH 6.8). We found that the density of CO2/H(+) chemoreceptive neuroepithelial cells (NECs) was increased in hypercapnia or isocapnic acidosis-exposed fish. Projection area (a measure of cell size) was unchanged. Acute exposure to progressive hypercapnia induced the fish to emerse (leave water) at water pH values ∼6.1, whereas addition of HCl to water caused a more variable response with a lower pH threshold (∼pH 5.5). These results support our hypothesis and suggest that aquatic hypercapnia provides an adequate stimulus for extant amphibious fishes to temporarily transition from aquatic to terrestrial habitats.

Peripheral chemoreceptors in fish: A brief history and a look ahead.

The story of control of cardiorespiratory reflexes by peripheral chemoreceptors includes a chapter on evolution in large part because of the work of Prof. William K. Milsom. Bill has reminded us to think comparatively about O2 and CO2/H(+) sensing. We present a brief review of the fish gill and O2 chemoreceptors, as well as recent results from our laboratory, that were discussed at a symposium in honour of Prof. Milsom's extensive career. In a series of papers from the Milsom laboratory from 1986 to 1995, it was demonstrated that the fish gill is a major site of chemosensory discharge during hypoxia, and that this response is sensitive to multiple neurochemicals involved in chemosensing. These and other more recent studies by Bill et al. are now fundamental and have helped to shape the field as it is today. At the cellular level, we have shown that chemosensitive neuroepithelial cells (NECs) of the gills may possess unique adaptations compared to their mammalian homologues. In addition, we used injection of the styryl dye, FM1-43, to identify gill NECs in zebrafish and demonstrate increased vesicular activity in NECs in vitro during acute stimulation. In vivo, we have identified 5-HT2, 5-HT3, dopaminergic and nicotinic receptor activity involved in the hyperventilatory response in developing zebrafish. With this model we have also traced the fate of mitotic cells in the gills, and demonstrated the regeneration of resected gill filaments and replacement of O2-sensitive NECs.

Cell proliferation and regeneration in the gill.

Seminal studies from the early 20th century defined the structural changes associated with development and regeneration of the gills in goldfish at the gross morphological and cellular levels using standard techniques of light and electron microscopy. More recently, investigations using cell lineage tracing, molecular biology, immunohistochemistry and single-cell RNA-sequencing have pushed the field forward and have begun to reveal the cellular and molecular processes that orchestrate cell proliferation and regeneration in the gills. The gill is a multifunctional organ that mediates an array of important physiological functions, including respiration, ion regulation and excretion of waste products. It is comprised of unique cell types, such as pavement cells, ionocytes, chemoreceptors and undifferentiated stem or progenitor cells that regulate growth and replenish cell populations. The gills develop from the embryonic endoderm and are rich in cell types derived from the neural crest. The gills have the capacity to remodel themselves in response to environmental change, such as in the case of ionocytes, chemoreceptors and the interlamellar cell mass, and can completely regenerate gill filaments and lamellae. Both processes of remodeling and regeneration invariably involve cell proliferation. Although gill regeneration has been reported in only a limited number of fish species, the process appears to have many similarities to regeneration of other organs in fish and amphibians. The present article reviews the studies that have described gill development and growth, and that demonstrate a suite of genes, transcription factors and other proteins involved in cell proliferation and regeneration in the gills.

A role for dopamine in control of the hypoxic ventilatory response via D2 receptors in the zebrafish gill.

Dopamine is a neurotransmitter involved in oxygen sensing and control of reflex hyperventilation. In aquatic vertebrates, oxygen sensing occurs in the gills via chemoreceptive neuroepithelial cells (NECs), but a mechanism for dopamine in autonomic control of ventilation has not been defined. We used immunohistochemistry and confocal microscopy to map the distribution of tyrosine hydroxylase (TH), an enzyme necessary for dopamine synthesis, in the gills of zebrafish. TH was found in nerve fibers of the gill filaments and respiratory lamellae. We further identified dopamine active transporter (dat) and vesicular monoamine transporter (vmat2) expression in neurons of the gill filaments using transgenic lines. Moreover, TH- and dat-positive nerve fibers innervated NECs. In chemical screening assays, domperidone, a D2 receptor antagonist, increased ventilation frequency in zebrafish larvae in a dose-dependent manner. When larvae were confronted with acute hypoxia, the D2 agonist, quinpirole, abolished the hyperventilatory response. Quantitative polymerase chain reaction confirmed expression of drd2a and drd2b (genes encoding D2 receptors) in the gills, and their relative abundance decreased following acclimation to hypoxia for 48 h. We localized D2 receptor immunoreactivity to NECs in the efferent gill filament epithelium, and a novel cell type in the afferent filament epithelium. We provide evidence for the synthesis and storage of dopamine by sensory nerve terminals that innervate NECs. We further suggest that D2 receptors on presynaptic NECs provide a feedback mechanism that attenuates the chemoreceptor response to hypoxia. Our studies suggest that a fundamental, modulatory role for dopamine in oxygen sensing arose early in vertebrate evolution.

Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish.

The chemosensory roles of gill neuroepithelial cells (NECs) in mediating the hyperventilatory response to hypoxia are not clearly defined in fish. While serotonin (5-HT) is the predominant neurotransmitter in O(2)-sensitive gill NECs, acetylcholine (ACh) plays a more prominent role in O(2) sensing in terrestrial vertebrates. The present study characterized the developmental chronology of potential serotonergic and cholinergic chemosensory pathways of the gill in the model vertebrate, the zebrafish (Danio rerio). In immunolabelled whole gills from larvae, serotonergic NECs were observed in epithelia of the gill filaments and gill arches, while non-serotonergic NECs were found primarily in the gill arches. Acclimation of developing zebrafish to hypoxia (P(O2)=75 mmHg) reduced the number of serotonergic NECs observed at 7 days post-fertilization (d.p.f.), and this effect was absent at 10 d.p.f. In vivo administration of 5-HT mimicked hypoxia by increasing ventilation frequency (f(V)) in early stage (7-10 d.p.f.) and late stage larvae (14-21 d.p.f.), while ACh increased f(V) only in late stage larvae. In time course experiments, application of ketanserin inhibited the hyperventilatory response to acute hypoxia (P(O2)=25 mmHg) at 10 d.p.f., while hexamethonium did not have this effect until 12 d.p.f. Cells immunoreactive for the vesicular acetylcholine transporter (VAChT) began to appear in the gill filaments by 14 d.p.f. Characterization in adult gills revealed that VAChT-positive cells were a separate population of neurosecretory cells of the gill filaments. These studies suggest that serotonergic and cholinergic pathways in the zebrafish gill develop at different times and contribute to the hyperventilatory response to hypoxia.

Functional prediction and physiological characterization of a novel short trans-membrane protein 1 as a subunit of mitochondrial respiratory complexes.

Mitochondrial respiration is mediated by a set of multisubunit assemblies of proteins that are embedded in the mitochondrial inner membranes. Respiratory complexes do not only contain central catalytic subunits essential for the bioenergetic transformation, but also many short trans-membrane subunits (sTMs) that are implicated in the proper assembly of complexes. Defects in sTMs have been discovered in some human neurodegenerative diseases. Here we identify a new subunit that we named Stmp1 and have characterized its function using both computational and experimental approaches. Stmp1 is a short trans-membrane protein, and sequence/structure analysis revealed that it shares common features like the small size, presence of a single or two TM region, and a COOH-terminal charged region, as many typical sTMs of respiratory complexes. In situ hybridization and RT-PCR assays showed that the Stmp1 expression is ubiquitous throughout zebrafish embryogenesis. In adults, Stmp1 expression was highest in the brain compared with muscle and liver. In zebrafish larvae (3-5 days postfertilization), antisense morpholino oligonucleotide-mediated knockdown of the Stmp1 gene (Stmp1-MO) resulted in a series of mild morphological defects, including abnormal shape of head and jaw and cardiac edema. Larvae injected with the Stmp1-MO had negligible responses to touch stimuli. By ventilation frequency analysis we found that Stmp1-MO-injected zebrafish displayed a severe dysfunction of ventilatory activities when exposed to hypoxic conditions, suggesting a defective mitochondrial activity induced by the loss of Stmp1. Phylogenetic profiling of known respiratory sTMs compared with Stmp1 revealed that all defined sTMs from four respiratory complexes have restricted or variable phyletic distribution, indicating that they are products of evolutionary innovations to fulfill lineage-related functional requirements for respiratory complexes. Thus, being present in animals, filasterea, choanoflagellida, amoebozoa, and plants, Stmp1 may have evolved to confer a new or complementary regulation of respiratory activities.

Serotonergic neuroepithelial cells of the skin in developing zebrafish: morphology, innervation and oxygen-sensitive properties.

In teleost fish, O(2) chemoreceptors of the gills (neuroepithelial cells or NECs) initiate cardiorespiratory reflexes during hypoxia. In developing zebrafish, hyperventilatory and behavioural responses to hypoxia are observed before development of gill NECs, indicating that extrabranchial chemoreceptors mediate these responses in embryos. We have characterised a population of cells of the skin in developing zebrafish that resemble O(2)-chemoreceptive gill NECs. Skin NECs were identified by serotonin immunolabelling and were distributed over the entire skin surface. These cells contained synaptic vesicles and were associated with nerve fibres. Skin NECs were first evident in embryos 24-26 h post-fertilisation (h.p.f.), and embryos developed a behavioural response to hypoxia between 24 and 48 h.p.f. The total number of NECs declined with age from approximately 300 cells per larva at 3 days post-fertilisation (d.p.f.) to ~120 cells at 7 d.p.f., and were rarely observed in adults. Acclimation to hypoxia (30 mmHg) or hyperoxia (300 mmHg) resulted in delayed or accelerated development, respectively, of peak resting ventilatory frequency and produced changes in the ventilatory response to hypoxia. In hypoxia-acclimated larvae, the temporal pattern of skin NECs was altered such that the number of cells did not decrease with age. By contrast, hyperoxia produced a more rapid decline in NEC number. The neurotoxin 6-hydroxydopamine degraded catecholaminergic nerve terminals that made contact with skin NECs and eliminated the hyperventilatory response to hypoxia. These results indicate that skin NECs are sensitive to changes in O(2) and suggest that they may play a role in initiating responses to hypoxia in developing zebrafish.