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PubMed2006

Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio).

Some chemicals have the potential to adversely affect sexual development through multiple endocrine actions. Prochloraz is an imidazole fungicide that displays diverse mechanisms of action, including inhibition of aromatase activity, inhibition of androgen synthesis, and antagonism of the androgen receptor. The objective of this study was to assess the effects of prochloraz on the sexual development of zebrafish (Danio rerio) in the Fish Sexual Development Test (FSDT) proposed as an OECD test guideline for detection of endocrine disruptors. Zebrafish were exposed to prochloraz (0, 16, 64 or 202 microg/L) for 60 days from 24 h post fertilization. Fish exposed to 202 microg/L prochloraz showed an increased proportion of males. Furthermore, the incidence of intersex and the stages of the gonads were altered in the treated fish compared to the control fish. A significant vitellogenin decrease was observed in both female and male zebrafish at an exposure concentration of 202 microg/L prochloraz. However, in the male fish, significantly increased vitellogenin concentrations were observed in the groups exposed to 16 or 64 microg/L prochloraz. This study serves as a part of the validation of the FSDT and indicates that the FSDT is suitable in detecting compounds with multiple endocrine actions. This is of importance in the assessment of the potential risk of existing and new chemicals.

Animals↗

PubMed1999

Disruption of zebrafish somite development by pharmacologic inhibition of Hsp90.

Members of the Hsp90 family of molecular chaperones play important roles in allowing some intracellular signaling molecules and transcription factors to reach and maintain functionally active conformations. In the present study, we have utilized the specific Hsp90-binding agent, geldanamycin, to examine the requirement for Hsp90 during zebrafish development. We show that geldanamycin interacts with both the alpha and the beta-isoforms of zebrafish Hsp90 and that geldanamycin-treated embryos consistently exhibit a number of defects in tissues which express either one of these genes. Within the somites, geldanamycin treatment results in the absence of eng-2-expressing muscle pioneer cells. However, early development of adaxial cells, which give rise to muscle pioneers and which strongly express the hsp90alpha gene shortly before muscle pioneer formation, appeared unaffected. Furthermore, development of the notochord, which provides many of the signals required for proper somite patterning and which does not express detectable levels of either hsp90alpha or hsp90beta mRNA, was similarly unaffected in geldanamycin-treated embryos. The data are consistent with there being a temporal and spatial requirement for Hsp90 function within somitic cells which is necessary for the formation of eng-2-expressing muscle pioneers and possibly other striated muscle fiber types.

Animals↗

PubMed2002

Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish.

The expression patterns of region-specific neuroectodermal genes and fate-map analyses in zebrafish gastrulae suggest that posterior neural development is initiated by nonaxial signals, distinct from organizer-derived secreted bone morphogenetic protein (BMP) antagonists. This notion is further supported by the misexpression of a constitutively active form of zebrafish BMP type IA receptor (CA-BRIA) in the zebrafish embryos. It effectively suppressed the anterior neural marker, otx2, but not the posterior marker, hoxb1b. Furthermore, we demonstrated that the cells in the presumptive posterior neural region lose their neural fate only when CA-BRIA and Xenopus dominant-negative fibroblast growth factor (FGF) receptors (XFD) are coexpressed. The indications are that FGF signaling is involved in the formation of the posterior neural region, counteracting the BMP signaling pathway within the target cells. We then examined the functions of Fgf3 in posterior neural development. Zebrafish fgf3 is expressed in the correct place (dorsolateral margin) and at the correct time (late blastula to early gastrula stages), the same point that the most precocious posterior neural marker, hoxb1b, is first activated. Unlike other members of the FGF family, Fgf3 had little mesoderm-inducing activity. When ectopically expressed, Fgf3 expands the neural region with suppression of anterior neural fate. However, this effect was mediated by Chordino (zebrafish Chordin), because Fgf3 induces chordino expression in the epiblast and Fgf3-induced neural expansion was substantially suppressed in dino mutants with mutated chordino genes. The results obtained in the present study reveal multiple actions of the FGF signal on neural development: it antagonizes BMP signaling within posterior neural cells, induces the expression of secreted BMP antagonists, and suppresses anterior neural fate.

Animals↗

PubMed2005

Expression of cyp26b1 during zebrafish early development.

We have cloned the zebrafish ortholog of the mammalian cyp26b1 gene. The predicted zebrafish cyp26b1 protein shares greater than 73% identity with mammalian homologues. cDNA transfection assays showed that like human cyp26b1, zebrafish cyp26b1 is involved in limiting the activity of retinoic acid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of embryonic RNAs suggested that no maternal cyp26b1 message is detectable. Zygotic cyp26b1 message could be detected at 75% epiboly by RT-PCR and localized to presumptive rhombomere 3 and rhombomere 4 at the early two-somite (2S) stage (10.5 hpf: hour post fertilization) by whole mount in situ hybridization. As development proceeds expression expands anteriorly to include rhombomere 2 at the 10S stage (14hpf). By 14S (16hpf) expression in the hindbrain has also expanded posteriorly and encompasses rhombomere 2 through rhombomere 6. At later stages, 24 through 48 hpf, additional expression was found in the eyes, diencephalon, midbrain-hindbrain boundary, cerebellum, pectoral fin and the pharyngeal arch primordia.

Amino Acid Sequence↗

PubMed2000

Effects of abnormal lighting on the development of zebrafish visual behavior.

Studies across several species have demonstrated that the developing visual system is influenced by environmental conditions. This study examined the effects of abnormal lighting environments on zebrafish visual behavior. Zebrafish were raised under different lighting conditions from fertilization to 6 days postfertilization (dpf). These conditions included a normal light/dark cycle, constant dark, constant light, as well as exposure to various lighting cycles with intense light. Visual acuity was tested using the optomotor response to black and white vertical stripes of various widths. The results showed that visual acuity of the normal fish improved with age. In addition, fish raised in constant light had significantly lower visual acuity than fish raised under normal lighting conditions. Subjects raised in constant dark showed somewhat lower visual acuity than normal subjects when tested at 12-14 dpf, but the deficits were not as severe as those found in fish raised in constant light. Intense light rearing did not have any substantial effects on visual development, unless it was constant. In conclusion, under normal conditions, zebrafish visual acuity improves with age, supporting previous work on zebrafish development. In addition, zebrafish visual behavior is altered by abnormal lighting conditions; the most severe decrements occur when raised under constant light.

Animals↗

PubMed2005

Analysis of early epidermal development in zebrafish.

The zebrafish (Danio rerio) is a member of the teleost (bony fish) lineage that diverged from the mammalian lineage 420 million years ago. Despite this ancient divergence, there are important similarities between the epidermis of fish and mammals. In addition, recent work suggests that the genes required for epidermal development in the zebrafish also are essential for this process in higher vertebrates, indicating that analysis of the zebrafish system will contribute to understanding mouse and human development. The zebrafish is a very useful genetic system, and many mutants with epidermal defects have been obtained in large-scale genetic screens. In addition, zebrafish embryos are transparent, develop externally, and can be collected in abundance. The aim of this chapter is to provide the researcher with basic protocols to examine gene expression, cell proliferation, and cell death in the developing zebrafish. Although only a few specific epidermal markers have been identified in the zebrafish thus far, we also provide a list of useful genes for epidermal analysis.

Animals↗

PubMed2001

Changes in NADPH diaphorase expression in the fish visual system during optic nerve regeneration and retinal development.

The various functions of nitric oxide (NO) in the nervous system are not fully understood, including its role in neuronal regeneration. The goldfish can regenerate its optic nerve after transection, making it a useful model for studying central nervous regeneration in response to injury. Therefore, we have studied the pattern of NO expression in the retina and optic tectum after optic nerve transection, using NADPH diaphorase histochemistry. NO synthesis was transiently up-regulated in the ganglion cell bodies, peaking during the period when retinal axons reach the tectum, between 20-45 days after optic nerve transection. Enzyme activity in the tectum was transiently down-regulated and then returned to control levels at 60 days after optic nerve transection, during synaptic refinement. To compare NO expression in the developing and regenerating retina, we have looked at NO expression in the developing zebrafish retina. In the developing zebrafish retina the pattern of staining roughly followed the pattern of development with the inner plexiform layer and horizontal cells having the strongest pattern of staining. These results suggest that NO may be involved in the survival of ganglion cells in the regenerating retina, and that it plays a different role in the developing retina. In the tectum, NO may be involved in synaptic refinement.

Animals↗

PubMed2003

Hedgehog signaling is directly required for the development of zebrafish dorsal root ganglia neurons.

Hedgehog (Hh) signal transduction is directly required in zebrafish DRG precursors for proper development of DRG neurons. Zebrafish mutations in the Hh signaling pathway result in the absence of DRG neurons and the loss of expression of neurogenin1 (ngn1), a gene required for determination of DRG precursors. Cell transplantation experiments demonstrate that Hh acts directly on DRG neuron precursors. Blocking Hh pathway activation at later stages of embryogenesis with the steroidal alkaloid, cyclopamine, further reveals that the requirement for a Hh signal response in DRG precursors correlates with the onset of ngn1 expression. These results suggest that Hh signaling may normally promote DRG development by regulating expression of ngn1 in DRG precursors.

Animals↗

PubMed1999

Maturation of neuromuscular transmission during early development in zebrafish.

We have examined the rapid development of synaptic transmission at the neuromuscular junction (NMJ) in zebrafish embryos and larvae by patch-clamp recording of spontaneous miniature endplate currents (mEPCs) and single acetylcholine receptor (AChR) channels. Embryonic (24-36 h) mEPCs recorded in vivo were small in amplitude (<50 pA). The rate of mEPCs increased in larvae (3.5-fold increase measured by 6 days), and these mEPCs were mostly of larger amplitude (10-fold on average) with (</=5-fold) faster kinetics. Intracellular labeling with Lucifer yellow indicated extensive coupling between muscle cells in both embryos and larvae (</=10 days). Blocking acetylcholinesterase (AChE) with eserine had no effect on mEPC kinetics in embryos at 1 day and only partially slowed (by approximately 1/2) the decay rate in larvae at 6 days. In acutely dissociated muscle cells, we observed the same two types of AChR with conductances of 45 and 60 pS and with similar, brief (<0.5 ms) mean open times in both embryos and larvae. We conclude that AChR properties are set early during development at these early stages; functional maturation of the NMJ is only partly shaped by expression of AChE and may also depend on postsynaptic AChR clustering and presynaptic maturation.

Animals↗

PubMed2025

Uncovering parental exposure risks of TCPP: Impaired development and metabolic homeostasis in zebrafish offspring.

As brominated flame retardants are phased out, tris (1‑chloro-2-propyl) phosphate (TCPP), a phosphorus-based flame retardant, has emerged as a prominent detectable flame retardant in the environment. However, TCPP has been found to exhibit endocrine-disrupting effects on organisms, raising significant safety concerns. In our study, we utilized the zebrafish model to explore the toxic effects of parental TCPP exposure on offspring and uncover its regulatory mechanisms through metabolomics analysis. Moreover, the impact on the nervous system and lipid metabolism was examined through behavioral analysis and specific staining. Our findings demonstrated that both embryonic and parental TCPP exposure induced developmental disorders in larvae, along with decreased locomotor activity and disordered lipid metabolism homeostasis. Parental exposure to TCPP, exhibiting stronger developmental toxicity than direct embryonic exposure, notably led to reductions in crucial energy substrates such as amino acids and carbohydrates. Meanwhile, embryonic TCPP exposure primarily affected the endogenous lipid-related metabolites including phospholipids, lipid-soluble vitamins, steroids and fatty acids, promoting lipid accumulation in larval liver and subcutaneous tissue. What's more, continuously parental and embryonic exposure showed the most pronounced effects on zebrafish development and metabolic regulation. Our study highlights the risk posed by parental exposure to TCPP on offspring zebrafish, underscoring the need for comprehensive consideration of the impact from parental exposure in pollutants regulation.

Animals↗

PubMed2006

Profiling of prostanoids in zebrafish embryonic development.

Prostanoids (PG) play important roles in vascular, pulmonary, reproductive and renal physiology. Little is known about their roles in the embryonic development. Using the oviparous zebrafish embryo as a model, we determined the temporal expression of PGs synthesized from exogenous prostaglandin H(2). Prostaglandin E(2) is the major PG throughout first 120 h post-fertilization (hpf), whereas prostaglandin F(2)(alpha) is at a lower but also a constant level. Reverse transcription-polymerase chain reaction (RT-PCR) showed that transcripts of cytosolic and membrane-bound PGE synthases were evident during the 120 hpf period. Compared with thromboxane A(2), the level of prostacyclin (PGI(2))is higher at first 24 hpf, the stage before the formation of blood vessel. RT-PCR showed that transcript of prostacyclin synthase appeared at 7 hpf whereas thromboxane synthase appeared at 48 hpf, suggesting that PGI(2) has additional functions besides hemostasis. Interestingly, level of prostaglandin D(2) (PGD(2)) followed an exponential decay over 120 hpf with a rate constant of 0.048 h(-1) and transcript of lipocalin-type PGD synthase was expressed at a higher level at early stage of development, suggesting that PGD(2) is highly regulated during embryogenesis.

Animals↗

PubMed2004

Time-lapse microscopy of brain development.

Zebrafish embryos represent an ideal vertebrate model organism for noninvasive intravital imaging because of their optical clarity, external embryogenesis, and fast development. Many different labeling techniques have been adopted from other model organisms or newly developed to address a wealth of different developmental questions directly inside the living organism. The parallel advancements in the field of optical imaging let us now observe dynamic processes at the cellular and subcellular resolution. Combined with the repertoire of available surgical and genetic manipulations, zebrafish embryos provide the powerful and almost unique possibility to observe the interplay of molecular signals with cellular, morphological, and behavioral changes directly within a living and developing vertebrate organism. A bright future for zebrafish is yet to come, let there be light.

Animals↗

PubMed2003

Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish.

Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.

Amino Acid Sequence↗

PubMed1982

Differentiation of muscle fiber types in the teleost Brachydanio rerio, the zebrafish. Posthatching development.

The trunk musculature of adult zebrafishes contains three major fiber types: adult red, intermediate, and white; and two minor populations: red muscle rim and scattered intermediate fibers. In this paper, the post hatching development of these muscle fiber types was studied by means of immunohistochemistry, using anti-myosin sera. Just hatched larvae contain two muscle fiber populations: embryonic red and white, which give rise to the red muscle rim and the intermediate fibers respectively. Adult red fibers arise post hatching as a new separate population with distinct myosin properties. The differentiation of these fiber types occurs within the first four weeks after fertilization, when the adult pattern of peripheral axon bundles has become established. Differences in the muscle fiber type composition between the midbody and the tail myotomes become apparent in two month old fries. The number of scattered intermediate fibers increases from rostral to caudal, the opposite holds for the red muscle rim fibers. The red and intermediate area is triangular in the midbody; in the tail part it is stretched out along the lateral surface of the myotomes. These changes are considered as adaptations to improve the efficiency of the swimming performance.

Animals↗

PubMed2005

Development of zebrafish (Danio rerio) pectoral fin musculature.

During posthatching development the fins of fishes undergo striking changes in both structure and function. In this article we examine the development of the pectoral fins from larval through adult life history stages in the zebrafish (Danio rerio), describing in detail their pectoral muscle morphology. We explore the development of muscle structure as a way to interpret the fins' role in locomotion. Genetic approaches in the zebrafish model are providing new tools for examining fin development and we take advantage of transgenic lines in which fluorescent protein is expressed in specific tissues to perform detailed three-dimensional, in vivo fin imaging. The fin musculature of larval zebrafish is organized into two thin sheets of fibers, an abductor and adductor, one on each side of an endoskeletal disk. Through the juvenile stage the number of muscle fibers increases and muscle sheets cleave into distinct muscle subdivisions as fibers orient to the developing fin skeleton. By the end of the juvenile period the pectoral girdle and fin muscles have reoriented to take on the adult organization. We find that this change in morphology is associated with a switch of fin function from activity during axial locomotion in larvae to use in swim initiation and maneuvering in adults. The examination of pectoral fins of the zebrafish highlights the yet to be explored diversity of fin structure and function in subadult developmental stages. J. Morphol. (c) 2005 Wiley-Liss, Inc.

Animals↗

PubMed2001

evx1 transcription in bony fin rays segment boundaries leads to a reiterated pattern during zebrafish fin development and regeneration.

The dermoskeleton of zebrafish fins is composed of actinotrichia and segmented bony rays, or lepidotrichia, which grow by successive addition of distal segments. The present study shows that evx1, a new zebrafish even-skipped related gene (Thaëron et al., 2000) displays during bony ray morphogenesis, a unique repetitive expression pattern along the proximodistal axis of the fin. Whole-mount in situ hybridization performed on larvae and adult regenerating fins show that evx1 signal appears as parallel dash lines crossing the width of each developing and regenerating rays, in a ladder-like fashion. Cytological studies show that a subpopulation of bone forming cells (scleroblasts) expresses evx1 at the level of the joint between two adjacent segments except in the apical part of the differentiating ray where evx1 expression precedes the formation of the joint. This distal transcription is turned on again only when the latest differentiating segment reached its final size and might label the putative next segment boundary. This suggests the existence of a molecular mechanism controlling the periodic expression of evx1 which could be involved in the establishment of segment boundaries during fin ray morphogenesis, and could play a key role during dermal skeleton patterning.

Amputation, Surgical↗

PubMed1998

Conservation of PDX-1 structure, function, and expression in zebrafish.

Development of the mammalian pancreas has been studied extensively in mice. The stages from budding of the pancreatic anlaga through endocrine and exocrine cell differentiation and islet formation have been described in detail. Recently, the homeodomain transcription factor PDX-1 has been identified as an important factor in the proliferation and differentiation of the pancreatic buds to form a mature pancreas. To evaluate the possibility of using zebrafish as a model for the genetic analysis of pancreas development, we have cloned and characterized PDX-1 from this organism. The deduced sequence of zebrafish PDX-1 contains 246 amino acids and is 95% identical to mammalian PDX-1 in the homeodomain. We also cloned zebrafish preproinsulin complementary DNA as a marker for islet tissue. By in situ hybridization we demonstrate that PDX-1 and insulin are coexpressed during embryonic development and in adults, although PDX-1 expression appears to be biphasic. Insulin expression apparently begins before 44 hpf, the earliest stage examined in this study. Additionally, very high levels of PDX-1 expression were observed in the pyloric caeca, the accessory digestive organs that also are derived from the proximal region of the intestine in teleosts. Finally, our data show that the evolutionary conservation of zebrafish PDX-1 extends to its DNA binding properties. Zebrafish PDX-1 was equally as effective as mouse PDX-1 in stimulating insulin gene transcription, and maximum promoter activation was dependent on the presence of four intact A elements. The demonstration of this capability suggests that transcriptional regulatory mechanisms that control pancreatic development and insulin gene expression have been conserved among vertebrates.

Amino Acid Sequence↗

PubMed2002

Molecular dissection of craniofacial development using zebrafish.

The zebrafish, Danio rerio, is a small, freshwater teleost that only began to be used as a vertebrate genetic model by the late George Streisinger in the early 1980s. The strengths of the zebrafish complement genetic studies in mice and embryological studies in avians. Its advantages include high fecundity, externally fertilized eggs and transparent embryos that can be easily manipulated, inexpensive maintenance, and the fact that large-scale mutagenesis screens can be performed. Here we review studies that have used the zebrafish as a model for craniofacial development. Lineage studies in zebrafish have defined the origins of the cranial skeleton at the single-cell level and followed the morphogenetic behaviors of these cells in skeletal condensations. Furthermore, genes identified by random mutational screening have now revealed genetic pathways controlling patterning of the jaw and other pharyngeal arches, as well as the midline of the skull, that are conserved between fish and humans. We discuss the potential impact of specialized mutagenesis screens and the future applications of this versatile, vertebrate developmental model system in the molecular dissection of craniofacial development.

Animals↗