Histological development and integration of the Zebrafish Weberian apparatus.

BACKGROUND: The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability. RESULTS: Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period. CONCLUSIONS: This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality.

PRPS polymerization influences lens fiber organization in zebrafish.

BACKGROUND: The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown. RESULTS: By generating novel alleles in the zebrafish PRPS paralogs, prps1a and prps1b, we gained new insight into the role of PRPS filaments during eye development. We found that mutations in prps1a alone are sufficient to generate abnormally small eyes along with defects in head size, pigmentation, and swim bladder inflation. Furthermore, a loss-of-function mutation that truncates the Prps1a protein resulted in the failure of PRPS filament assembly. Lastly, in mutants that fail to assemble PRPS filaments, we observed disorganization of the actin network in the lens fibers. CONCLUSIONS: The truncation of Prps1a blocked PRPS filament formation and resulted in a disorganized lens fiber actin network. Altogether, these findings highlight a potential role for PRPS filaments during lens fiber organization in zebrafish.

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes.

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe-finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray-finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.

Dorsoventral inversion of the air-filled organ (lungs, gas bladder) in vertebrates: RNAsequencing of laser capture microdissected embryonic tissue.

How modification of gene expression generates novel traits is key to understanding the evolutionary process. We investigated the genetic basis for the origin of the piscine gas bladder from lungs of ancestral bony vertebrates. Distinguishing these homologous organs is the direction of budding from the foregut during development; lungs bud ventrally and the gas bladder buds dorsally.

Sex-specific endocrine-disrupting effects of three halogenated chemicals in Japanese medaka.

Several halogenated chemicals are found in an array of products that can cause endocrine disruption. Human studies have shown that endocrine responses are sex specific, with females more likely to develop hypothyroidism and males more likely to have reproductive impairment. The objective of this study was to assess sex differences on thyroid and estrogenic effects after exposure of Japanese medaka (Oryzias latipes, SK2MC) to halogenated compounds. This strain is an excellent model for these studies as sex can be determined non-destructively a few hours postfertilization. Medaka embryos were exposed to sublethal concentrations of Tris(1,3-dichloro-2-propyl) phosphate (TDCPP, 0.019 mg/L), perfluorooctanoic acid (PFOA, 4.7 mg/L) and its next generation alternative, perfluorobutyric acid (PFBA, 137 mg/L). Methimazole (inhibits thyroid hormone synthesis) and the thyroid hormone triiodothyronine served as reference controls. Fish were exposed throughout embryo development until 10 days postfertilization. Females displayed significantly larger swim bladders (which are under thyroid hormone control) after exposure to all chemicals with the exception of triiodothyronine, which caused the opposite effect. Females exposed to TDCPP and PFOA had increased expression of vitellogenin and exposure to PFOA upregulated expression of multiple thyroid-related genes. Upregulation of estrogenic-regulated genes after exposure to TDCPP, PFOA and methimazole was only observed in males. Overall, our results suggest that females and males show an estrogenic response when exposed to these halogenated chemicals and that females appear more susceptible to thyroid-induced swim bladder dysfunction compared with males. These results further confirm the importance of considering sex effects when assessing the toxicity of endocrine-disrupting compounds.

The Air Sac Primordium of Drosophila: A Model for Invasive Development.

The acquisition of invasive properties preceding tumor metastasis is critical for cancer progression. This phenomenon may result from mutagenic disruption of typical cell function, but recent evidence suggests that cancer cells frequently co-opt normal developmental programs to facilitate invasion as well. The signaling cascades that have been implicated present an obstacle to identifying effective therapeutic targets because of their complex nature and modulatory capacity through crosstalk with other pathways. Substantial efforts have been made to study invasive behavior during organogenesis in several organisms, but another model found in Drosophilamelanogaster has not been thoroughly explored. The air sac primordium (ASP) appears to be a suitable candidate for investigating the genes and morphogens required for invasion due to the distinct overlap in the events that occur during its normal growth and the development of metastatic tumor cells. Among these events are the conversion of larval cells in the trachea into a population of mitotically active cells, reduced cell⁻cell contact along the leading edge of the ASP, and remodeling of the extracellular matrix (ECM) that surrounds the structure. Here, we summarize the development of ASPs and invasive behavior observed therein.

Histological Aspects of the Early Development of the Digestive System of Burbot Lota lota L. (Lotidae, Gadiformes).

The ontogeny of the digestive tract was studied histologically in burbot, Lota lota L., from hatching to 42 days post-hatch (dph). At hatching, the digestive tract consisted of a straight tube with discernible digestive accessory glands (the liver and the pancreas) dorsally attached to the yolk sac. Most of the yolk sac reserves were consumed during the first 12 days and were completely depleted by 17 dph. The first PAS-positive goblet cells appeared at 6 dph, dispersed within the epithelium of the oesophagus and increasing substantially in number and distribution as development progressed. At 12 dph, the first vacuoles (neutral lipids) appeared in the intestine, indicating the functional absorption of nutrients from food. Differentiation of gastric glands was first noticed at 17 dph and was extensive by 27 dph. L. lota larvae have a morphologically complete digestive tract by 32 dph. These findings on the development of the digestive system in L. lota may contribute to a better understanding of its ontogeny and can be useful for improvement of the larval rearing techniques of this promising species for freshwater aquaculture diversification.

The transcription factor Atonal homolog 8 regulates Gata4 and Friend of Gata-2 during vertebrate development.

GATA and Friend of GATA (FOG) form a transcriptional complex that plays a key role in cardiovascular development in both fish and mammals. In the present study we demonstrate that the basic helix-loop-helix transcription factor Atonal homolog 8 (Atoh8) is required for development of the heart in fish but not in mice. Genetic studies reveal that Atoh8 interacts specifically with Gata4 and Fog1 during development of the heart and swim bladder in the fish. Biochemical studies reveal that ATOH8, GATA4, and FOG2 associate in a single complex in vitro. In contrast to fish, ATOH8-deficient mice exhibit normal cardiac development and loss of ATOH8 does not alter cardiac development in Gata4(+/-) mice. This species difference in the role of ATOH8 is explained in part by LacZ and GFP reporter alleles that reveal restriction of Atoh8 expression to atrial but not ventricular myocardium in the mouse. Our findings identify ATOH8 as a novel regulator of GATA-FOG function that is required for cardiac development in the fish but not the mouse. Whether ATOH8 modulates GATA-FOG function at other sites or in more subtle ways in mammals is not yet known.

Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants.

Wnt signaling plays critical roles in development of both tetrapod lung and fish swimbladder, which are the two evolutionary homologous organs. Our previous data reveal that down-regulation of Wnt signaling leads to defective swimbladder development. However, the effects of up-regulation of Wnt signaling on swimbladder development remain unclear. By knockdown of the Wnt protein inhibitory gene wif1, we demonstrated that up-regulation of Wnt signaling also resulted in perturbed development of the swimbladder. Specifically, the growth of epithelium and mesenchyme was greatly inhibited, the smooth muscle differentiation was abolished, and the organization of mesothelium was disturbed. Furthermore, our data reveal that it is the reduced cell proliferation, but not enhanced apoptosis, that contributes to the disturbance of swimbladder development in wif1 morphants. Blocking Wnt signaling by the Wnt antagonist IWR-1 did not affect wif1 expression in the swimbladder, but complete suppression of Hedgehog signaling in smo-/- mutants abolished wif expression, consistent with our earlier report of a negative feedback regulation of Wnt signaling in the swimbladder by the Hedgehog signaling. Our works established the importance of proper level of Wnt signaling for normal development of swimbladder in zebrafish.

The interaction of epithelial Ihha and mesenchymal Fgf10 in zebrafish esophageal and swimbladder development.

Developmental patterning and growth of the vertebrate digestive and respiratory tracts requires interactions between the epithelial endoderm and adjacent mesoderm. The esophagus is a specialized structure that connects the digestive and respiratory systems and its normal development is critical for both. Shh signaling from the epithelium regulates related aspects of mammalian and zebrafish digestive organ development and has a prominent effect on esophageal morphogenesis. The mechanisms underlying esophageal malformations, however, are poorly understood. Here, we show that zebrafish Ihha signaling from the epithelium acting in parallel, but independently of Shh, controls epithelial and mesenchymal cell proliferation and differentiation of smooth muscles and neurons in the gut and swimbladder. In zebrafish ihha mutants, the esophageal and swimbladder epithelium is dysmorphic, and expression of fgf10 in adjacent mesenchymal cells is affected. Analysis of the development of the esophagus and swimbladder in fgf10 mutant daedalus (dae) and compound dae/ihha mutants shows that the Ihha-Fgf10 regulatory interaction is realized through a signaling feedback loop between the Ihha-expressing epithelium and Fgf10-expressing mesenchyme. Disruption of this loop further affects the esophageal and swimbladder epithelium in ihha mutants, and Ihha acts in parallel to but independently of Shha in this process. These findings contribute to the understanding of epithelial-mesenchymal interactions and highlight an interaction between Hh and Fgf signaling pathways during esophagus and swimbladder development.

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish.

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on ß-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

Early development of New Zealand hapuku Polyprion oxygeneios eggs and larvae.

This study describes for the first time the normal development of New Zealand hapuku Polyprion oxygeneios embryos and larvae reared from fertilization to 11 days post-hatch (dph) at a constant temperature. Fertilized eggs were obtained from natural spawnings from communally reared captive wild broodstock. Eggs averaged 2 mm in diameter and had single or multiple oil globules. Embryos developed following the main fish embryological stages and required an average of 1859·50 degree hours post-fertilization (dhpf) to hatch. The newly hatched larvae (4·86 mm mean total length, L(T) ) were undifferentiated, with unpigmented eyes, a single and simple alimentary tube and a finfold that covered the entire body. Larvae relied on the energy from the yolk-sac reserves until 11 dph (7·33 mm mean L(T) ), when yolk-sac reabsorption was almost completed. Some of the major developmental stages from hatching to yolk-sac reabsorption were eye pigmentation (5 dph), upper jaw formation (7 dph), lower jaw formation (8 dph) and mouth opening (8-9 dph). By 9 dph, the digestive system consisted of pancreas, liver, primordial stomach, anterior and posterior gut; therefore, P. oxygeneios larvae would be capable of feeding on live prey. The developmental, morphological and histological data described constitutes essential baseline information on P. oxygeneios biology and normal development.

Pbx1 is essential for growth of zebrafish swim bladder.

pbx1, a TALE (three-amino acid loop extension) homeodomain transcription factor, is involved in a diverse range of developmental processes. We examined the expression of pbx1 during zebrafish development by in situ hybridization. pbx1 transcripts could be detected in the central nervous system and pharyngeal arches from 24 hpf onwards. In the swim bladder anlage, pbx1 was detected as early as 28 hpf, making it the earliest known marker for this organ. Morpholino-mediated gene knockdown of pbx1 revealed that the swim bladder failed to inflate, with eventual lethality occurring by 8 dpf. The knockdown of pbx1 did not perturb the expression of prdc and foxA3, with both early swim bladder markers appearing normally at 36 and 48 hpf, respectively. However, the expression of anxa5 was completely abolished by pbx1 knockdown at 60 hpf suggesting that pbx1 may be required during the late stage of swim bladder development.

Regulation of Drosophila matrix metalloprotease Mmp2 is essential for wing imaginal disc:trachea association and air sac tubulogenesis.

The Drosophila Dorsal Air Sac Primordium (ASP) is a tracheal tube that grows toward Branchless FGF-expressing cells in the wing imaginal disc. We show that the ASP arises from a tracheal branch that invades the basal lamina of the disc to juxtapose directly with disc cells. We examined the role of matrix metalloproteases (Mmps), and found that reducing Mmp2 activity perturbed disc-trachea association, altered peritracheal distributions of collagen IV and Perlecan, misregulated ASP growth, and abrogated development of the dorsal air sacs. Whereas the function of the membrane-tethered Mmp2 in the ASP is non-cell autonomous we find that it may have distinct tissue-specific roles in the ASP and disc. These findings demonstrate a critical role for Mmp2 in tubulogenesis post-induction, and implicate Mmp2 in regulating dynamic and essential changes to the extracellular matrix.

Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers.

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)(sq33-2) and Et(krt4:EGFP)(sqet3) that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.

The role of vasculature and blood circulation in zebrafish swimbladder development.

BACKGROUND: Recently we have performed a detailed analysis of early development of zebrafish swimbladder, a homologous organ of tetrapod lung; however, the events of swimbladder development are still poorly characterized. Many studies have implicated the role of vascular system in development of many organs in vertebrates. As the swimbladder is lined with an intricate network of blood capillaries, it is of interest to investigate the role of the vascular system during early development of swimbladder. RESULTS: To investigate the role of endothelial cells (ECs) and blood circulation during development of the swimbladder, phenotypes of swimbladder were analysed at three different stages (approximately 2, 3 and 5 dpf [day postfertilization]) in cloche (clo) mutant and Tnnt2 morphants, in the background of transgenic lines Et(krt4:EGFP)sq33-2 and Et(krt4:EGFP)sqet3 which express EGFP in the swimbladder epithelium and outer mesothelium respectively. Analyses of the three tissue layers of the swimbladder were performed using molecular markers hb9, fgf10a, acta2, and anxa5 to distinguish epithelium, mesenchyme, and outer mesothelium. We showed that the budding stage was independent of ECs and blood flow, while early epithelial growth, mesenchymal organization and its differentiation into smooth muscle, as well as outer mesothelial organization, were dependent on ECs. Blood circulation contributed to later stage of epithelial growth, smooth muscle differentiation, and organization of the outer mesothelium. Inflation of the swimbladder was also affected as a result of absence of ECs and blood flow. CONCLUSION: Our data demonstrated that the vascular system, though not essential in swimbladder budding, plays an important role in the development of the swimbladder starting from the early growth stage, including mesenchyme organization and smooth muscle differentiation, and outer mesothelial organization, which in turn may be essential for the function of the swimbladder as reflected in its eventual inflation.

The expression of zebrafish NAD(P)H:quinone oxidoreductase 1(nqo1) in adult organs and embryos.

NQO1, NAD(P)H: quinone oxidoreductase 1, was first identified in rat and its role has been extensively studied. Even the roles of NQO1 in the maintenance of physiological function and disease were largely addressed, whether the tissue specific functions of the NQO1 in organ development remains unknown. In the current study, we identified two NQO1 isoforms (isoform 1 and isoform 2) and examined the expression of nqo1 variants in adult zebrafish organs and embryos at different stages. In adult organs, RT-PCR result indicated that nqo1 variant 1 was mainly expressed in stomach and intestine, while nqo1 variant 2 was expressed in all organs investigated except for heart. Further, RT-PCR result showed that the nqo1 variant 1 and variant 2 were expressed at all the embryonic stages, but nqo1 variant 1 expression level was much lower than that of nqo1 variant 2. To specifically examine the expression pattern of these two different nqo1 variants, we did whole mount in situ hybridization and the results demonstrated that, both of them were maternally expressed at 8-cell stage, and they were all expressed ubiquitously at early stage. At 24 hpf, nqo1 variant 2 was mainly expressed in yolk cells, and slightly in head and eyes. At 48 hpf, nqo1 variant 2 was restricted in lateral line neuromasts. From 72 hpf to 144 hpf, nqo1 variant 2 was mainly restricted in branchial arch, liver, swimming bladder and lateral line neuromasts, while from 124 hpf to 192 hpf, nqo1 variant 2 only restricted in liver, and disappeared in lateral line neuromasts. On the contrary, at the late embryonic stage, nqo1 variant 1 was only expressed in liver and swimming bladder while not in branchial arch and lateral line neuromasts. In conclusion, we systematically analyzed the expression pattern of nqo1 variant 1 and variant 2 in zebrafish at different embryonic stages, and our data implied the possible role of nqo1 in regulating liver, branchial arch and lateral neuromasts development.

Evolution of Shh endoderm enhancers during morphological transition from ventral lungs to dorsal gas bladder.

Shh signalling plays a crucial role for endoderm development. A Shh endoderm enhancer, MACS1, is well conserved across terrestrial animals with lungs. Here, we first show that eliminating mouse MACS1 causes severe defects in laryngeal development, indicating that MACS1-directed Shh signalling is indispensable for respiratory organogenesis. Extensive phylogenetic analyses revealed that MACS1 emerged prior to the divergence of cartilaginous and bony fishes, and even euteleost fishes have a MACS1 orthologue. Meanwhile, ray-finned fishes evolved a novel conserved non-coding sequence in the neighbouring region. Transgenic assays showed that MACS1 drives reporter expression ventrally in laryngeal epithelium. This activity has been lost in the euteleost lineage, and instead, the conserved non-coding sequence of euteleosts acquired an enhancer activity to elicit dorsal epithelial expression in the posterior pharynx and oesophagus. These results implicate that evolution of these two enhancers is relevant to the morphological transition from ventral lungs to dorsal gas bladder.

Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish.

Endocrine disrupting chemicals (EDCs) can alter thyroid function and adversely affect growth and development. Halogenated compounds, such as perfluorinated chemicals commonly used in food packaging, and brominated flame retardants used in a broad range of products from clothing to electronics, can act as thyroid disruptors. Due to the adverse effects of these compounds, there is a need for the development of safer next generation chemicals. The objective of this study was to test the thyroid disruption potential of old use and next generation halogenated chemicals. Zebrafish embryos were exposed to three old use compounds, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) and two next generation chemicals, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxdie (DOPO) and perfluorobutyric acid (PFBA). Sub-chronic (0-6days post fertilization (dpf)) and chronic (0-28dpf) exposures were conducted at 1% of the concentration known to kill 50% (LC50) of the population. Changes in the surface area of the swim bladder as well as in expression levels of genes involved in the thyroid control of swim bladder inflation were measured. At 6dpf, zebrafish exposed to all halogenated chemicals, both old use and next generation, had smaller posterior swim bladder and increased expression in the gene encoding thyroid peroxidase, tpo and the genes encoding two swim bladder surfactant proteins, sp-a and sp-c. These results mirrored the effects of thyroid hormone-exposed positive controls. Fish exposed to a TPO inhibitor (methimazole, MMI) had a decrease in tpo expression levels at 28dpf. Effects on the anterior swim bladder at 28dpf, after exposure to MMI as well as both old and new halogenated chemicals, were the same, i.e., absence of SB in ∼50% of fish, which were also of smaller body size. Overall, our results suggest thyroid disruption by the halogenated compounds tested via the swim bladder surfactant system. However, with the exception of TBBPA and TDCPP, the concentrations tested (∼5-137ppm) are not likely to be found in the environment.

Copper impairs zebrafish swimbladder development by down-regulating Wnt signaling.

Copper nanoparticles (CuNPs) are used widely in different fields due to their attractive and effective abilities in inhibiting bacteria and fungi, but little information is available about their biological effects and potential molecular mechanisms on fish development. Here, CuNPs and copper (II) ions (Cu2+) were revealed to inhibit the specification and formation of three layers of zebrafish embryonic posterior swimbladder and impair its inflation in a stage-specific manner. CuNPs and Cu2+ were also revealed to down-regulate Wnt signaling in embryos. Furthermore, Wnt agonist 6-Bromoindirubin-3'-oxime (BIO) was found to neutralize the inhibiting effects of CuNPs or Cu2+ or both on zebrafish swimbladder development. The integrated data here provide the first evidence that both CuNPs and Cu2+ act on the specification and growth of the three layers of swimbladder and inhibit its inflation by down-regulating Wnt signaling in a stage-specific manner during embryogenesis.