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PubMed2006

Time course and development of light adaptation processes in the outer zebrafish retina.

Retinomotor movements are morphological changes in the outer retina in response to changing light conditions. They can be separated into two components: Migration of pigment granules within the microvilli of the retinal pigment epithelium (RPE) and positional changes in photoreceptor cells. These positional changes optimize exposure of the cone and rod photoreceptors to light. The aim of this study was to analyze both the time course of retinomotor movements in the adult zebrafish and the maturation of these processes in the developing fish. We show that retinomotor movements are used as a dark/light adaptation mechanism in zebrafish. In adult zebrafish, melanin granules of the RPE migrate with constant speed and reach the fully light adapted (LA) state approximately after 1 h. In contrast, about two thirds of double cone outer segment movements are finished in 5 min, and are fully completed in 10 to 20 min. During development there are three crucial stages leading to mature retinomotor movements in response to light: at 5 dpf (days post fertilization) the migration of pigment granules begins, at 20 dpf the pigment granules condense in the apical part of the RPE microvilli, and at 28 dpf, concomitant with the functional maturation of rods, the double cones contract as in adult retinas.

Adaptation, Ocular↗

PubMed2006

Zebrafish as a model for developmental neurotoxicity testing.

BACKGROUND: To establish zebrafish as a developmental toxicity model, we used 7 well-characterized compounds to examine several parameters of neurotoxicity during development. METHODS: Embryos were exposed by semistatic immersion from 6 hrs postfertilization (hpf). Teratogenicity was assessed using a modified method previously developed by Phylonix. Dying cells in the brain were assessed by acridine orange staining (these cells are likely to be apoptotic). Motor neurons were assessed by antiacetylated tubulin staining and catecholaminergic neurons were visualized by antityrosine hydroxylase staining. RESULTS: Atrazine, dichlorodiphenyltrichloroethane (DDT), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were primarily teratogenic and not specifically neurotoxic. 2,4-dichlorophenoxyacetic acid (2,4-D), dieldrin, and nonylphenol showed specific neurotoxicity; dieldrin and nonylphenol were specifically toxic to catecholaminergic neurons. Malathion, although not teratogenic, showed some nonspecific toxicity. CONCLUSIONS: Teratogenicity measured in 96-hpf zebrafish is predictive of mammalian teratogenicity and is useful in determining whether a compound causes specific neurotoxicity or general developmental toxicity. Induction of apoptosis or necrosis is an indicator of neurotoxicity. An effect on motor neurons in the caudal third of the embryo correlates with expected defects in motility. Overall, our results showed a strong correlation with mammalian data and suggest that zebrafish is a predictive animal model for neurotoxicity screening.

Animals↗

PubMed2004

In vivo biofluid dynamic imaging in the developing zebrafish.

Flow-structure interactions are ubiquitous in nature, and are important factors in the proper development of form and function in living organisms. In order to uncover the mechanisms by which flow-structure interactions affect vertebrate development, we first need to establish the techniques necessary to quantitatively describe the fluid flow environment within the embryo. To do this, we must bring dynamic, in vivo imaging methods to bear on living systems. Traditional avian and mammalian model systems can be problematic in this regard. The zebrafish (Danio rerio) is widely accepted as an excellent model organism for the study of vertebrate biology, as it shows substantial anatomical and genetic conservation with higher vertebrates, including humans. Their small size, optical transparency, and external development make zebrafish the ideal model system for dynamic imaging. This article reviews the current state of research in imaging biofluid flow within and around developing zebrafish embryos, with an emphasis on dynamic imaging modalities.

Animals↗

PubMed2002

Conserved and divergent patterns of Reelin expression in the zebrafish central nervous system.

The protein Reelin is suggested to function in cell-cell interactions and in mediating neuronal migrations in layered central nervous system structures. With the aim of shedding light on the development of the teleost telencephalon, which forms through the process of eversion and results in the formation of a nonlaminar pallium, we isolated a zebrafish ortholog of the reelin gene and studied its expression in developing and adult brain. The pattern of expression is highly dynamic during the first 24-72 hours of development. By 5 days postfertilization, high amounts of reelin mRNA are found in the dorsal telencephalon, thalamic and hypothalamic regions, pretectal nuclei, optic tectum, cerebellum, hindbrain, reticular formation, and spinal cord, primarily confined to postmitotic neurons. This pattern persists in 1- to 3-month-old zebrafish. This study, together with reports on reelin expression in other vertebrates, shows that reelin mRNA distribution is conserved in many regions of the vertebrate brain. A major exception is that reelin is expressed in the majority of the cells of the dorsal regions of the everted telencephalon in zebrafish embryos, whereas it is restricted to specific neuronal populations in the developing telencephalon of amniotes. To better understand the origin of these differences, we analyzed reelin expression in the telencephalon of an amphibian. Telencephalic reelin expression in Xenopus laevis shows more similarities with the sauropsidian than with the teleostean pattern. Thus, the differences in the telencephalic expression of reelin between teleosts and tetrapods are likely to be due to different roles for Reelin during eversion, a process that is specific for the teleost telencephalon.

Aging↗

PubMed2002

Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of the zebrafish (Danio rerio).

The distribution of thyrotropin-releasing hormone (TRH) in the brain of the adult zebrafish was studied with immunohistochemical techniques. In the telencephalon, abundant TRH-immunoreactive (TRHir) neurons were observed in the central, ventral, and supra- and postcommissural regions of the ventral telencephalic area. In the diencephalon, TRHir neurons were observed in the anterior parvocellular preoptic nucleus, the suprachiasmatic nucleus, the lateral hypothalamic nucleus, the rostral parts of the anterior tuberal nucleus and torus lateralis, and the posterior tuberal nucleus. Some TRHir neurons were also observed in the central posterior thalamic nucleus and in the habenula. The mesencephalon contained TRHir cells in the rostrodorsal tegmentum, the Edinger-Westphal nucleus, the torus semicircularis, and the nucleus of the lateral lemniscus. Further TRHir neurons were observed in the interpeduncular nucleus. In the rhombencephalon, TRHir cells were observed in the nucleus isthmi and the locus coeruleus, rostrally, and in the vagal lobe and vagal motor nucleus, caudally. In the forebrain, TRHir fibers were abundant in several regions, including the medial and caudodorsal parts of the dorsal telencephalic area, the ventral and commissural parts of the ventral telencephalic area, the preoptic area, the posterior tubercle, the anterior tuberal nucleus, and the posterior hypothalamic lobe. The dorsal thalamus exhibited moderate TRHir innervation. In the mesencephalon, the optic tectum received a rich TRHir innervation between the periventricular gray zone and the stratum griseum centrale. A conspicuous TRHir longitudinal tract traversed the tegmentum and extended to the rhombencephalon. The medial and lateral mesencephalic reticular areas and the interpeduncular nucleus were richly innervated by TRHir fibers. In the rhombencephalon, the secondary gustatory nucleus received abundant TRHir fibers. TRHir fibers moderately innervated the ventrolateral and ventromedial reticular area and richly innervated the vagal lobe and Cajal's commissural nucleus. Some TRHir fibers coursed in the lateral funiculus of the spinal cord. Some TRHir amacrine cells were observed in the retina. The wide distribution of TRHir neurons and fibers observed in the zebrafish brain suggests that TRH plays different roles. These results in the adult zebrafish reveal a number of differences with respect to the TRHir systems reported in other adult teleosts but were similar to those found during late developmental stages of trout (Díaz et al., 2001).

Animals↗

PubMed2003

Neuroepithelial cells and associated innervation of the zebrafish gill: a confocal immunofluorescence study.

Peripheral chemoreceptors responsive to hypoxia have been well characterized in air-breathing vertebrates, but poorly in water-breathers. The present study examined the distribution of five populations of neuroepithelial cells (NECs), putative O(2) chemoreceptors, and innervation patterns in the zebrafish gill using whole-mounts and confocal immunofluorescence. Nerve bundles and fibers of the gill were labeled with zn-12 (a zebrafish-specific neuronal marker) and SV2 antisera and NECs were characterized by serotonin (5-HT) immunoreactivity (IR), SV2-IR and the purinoceptor P2X(3)-IR. A zn-12-IR nerve bundle extended the length of the gill filament and gave rise to a nerve plexus surrounding the efferent filament artery (eFA) and a rich network of fibers that innervated both serotonergic and nonserotonergic NECs of the filament and lamellar epithelium. Three populations of serotonergic, SV2-IR neurons intrinsic to the gill filaments are described, one of which provided innervation to NECs of the filament epithelium. Degeneration of nerve fibers in gill arches maintained in explant culture for 2 days revealed the extrinsic origin of nerve fibers of the plexus and lamellae and the innervation of filament NECs by both intrinsic and extrinsic fibers. Intrinsic innervation surrounding the eFA survived in explant cultures, suggesting a mechanism of local vascular control within the gill. In addition, NECs survived in explants after degeneration of extrinsic nerve fibers. Thus, NECs of the zebrafish gill are organized in a manner reminiscent of O(2) chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation.

Animals↗

PubMed2004

Cellular patterns in the inner retina of adult zebrafish: quantitative analyses and a computational model of their formation.

The mechanisms that control cellular pattern formation in the growing vertebrate central nervous system are poorly understood. In an effort to reveal mechanistic rules of cellular pattern formation in the central nervous system, quantitative spatial analysis and computational modeling techniques were applied to cellular patterns in the inner retina of the adult zebrafish. All the analyzed cell types were arrayed in nonrandom patterns tending toward regularity; specifically, they were locally anticlustered. Over relatively large spatial scales, only one cell type exhibited consistent evidence for pattern regularity, suggesting that cellular pattern formation in the inner retina is dominated by local anticlustering mechanisms. Cross-correlation analyses revealed independence between the patterns of different cell types, suggesting that cellular pattern formation may involve multiple, independent, homotypic anticlustering mechanisms. A computational model of cellular pattern formation in the growing zebrafish retina was developed, which featured an inhibitory, homotypic signaling mechanism, arising from differentiated cells, that controlled the spatial profile of cell fate decisions. By adjusting the spatial profile of this decaying-exponential signal, the model provided good estimates of all the cellular patterns that were observed in vivo, as objectively judged by quantitative spatial pattern analyses. The results support the hypothesis that cellular pattern formation in the inner retina of zebrafish is dominated by a set of anticlustering mechanisms that may control events at, or near, the spatiotemporal point of cell fate decision.

Animals↗

PubMed2001

A confocal study of spinal interneurons in living larval zebrafish.

We used confocal microscopy to examine the morphology of spinal interneurons in living larval zebrafish with the aim of providing a morphological foundation for generating functional hypotheses. Interneurons were retrogradely labeled by injections of fluorescent dextrans into the spinal cord, and the three-dimensional morphology of living cells was reconstructed from confocal optical sections through the transparent fish. At least eight types of interneurons are present in the spinal cord of larval zebrafish; four of these are described here for the first time. The newly discovered cell types include classes of commissural neurons with axons that ascend, descend, and bifurcate in the contralateral spinal cord. Our reexamination of previously described cell types revealed functionally relevant features of their morphology, such as undescribed commissural axons, as well as the relationships between the trajectories of the axons of interneurons and the descending Mauthner axons. In addition to describing neurons, we surveyed their morphology at multiple positions along the spinal cord and found longitudinal changes in their distribution and sizes. For example, some cell types increase in size from rostral to caudal, whereas others decrease. Our observations lead to predictions of the roles of some of these interneurons in motor circuits. These predictions can be tested with the combination of functional imaging, single-cell ablation, and genetic approaches that make zebrafish a powerful model system for studying neuronal circuits.

Animals↗

PubMed2001

Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio).

Although the zebrafish has become an important model for genetic analysis of the vertebrate auditory system, a comprehensive description of the zebrafish ear has been provided for embryonic and larval development only (Haddon and Lewis [1996] J. Comp. Neurol. 365:113). Here we describe the development of sensory maculae in juvenile fish and the morphology of the adult zebrafish ear. This description was obtained via three-dimensional reconstruction of serial sections and confocal microscopy of immunolabeled preparations and includes the Weberian ossicles and fluid spaces. Phalloidin staining, which labels actin filaments of stereocilia, was used to delineate the sensory epithelia, to visualize the distribution of hair cells, to estimate their density in different areas of the maculae, and to perform hair cell counts. Morphology of ciliary bundles in different regions of the lagena, saccule, utricle, macula neglecta, and cristae was characterized with an anti-acetylated tubulin antibody and by phalloidin staining. We have identified two antibodies characterized by region-specific staining patterns in the inner ear epithelia. Zn-1 antibody staining largely correlates with the presence of short-bundle hair cells in the peripheral regions of sensory epithelia. Zn-4 antibody, on the other hand, labels a zone of epithelial cells surrounding the sensory maculae. These analyses extend previous observations of cell-type heterogeneity in both sensory and nonsensory epithelia of the fish ear.

Age Factors↗

PubMed2004

Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina.

Horizontal, bipolar, and amacrine cells in the zebrafish retina were morphologically characterized using DiOlistic techniques. In this method, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-coated microcarriers are shot at high speed onto the surfaces of living retinal slices where the DiI then delineates axons, somata, and dendrites of isolated neurons. Zebrafish retinal somata were 5-10 microm in diameter. Three horizontal cell types (HA-1, HA-2, and HB) were identified; dendritic tree diameters averaged 25-40 microm. HA somata were round. Cells classified as HA-2 were larger than HA-1 cells and possessed an axon. HB somata were flattened, without an axon, although short fusiform structure(s) projected from the soma. Bipolar cells were separated into 17 morphological types. Dendritic trees ranged from 10 to 70 microM. There were six B(on) types with axon boutons only in the ON sublamina of the inner plexiform layer (IPL), and seven B(off) types with axon boutons or branches only in the OFF sublamina. Four types of bistratified bipolar cells displayed boutons in both ON and OFF layers. Amacrine cells occurred in seven types. A(off) cells (three types) were monostratified and ramified in the IPL OFF sublamina. Dendritic fields were 60-150 microM. A(on) pyriform cells (three types) branched in the ON sublamina. Dendritic fields were 50-170 microM. A(diffuse) cells articulated processes in all IPL strata. Dendritic fields were 15-90 microM. These findings are important for studies examining signal processing in zebrafish retina and for understanding changes in function resulting from mutations and perturbations of retinal organization.

Amacrine Cells↗

PubMed2004

Differential expression of cadherins in the developing and adult zebrafish olfactory system.

Cadherins are cell adhesion molecules that play important roles in development of a variety of tissues and maintenance of adult structures. Although cadherin expression has been studied in detail in the central nervous system of several vertebrate species, little is known of their distribution in the developing and adult olfactory structures, and there is no published report, to our knowledge, of cadherin expression in fish olfactory system. In this study, we examined expression patterns of three cadherins, cadherin-1 (E-cadherin), cadherin-2 (N-cadherin), and cadherin-4 (R-cadherin), in the olfactory system of developing and adult zebrafish by using both in situ hybridization and immunocytochemical methods. Cadherin-1 is detected in the newly formed olfactory placode, and its expression is maintained in the developing and adult olfactory epithelium and olfactory nerve. Cadherin-2 is expressed in the olfactory epithelium, olfactory nerve, and olfactory bulb of the embryonic and larval zebrafish, and its expression is reduced in the adult olfactory system. In contrast to the cadherin-1 and cadherin-2 expression, cadherin-4 is not found in the olfactory epithelium, but it is detected in the larval and adult olfactory bulb, in the olfactory tract, and its targets in the telencephalon. We hypothesize that the differential expression of these three cadherins in the developing zebrafish major olfactory structures reflects functionally different roles in the development of the vertebrate olfactory system.

Age Factors↗

PubMed2005

Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene.

Compared with adult mammals, the brain of teleost fish is characterized by an extremely high capacity to aromatize androgens into estrogens, and this metabolic activity results from the expression of a specific brain aromatase (AroB) generated by the cyp19b gene. In this study, we first generated antibodies to zebrafish AroB and used them to map AroB-positive structures in the brain of adult zebrafish. We show that AroB is exclusively expressed in radial glial cells, mainly in the olfactory bulbs, telencephalon, preoptic area, and hypothalamus. Second, we investigated in vivo and in vitro the mechanisms involved in the estradiol (E2) regulation of the cyp19b gene. By means of whole-mount hybridization and immunohistochemistry on zebrafish embryos and larvae, we confirmed the E2-dependent upregulation of the cyp19b gene, and we show that E2 triggers AroB expression in radial glial cells mainly in the preoptic area and mediobasal hypothalamus of 48 hpf (hours post fertilization) and 108 hpf larvae. In addition, an in vitro analysis of 0.5 kb of the promoter region of the cyp19b gene demonstrated that this E2-dependent regulation involves a direct transcriptional action of estrogen receptors requiring estrogen-responsive elements. However, the data obtained on different cell lines demonstrate that a glial cell context is necessary for full E2 induction. The correlation between our in vivo and in vitro data suggests that the E2-dependent upregulation of AroB is favored by a glial cell context.

Animals↗

PubMed2006

Mitral cells in the olfactory bulb of adult zebrafish (Danio rerio): morphology and distribution.

The mitral cell is the primary output neuron and central relay in the olfactory bulb of vertebrates. The morphology of these cells has been studied extensively in mammalian systems and to a lesser degree in teleosts. This study uses retrograde tract tracing and other techniques to characterize the morphology and distribution of mitral cells in the olfactory bulb of adult zebrafish, Danio rerio. These output neurons, located primarily in the glomerular layer and superficial internal cell layer, had variable-shaped somata that ranged in size from 4-18 microm in diameter and 31-96 microm2 in cross-sectional area. The mitral cells exhibited two main types of morphologies with regard to their dendrites: the unidendritic morphology was a single primary dendrite with one or more tufts, but multidendritic cells with several dendritic projections also were seen. The axons of these cells projected to either the medial or the lateral olfactory tract and, in general, the location of the cell on the medial or lateral side of the bulb was indicative of the tract to which it would project. Further, this study shows that the majority of zebrafish mitral cells likely innervate a single glomerulus rather than multiple glomeruli. This information is contrary to the multiple innervation pattern suggested for all teleost mitral cells. Our findings suggest that mitral cells in zebrafish may be more similar to mammalian mitral cells than previously believed, despite variation in size and structure. This information provides a revised anatomical framework for olfactory processing studies in this key model system.

Animals↗

PubMed2007

Dynamic expression of the basic helix-loop-helix transcription factor neuroD in the rod and cone photoreceptor lineages in the retina of the embryonic and larval zebrafish.

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for determining neuronal cell fate and regulating withdrawal from the cell cycle. We showed previously that, in goldfish, neuroD is expressed in the rod photoreceptor lineage, and we inferred that neuroD is also expressed in a subset of amacrine cells and nascent cone photoreceptors. Here we extended that study by examining the temporal and spatial expression pattern of neuroD in the embryonic and larval zebrafish and by identifying the cell types that express this gene. NeuroD expression in the developing zebrafish retina is dynamic, spanning early retinogenesis and the maturation of cone photoreceptors. In early retinogenesis neuroD expression expands from a small patch in the ventronasal retina, through the remaining retinal neuroepithelium. As retinogenesis progresses, neuroD expression becomes restricted to amacrine cells, immature cones, and cells of rod and cone lineages. This expression achieves an adult pattern by 96 hours postfertilization (hpf), whereupon the temporal pattern of neuroD expression in central retina is spatially recapitulated at the germinative margin. The cellular pattern of expression suggests that neuroD regulates aspects of rod and cone genesis, but through separate cellular lineages. Furthermore, neuroD is coexpressed with the cone-rod-homeobox transcription factor (Crx) in putative cone progenitors and nascent cone photoreceptors, suggesting that, in the zebrafish retina, as in other vertebrate retinas, similar genetic cascades regulate photoreceptor genesis and maturation.

Animals↗

PubMed1994

Development of the retinofugal projections in the embryonic and larval zebrafish (Brachydanio rerio).

Studies of the projection from the vertebrate retina have contributed significantly to current concepts of neural development. The zebrafish has recently become a favored system for the study of development in general and neural development in particular. Although the development of both the optic nerve and the retinotectal projection of the zebrafish has been described, the retinofugal projection in its entirety has not. This paper describes it and also addresses the issue of projectional exuberance: i.e., transient projections to targets that are not innervated in the adult. The retinofugal projection of embryonic and larval zebrafish (32 hours to 7 days post-fertilization) was labeled by intraocular injection of DiI (1,1'-dioctadecyl-3,3,3',3',tetramethylindocarbocyanine perchlorate) and then studied in wholemounts and sections. The first optic axons crossed the chiasm at 32 hours post-fertilization and projected in a straight line to reach the tectum at about 44 hours. At 48 hours, a few optic axons deviated along either the tract of the posterior commissure or the tract of the postoptic commissure. By 72 hours (about the time of hatching) optic axons arborized in ten distinct regions, termed arborization fields. At 6-7 days post-fertilization, the same ten arborization fields (nine contralateral, one bilateral) were evident. Most of the arborization fields were located in the superficial neuropil and were not associated with morphologically identifiable clusters of somata. On the basis of various landmarks, the ten arborization fields are identified as precursors of retinorecipient nuclei previously described in other adult cypriniform fishes. The development was characterized by the nearly complete absence of any transient projections. Thus, the idea that axonal outgrowth is initially exuberant and trimmed back later is not supported by these results.

Afferent Pathways↗

PubMed1995

Developmental patterning of rod and cone photoreceptors in embryonic zebrafish.

Cone photoreceptors in the zebrafish retina are arranged in a crystalline lattice, with each spectral subtype at a specific position in the array; rod photoreceptors are inserted around the cones. Patterning events and developmental mechanisms that lead to the formation of the cone mosaic are not known. To begin investigating this issue, we examined the initial stages of opsin expression in zebrafish embryos by in situ hybridization with goldfish opsin cRNA probes to determine how and when the cone mosaic pattern arises. We found both differences and similarities in the spatiotemporal patterns of rod and cone development, which suggest the following: 1) Expression of opsin message (including rod opsin, blue and red cone opsins) was found in a ventral patch of retina located nasal to the choroid fissure. 2) The cone mosaic pattern was generated by a crystallization-like process initiated in the precocial ventral patch and secondarily in nasal retina, which then swept like a wave into dorsotemporal retina. 3) The remainder of the retina, suggesting that these precocial rods might differ from typical rods. 4) Developmental maturation of rods in zebrafish, as reflected by expression of opsin, may be accelerated compared to cones, which are thought to become postmitotic before rods. These data are consistent with a model in which lateral inductive interactions among differentiating photoreceptors lead to patterning of the array.

Animals↗

PubMed2002

Morphologic transformation of the thymus in developing zebrafish.

The morphologic transformation of the developing zebrafish thymus from 1 week postfertilization (wpf) to 15 wpf is described. The thymus overall morphology changed from a small pouch-like shape at 1 wpf to a conical shape between 2 and 3 wpf before acquiring a more complex shape from 4 wpf onward. Rapid growth rate along the lateral axis at the region near the pharyngeal epithelium occurred between 1 and 2 wpf, whereas rapid growth rate along the dorsal-ventral axis occurred between 3 and 6 wpf. Expansion of thymocyte population beginning from 1 wpf became more evident by 2 to 3 wpf, as indicated by the apparent increase of different sizes of lymphocytes, recombination activating gene-1 (rag-1), and T-cell antigen receptor alpha chain constant region (TCRAC) -positive cells. Tissue section in situ hybridization (ISH) analysis with rag-1 probe reveals that cortex-medullary regionalization has begun between 1 and 2 wpf as rag-1 expression clearly demarcated the cortex, whereas the medulla was rag-1 negative. The presence of TCRAC-positive cells in the medulla by 2-3 wpf, suggests that the thymic selection processes had begun. The zebrafish thymus is morphologically mature by 3 wpf. Early signs of thymic involution were observed in zebrafish aged 15 wpf.

Animals↗

PubMed2002

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

Animals↗