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Background: Pharyngeal arches (PA) are sequentially generated in an anterior-to-posterior order. Ripply3 is essential for posterior PA development in mouse embryos and its expression is sequentially activated in ectoderm and endoderm prior to formation of each PA. Since the PA phenotype of Ripply3 knockout (KO) mice is similar to that of retinoic acid (RA) signal-deficient embryos, we investigated the relationship between RA signaling and Ripply3 in mouse embryos., Results: In BMS493 (pan-RAR antagonist) treated embryos, which are defective in third and fourth PA development, Ripply3 expression is decreased in the region posterior to PA2 at E9.0. This expression remains and its distribution is expanded posteriorly at E9.5. Conversely, high dose RA exposure does not apparently change its expression at E9.0 and 9.5. Knockout of retinaldehyde dehydrogenase 2 (Raldh2), which causes more severe PA defect, attenuates sequential Ripply3 expression at PA1 and reduces its expression level. EGFP reporter expression driven by a 6 kb Ripply3 promoter fragment recapitulates the endogenous Ripply3 mRNA expression during PA development in wild-type, but its distribution is expanded posteriorly in BMS493-treated and Raldh2 KO embryos., Conclusion: Spatio-temporal regulation of Ripply3 expression by RA signaling is indispensable for the posterior PA development in mouse., (© 2021 American Association of Anatomists.)

Information flow through neural circuits often requires their organization into topographic maps in which the positions of cell bodies and synaptic targets correspond. To understand how topographic map development is controlled, we examine the mechanism underlying targeting of vagus motor axons to the pharyngeal arches in zebrafish. We reveal that retinoic acid organizes topography by specifying anterior-posterior identity in vagus motor neurons. We then show that chemoattractant signaling between Hgf and Met is required for vagus innervation of the pharyngeal arches. Finally, we find that retinoic acid controls the spatiotemporal dynamics of Hgf/Met signaling to coordinate axon targeting with the developmental progression of the pharyngeal arches and show that experimentally altering the timing of Hgf/Met signaling is sufficient to redirect axon targeting and disrupt the topographic map. These findings establish a mechanism of topographic map development in which the regulation of chemoattractant signaling in space and time guides axon targeting., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aquaporins (AQPs) facilitate transmembrane water and solute transport, and in addition to contributing to transepithelial water transport, they safeguard cell volume homeostasis. This study examined the expression and localization of AQP1 and AQP3 in the gills of Japanese medaka ( Oryzias latipes) in response to osmotic challenges and osmoregulatory hormones, cortisol, and prolactin (PRL). AQP3 mRNA was inversely regulated in response to salinity with high levels in ion-poor water (IPW), intermediate levels in freshwater (FW), and low levels in seawater (SW). AQP3 protein levels decreased upon SW acclimation. By comparison, AQP1 expression was unaffected by salinity. In ex vivo gill incubation experiments, AQP3 mRNA was stimulated by PRL in a time- and dose-dependent manner but was unaffected by cortisol. In contrast, AQP1 was unaffected by both PRL and cortisol. Confocal microscopy revealed that AQP3 was abundant in the periphery of gill filament epithelial cells and co-localized at low intensity with Na + ,K + -ATPase in ionocytes. AQP1 was present at a very low intensity in most filament epithelial cells and red blood cells. No epithelial cells in the gill lamellae showed immunoreactivity to AQP3 or AQP1. We suggest that both AQPs contribute to cellular volume regulation in the gill epithelium and that AQP3 is particularly important under hypo-osmotic conditions, while expression of AQP1 is constitutive.

Feminizing endocrine disrupting compounds (EDCs) affect the growth and development of teleost fishes. The major regulator of growth performance, the growth hormone (Gh)/insulin-like growth-factor (Igf) system, is sensitive to estrogenic compounds and mediates certain physiological and potentially behavioral consequences of EDC exposure. Igf binding proteins (Igfbps) are key modulators of Igf activity, but their alteration by EDCs has not been examined. We investigated two life-stages (fry and smolts) of Atlantic salmon (Salmo salar), and characterized how the Gh/Igf/Igfbp system responded to waterborne 17α-ethinylestradiol (EE 2 ), 17β-estradiol (E 2 ) and 4-nonylphenol (NP). Fry exposed to EE 2 and NP for 21 days had increased hepatic vitellogenin (vtg) mRNA levels while hepatic estrogen receptor α (erα), gh receptor (ghr), igf1 and igf2 mRNA levels were decreased. NP-exposed fry had reduced body mass and total length compared to controls. EE 2 and NP reduced hepatic igfbp1b1, -2a, -2b1, -4, -5b2 and -6b1, and stimulated igfbp5a. In smolts, hepatic vtg mRNA levels were induced following 4-day exposures to all three EDCs, while erα only responded to EE 2 and E 2 . EDC exposures did not affect body mass or fork length; however, EE 2 diminished plasma Gh and Igf1 levels in parallel with reductions in hepatic ghr and igf1. In smolts, EE 2 and E 2 diminished hepatic igfbp1b1, -4 and -6b1, and stimulated igfbp5a. There were no signs of compromised ionoregulation in smolts, as indicated by unchanged branchial ion pump/transporter mRNA levels. We conclude that hepatic igfbps respond (directly and/or indirectly) to environmental estrogens during two key life-stages of Atlantic salmon, and thus may modulate the growth and development of exposed individuals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies/deafness (CHARGE) syndrome is a congenital disorder affecting multiple organs and mainly caused by mutations in CHD7, a gene encoding a chromatin-remodeling protein. Immunodeficiency and reduced T cells have been noted in CHARGE syndrome. However, the mechanisms underlying T lymphopenia are largely unexplored. Herein, we observed dramatic decrease of T cells in both chd7knockdown and knockout zebrafish embryos. Unexpectedly, hematopoietic stem and progenitor cells and, particularly, lymphoid progenitor cells were increased peripherally in nonthymic areas in chd7-deficient embryos, unlikely to contribute to the T-cell decrease. Further analysis demonstrated that both the organogenesis and homing function of the thymus were seriously impaired. Chd7 might regulate thymus organogenesis through modulating the development of both neural crest cell-derived mesenchyme and pharyngeal endoderm-derived thymic epithelial cells. The expression of foxn1, a central regulator of thymic epithelium, was remarkably down-regulated in the pharyngeal region in chd7-deficient embryos. Moreover, the T-cell reduction in chd7-deficient embryos was partially rescued by overexpressing foxn1, suggesting that restoring thymic epithelium may be a potential therapeutic strategy for treating immunodeficiency in CHARGE syndrome. Collectively, the results indicated that chd7 was critical for thymic development and T-lymphopenia in CHARGE syndrome may be mainly attributed to the defects of thymic organogenesis. The current finding may benefit the diagnosis and therapy of T lymphopenia and immunodeficiency in CHARGE syndrome., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Defects in development of the secondary palate, which arise from the embryonic first branchial arch (1-BA), can cause cleft palate (CP). Administration of 5-Aza-2'-deoxycytidine (AzaD), a demethylating agent, to pregnant mice on gestational day 9.5 resulted in complete penetrance of CP in fetuses. Several genes critical for normal palatogenesis were found to be upregulated in 1-BA, 12h after AzaD exposure. MethylCap-Seq (MCS) analysis identified several differentially methylated regions (DMRs) in DNA extracted from AzaD-exposed 1-BAs. Hypomethylated DMRs did not correlate with the upregulation of genes in AzaD-exposed 1-BAs. However, most DMRs were associated with endogenous retroviral elements. Expression analyses suggested that interferon signaling was activated in AzaD-exposed 1-BAs. Our data, thus, suggest that a 12-h in utero AzaD exposure demethylates and activates endogenous retroviral elements in the 1-BA, thereby triggering an interferon-mediated response. This may result in the dysregulation of key signaling pathways during palatogenesis, causing CP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dissolved organic matter (DOM) is both ubiquitous and diverse in composition in natural waters, but its effects on the branchial physiology of aquatic organisms have received little attention relative to other variables (e.g. pH, hardness, salinity, alkalinity). Here, we investigated the effects of four chemically distinct DOM isolates (three natural, one commercial, ranging from autochthonous to highly allochthonous, all at ∼6 mg C l(-1)) on the physiology of gill ionoregulation and nitrogenous waste excretion in zebrafish acclimated to either circumneutral (7.0-8.0) or acidic pH (5.0). Overall, lower pH tended to increase net branchial ammonia excretion, net K(+) loss and [(3)H]PEG-4000 clearance rates (indicators of transcellular and paracellular permeability, respectively). However, unidirectional Na(+) efflux, urea excretion and drinking rates were unaffected. DOM sources tended to stimulate unidirectional Na(+) influx rate and exerted subtle effects on the concentration-dependent kinetics of Na(+) uptake, increasing maximum transport capacity. All DOM sources reduced passive Na(+) efflux rates regardless of pH, but exerted negligible effects on nitrogenous waste excretion, drinking rate, net K(+) loss or [(3)H]PEG-4000 clearance, so the mechanism of Na(+) loss reduction remains unclear. Overall, these actions appear beneficial to ionoregulatory homeostasis in zebrafish, and some may be related to physico-chemical properties of the DOM sources. They are very different from those seen in a recent parallel study on Daphnia magna using the same DOM isolates, indicating that DOM actions may be both species and DOM specific., (© 2016. Published by The Company of Biologists Ltd.)

Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80μg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study examined the distribution and orientation of gill O(2) chemoreceptors in Oreochromis niloticus and their role in cardiorespiratory responses to graded hypoxia. Intact fish, and a group with the first gill arch excised (operated), were submitted to graded hypoxia and their cardiorespiratory responses (oxygen uptake - V˙O(2) , breathing frequency - fR, ventilatory stroke volume - VT, gill ventilation - V˙G, O(2) extraction from the ventilatory current - EO(2) , and heart rate - fH) were compared. Their responses to bolus injections of NaCN into the bloodstream (internal) or ventilatory water stream (external) were also determined. The V˙O(2) of operated fish was significantly lower at the deepest levels of hypoxia. Neither reflex bradycardia nor ventilatory responses were completely abolished by bilateral excision of the first gill arch. EO(2) of the operated group was consistently lower than the intact group. The responses to internal and external NaCN included transient decreases in fH and increases in fR and Vamp (ventilation amplitude). These cardiorespiratory responses were attenuated but not abolished in the operated group, indicating that chemoreceptors are not restricted to the first gill arch, and are sensitive to oxygen levels in both blood and water., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The purpose of the present study was to identify the potential effect of prenatal vitamin B12 administration on retinoic acid (RA)-induced early craniofacial abnormalities in mice and to investigate the possible mechanisms by which vitamin B12 reduces malformations., Design: In our study, whole embryo culture was used to explore the effect of vitamin B12 on mouse embryos during the critical period of organogenesis. All embryos were exposed to 0.4 µM RA and different concentrations of vitamin B12 and scored for their growth in the branchial region at the end of a 48-hour culture period. The endothelin-1 (ET-1)/dHAND protein expression levels in the first branchial arch were investigated using an immunohistochemical method., Results: In the whole embryo culture, 100 and 10 µM vitamin B12 dose-dependently prevented branchial region malformations and decreased craniofacial defects by 90.5% and 77.3%, respectively. ET-1 and dHAND protein levels were significantly increased in vitamin B12-supplemented embryos compared to the RA-exposed group in embryonic branchial region., Conclusions: These results suggest that vitamin B12 may prevent RA-induced craniofacial abnormalities via prevention of an RA-induced decrease of ET-1 and dHAND protein levels in the branchial region during the organogenic period. This study may shed new light on preventing craniofacial abnormalities.

Three groups of compounds: (i) active peroxides (artemisinin and arterolene), (ii) inactive non-peroxidic derivatives (deoxyartemisinin and carbaOZ277) and (iii) inactive peroxide (OZ381) were tested by WEC system to provide insights into the relationship between chemical structure and embryotoxic potential, and to assess the relationship between embryotoxicity and antimalarial activity. Deoxyartemisinin, OZ381 and carbaOZ277 did not affect rat embryonic development. Artemisinin and arterolane affected primarily nucleated red blood cells (RBCs), inducing anemia and subsequent tissue damage in rat embryos, with NOELs for RBC damage at 0.1 and 0.175μg/mL, respectively. These data support the idea that only active antimalarial peroxides are able to interfere with normal embryonic development. In an attempt to establish whether and to what extent activity as antimalarials and embryotoxicity can be divorced, IC(50)s for activity in Plasmodium falciparum strains and the NOELs for RBCs were compared. From this comparison, arterolane showed a better safety margin than artemisinin., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Zebrafish tgfbeta3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfbeta3 in head skeletal formation, we knocked down tgfbeta3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfbeta3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfbeta3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfbeta3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfbeta3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfbeta3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfbeta3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfbeta3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfbeta3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfbeta3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfbeta3 protein levels in zebrafish., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

In this study the effects of growth hormone (GH) on silver sea bream branchial heat-shock protein 70 (HSP70) expression was investigated using in-vivo and in-vitro experiments. For in-vivo experiments, sea bream were administered recombinant bream GH or the GH secretagogue hexarelin. Pituitary levels of GH were unchanged in fish administered exogenous GH but decreased on hexarelin administration, in comparison with saline controls. Levels of HSP70 were measured using immunoanalysis and it was found that both GH and hexarelin administration caused a significant decrease in branchial HSP70 abundance. For in-vitro analysis, branchial filaments were exposed to a range of GH concentrations (1, 10, and 100 ng/ml) and it was found that HSP70 levels were significantly lowered in all cases. This study adds to the growing body of evidence surrounding the importance of hormones in regulating heat-shock protein expression in fish.

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-β1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.

Background: Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos., Methods: To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction., Results: Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos., Conclusions: Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high-affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site-directed short-interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long-term consequences for the development of the PAA and outflow tract.

Retinoic acid (RA) signaling regulates multiple aspects of vertebrate embryonic development and tissue patterning, in part through the local availability of nuclear hormone receptors called retinoic acid receptors (RARs) and retinoid receptors (RXRs). RAR/RXR heterodimers transduce the RA signal, and loss-of-function studies in mice have demonstrated requirements for distinct receptor combinations at different stages of embryogenesis. However, the tissue-specific functions of each receptor and their individual contributions to RA signaling in vivo are only partially understood. Here we use morpholino oligonucleotides to deplete the four known zebrafish RARs (raraa, rarab, rarga, and rargb). We show that while all four are required for anterior-posterior patterning of rhombomeres in the hindbrain, there are unique requirements for rarga in the cranial mesoderm for hindbrain patterning, and rarab in lateral plate mesoderm for specification of the pectoral fins. In addition, the alpha subclass (raraa, rarab) is RA inducible, and of these only raraa expression is RA-dependent, suggesting that these receptors establish a region of particularly high RA signaling through positive-feedback. These studies reveal novel tissue-specific roles for RARs in controlling the competence and sensitivity of cells to respond to RA.

We have used zebrafish and 3,3',4,4',5-pentachlorobiphenyl (PCB126) to investigate the developmental toxicity of polychlorinated biphenyls (PCBs) that exert their effects through the aryl hydrocarbon receptor (AHR). We found that cardiac and neural crest (NC)-derived jaw and branchial cartilages are specifically targeted early in development. The suite of malformations, which ultimately leads to circulatory failure, includes a severely dysmorphic heart with a reduced bulbus arteriosus and abnormal atrioventricular and outflow valve formation. Early NC migration and patterning of the jaw and branchial cartilages was normal. However, the jaw and branchial cartilages failed to grow to normal size. In the heart, the ventricular myocardium showed a reduction in cell number and size. The heart and jaw/branchial phenotype could be rescued by pifithrin-alpha, a blocker of p53. However, the function of pifithrin-alpha in this model may act as a competitive inhibitor of PCB at the AHR and is likely independent of p53. Morpholinos against p53 did not rescue the phenotype, nor were zebrafish with a mutant p53-null allele resistant to PCB126 toxicity. Morpholino knockdown of cardiac troponin T, which blocks the onset of cardiac function, prevented the PCB126-induced cardiac dysmorphogenesis but not the jaw/branchial phenotype. The cardiovascular characteristics appear to be similar to hypoplastic left heart syndrome (HLHS) and introduce the potential of zebrafish as a model to study this environmentally induced cardiovascular malformation. HLHS is a severe congenital cardiovascular malformation that has previously been linked to industrial releases of dioxins and PCBs.

Background: Among Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role of zebrafish Myc genes., Principal Findings: We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain., Significance: Mych is a novel factor required for neural crest cell survival in zebrafish.

Fathead minnows exposed to sublethal Cu concentrations may undergo branchial damage followed by repair, and may also develop enhanced Cu tolerance. The primary objective of this study was to determine whether the cycle of damage and repair was necessary for the development of enhanced Cu tolerance. Inferences regarding damage and repair were made from changes in the whole body Na(+) of juvenile (0.5 g) fathead minnows (Pimephales promelas). Two experiments were conducted in which juvenile minnows were exposed to various sublethal Cu concentrations for 16 d. In the first experiment, fish were exposed to one of four different Cu concentrations (0, 73, 118, 189 microg/L Cu) then challenged with an 8-d exposure at 302 microg Cu/L. Only the fish exposed to the highest Cu dose experienced enhanced Cu tolerance relative to the other three doses. In the second experiment, whole body Na(+) was monitored in fish exposed to one of five different Cu exposures (0, 70, 127, 202, 289 microg/L Cu). Fish exposed to 70 microg/L Cu did not experience a significant decline in whole body Na(+) at any point during the 16-d exposure period. The reduction in whole body Na(+) was short lived and moderate (17%) in the fish exposed to 127 microg/L Cu, but more severe (>30%) and longer lasting in the fish exposed to 202 microg/L Cu. At 289 microg/L Cu, the fish experienced irreversible reductions in whole body Na(+) and ultimately died. When taken together, results from these two experiments suggest that enhanced tolerance will only develop in fathead minnows that have experienced a pronounced, relatively long-term cycle of branchial damage and repair.

The clinically used antimycotic fluconazole (fluco) is teratogenic in rodents. Exposure in vitro to fluco, other investigated azoles (triadimefon, triadimenol, flusilazole, ketoconazole and imazalil) or retinoic acid (RA), is correlated to branchial arch abnormalities. Inhibition of RA degradation has been suggested as the azole-related mechanism. Citral is a RA synthesis inhibitor. E9.5 rat embryos were cultured for 48 h in normal serum or exposed in vitro to fluco 125 microM, citral 200 microM or co-exposed to the two molecules to test the hypothesis that citral attenuates fluco-related teratogenic effects. Some embryos were cultured for 12 extra hours, and cranial nerves immunodetected. Fluco induced typical abnormalities, including branchial arch and cranial nerve defects. The co-exposure to fluco+citral was significantly effective in reducing branchial arch and cranial nerve defects, supporting the hypothesis that citral balances the fluco-induced RA concentration increase. However, other fluco-related effects were unalterated by citral.

Branchial binding kinetics and gastro-intestinal uptake of copper and cadmium where examined in yellow perch (Perca flavescens) from a metal-contaminated lake (Hannah Lake, Sudbury, Ontario, Canada) and an uncontaminated lake (James Lake, North Bay, Ontario, Canada). An in vivo approach was taken for gill binding comparisons while an in vitro gut binding assay was employed for gastro-intestinal tract (GIT) uptake analysis. By investigating metal uptake at the gill and the gut we cover the two main routes of metal entry into fish. Comparisons of water and sediment chemistries, metal burdens in benthic invertebrate, and metal burdens in the livers of perch from the two study lakes clearly show that yellow perch from Hannah L. are chronically exposed to a highly metal-contaminated environment compared to a reference lake. We found that metal-contaminated yellow perch showed no significant difference in gill Cd binding compared to reference fish, but they did show significant decreases in new Cd binding and absorption in their GITs. The results show that gill Cd binding may involve low-capacity, high-affinity binding sites, while gastro-intestinal Cd uptake involves binding sites that are high-capacity, low-affinity. From this we infer that Cd may be more critically controlled at the gut rather than gills. Significant differences in branchial Cu binding (increased binding) were observed in metal-contaminated yellow perch. We suggest that chronic waterborne exposure to Cu (and/or other metals) may be the dominant influence in gill Cu binding rather than chronic exposure to high Cu diets. We give supporting evidence that Cd is taken up in the GIT, at least in part, by a similar pathway as Ca(2+), principally that elevated dietary Ca(2+) reduces Cd binding and uptake. Overall our study reveals that metal pre-exposure via water and diet can alter uptake kinetics of Cu and Cd at the gill and/or the gut.

Zinc is a vital micronutrient to all organisms, but is also a toxicant to aquatic species. It is therefore of importance to determine the mechanisms by which zinc uptake is modulated. In the present study, we investigated the regulatory effects of the vitamin D metabolite, 1alpha,25-(OH)(2)D(3), on branchial zinc influx in rainbow trout, Oncorhynchus mykiss. Our results showed that branchial zinc uptake in rainbow trout was stimulated 7 days after a single intraperitoneal injection of 1alpha,25-(OH)(2)D(3) (0.01 microg/g fish). To understand the molecular components of zinc uptake regulation by 1alpha,25-(OH)(2)D(3), a ZIP zinc transporter (OmSLC39A1) and a partial vitamin D receptor (OmVDR) were molecularly cloned from rainbow trout gill, and the transcriptional expression of OmSLC39A1, epithelial calcium channel (OmECaC) and OmVDR genes in the gill was subsequently analyzed in response to 1alpha,25-(OH)(2)D(3). OmECaC, OmSLC39A1 and OmVDR were all upregulated following treatment with 1alpha,25-(OH)(2)D(3), but the effect was observed at different time points. OmECaC expression was significantly increased by 1alpha,25-(OH)(2)D(3) on Days 3 and 5 after the injection, and expression of OmVDR was stimulated on Day 5. There was also an increased abundance of OmSLC39A1 mRNA on Day 7 following the injection with 1alpha,25-(OH)(2)D(3), but given the late response the effect of 1alpha,25-(OH)(2)D(3) on this gene might be indirect. The results from the present study provide strong evidence that administration of 1alpha,25-(OH)(2)D(3) results in enhanced zinc uptake across rainbow trout gill and that this effect is associated with an increased expression of transporters that mediate zinc uptake. The implications of our findings, in terms of aquatic toxicology, are that vitamin D status influences zinc accumulation in gill and body of fish.

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.

Chloride transport mechanisms in the gills of the estuarine spotted green pufferfish (Tetraodon nigroviridis) were investigated. Protein abundance of Na(+)/K(+)-ATPase (NKA) and the other four chloride transporters, i.e., Na(+)/K(+)/2Cl(-) cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), Cl(-)/HCO(3)(-) anion exchanger 1 (AE1), and chloride channel 3 (CLC-3) in gills of the seawater- (SW; 35 per thousand) or freshwater (FW)-acclimatized fish were examined by immunoblot analysis. Appropriate negative controls were used to confirm the specificity of the antibodies to the target proteins. The relative protein abundance of NKA was higher (i.e., 2-fold) in gills of the SW group compared to the FW group. NKCC and CFTR were expressed in gills of the SW group but not in the FW group. In contrast, the levels of relative protein abundance of branchial AE1 and CLC-3 in the FW group were 23-fold and 2.7-fold higher, respectively, compared to those of the SW group. This study is first of its kind to provide direct in vivo evidence of the protein expression of CLC-3 in teleostean gills, as well as to examine the simultaneous protein expression of the Cl(-) transporters, especially AE1 and CLC-3 of FW- and SW-acclimatized teleosts. The differential protein expression of NKA, chloride transporters in gills of the FW- and SW-acclimatized T. nigroviridis observed in the present study shows their close relationship to the physiological homeostasis (stable blood osmolality), as well as explains the impressive ionoregulatory ability of this euryhaline species in response to salinity challenges.

Objective: DiGeorge/del22q11 syndrome is one of the most common genetic causes of outflow tract and aortic arch defects in human. DiGeorge/del22q11 is thought to involve an embryonic defect restricted to the pharyngeal arches and the corresponding pharyngeal pouches. Previous studies have evidenced that retinoic acid (RA) signaling is definitely indispensable for the development of the pharyngeal arches. Tbx1, one of the T-box containing genes, is proved to be the most attractive candidate gene for DiGeorge/del22q11 syndrome. However, the interaction between RA and Tbx1 has not been fully investigated. Exploring the interaction will contribute to discover the molecular pathways disrupted in DiGeorge/del22q11 syndrome, and will also be essential for understanding genetic basis for congenital heart disease. It now seems possible that genes and molecular pathways disrupted in DiGeorge syndrome will also account for some isolated cases of congenital heart disease. Accordingly, the present study aimed to extensively study the effects of external RA on the cardiac development and Tbx1 expression during zebrafish embryogenesis., Methods: The chemical genetics approach was applied by treating zebrafish embryos with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA at 12.5 hour post fertilization (hpf). The expression patterns of Tbx1 were monitored by whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively., Results: The zebrafish embryos treated with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA for 1.5 h at 12.5 hpf exhibited selective defects of abnormal heart tube. The results of whole-mount in situ hybridization with Tbx1 RNA probe showed that Tbx1 was expressed in cardiac region, pharyngeal arches and otic vesicle during zebrafish embryogenesis. RA treatment led to a distinct spatio-temporal expression pattern for Tbx1 from that in wild type embryo. The real-time PCR analysis showed that Tbx1 expression levels were markedly reduced by RA treatment. Tbx1 expression in the pharyngeal arches and heart were obviously down regulated compared to the wild type embryos. In contrast to 5 x 10(-8) mol/L RA-treated groups, 10(-7) mol/L RA caused a more severe effect on the Tbx1 expression level., Conclusion: These results suggested that there was a genetic link between RA and Tbx1 during development of zebrafish embryo. RA could produce an altered Tbx1 expression pattern in zebrafish. RA may regulate the Tbx1 expression in a dose-dependant manner. RA could represent a major epigenetic factor to cause abnormal expression of Tbx1, secondarily, disrupt the pharyngeal arch and heart development.

Background: All-trans-retinoic acid (RA) can produce branchial arch abnormalities in postimplantation rodent embryos cultured in vitro. Platelet-derived growth factor C (PDGF-C) was recently identified as a member of the PDGF ligand family. Many members of the PDGF family are essential for branchial arch morphogenesis and can be regulated by RA. The roles of PDGF-C in branchial arch malformations induced by RA and possible mechanisms were investigated., Methods: In whole embryo culture (WEC), mouse embryos were exposed to RA at 0, 0.1, 0.4, 1.0, or 10.0 microM, PDGF-C at 25, 50, or 75 ng/mL, or PDGF-C at 25, 50, or 75 ng/mL containing 0.4 microM RA. After 48 h of culture, mouse embryos were examined for dysmorphogenesis, and whole-mount immunohistochemistry was applied to PDGF-C. In explant cultures, explants were exposed to the same doses of RA and PDGF-C as WEC. Semiquantitative RT-PCR, zymography, and reverse zymography were used to evaluate the expressions and activities of matrix metalloproteinase (MMP)-2, MMP-14, and tissue inhibitor of metalloproteinase (TIMP)-2., Results: PDGF-C was reduced by RA, and exogenous PDGF-C rescued the branchial arch malformations induced by RA. Moreover, PDGF-C prevented RA-induced inhibition of the migratory ability of mesenchymal cells in the first branchial arch, by regulating the expressions of MMP-2, MMP-14, and TIPM-2., Conclusions: Our results suggest that RA exposure reduces the expression of PDGF-C. The branchial arch malformations resulting from fetal RA exposure are caused at least partially by loss of PDGF-C and subsequent misregulations of the expressions of MMP-2, MMP-14, and TIMP-2.

We previously reported that long-term (54 days), repeated intraperitoneal exposure to low doses of tributyltin (TBT; 0.3 mg/kg) inhibited the metabolic activation of co-administered benzo[a]pyrene (BaP; 3 mg/kg) in the Arctic charr (Salvelinus alpinus); BaP, in turn, stimulated the metabolism and/or excretion of TBT. Here, we report the results of histopathological examinations of liver, kidney and pseudobranch tissue samples originating from these same fish. The results revealed higher lesion incidences at all sampling time points (Days 8, 32 and 56) among BaP-exposed fish compared with fish exposed to either TBT alone or combined with BaP. The severity of lesions like necrosis was also higher in BaP-exposed fish. Moreover, hepatic basophilic foci were observed exclusively in fish exposed to BaP alone. Together, these results provide new evidences that TBT can antagonize BaP toxicity in fish exposed to both pollutants under controlled laboratory conditions. In contrast, BaP does not appear to provide protection against TBT toxicity.

Retinoic acid (RA) is a teratogen that induces a variety of craniofacial abnormalities, including branchial arch deformities and cleft palate. Platelet-derived growth factor C (PDGF-C) is a recently identified member of the PDGF family. PDGF-C contributes to normal development of the heart, central nervous system, kidney and palatogenesis. But the roles of PDGF-C in branchial arches development and the relationship between PDGF-C and RA-induced branchial arches abnormalities are poorly understood. We examined the effects of RA on PDGF-C and its receptor PDGFR-alpha expressions. We demonstrated that administration of RA to mouse embryos resulted in dramatic losses of PDGF-C and its receptor PDGFR-alpha. Furthermore, we confirmed that blocking PDGF-C signaling by anti-PDGF-C neutralization antibody led to branchial arch malformations similar to that of RA induced, both hypoplastic branchial arches and FBA. These findings suggest the down-regulation of PDGF-C may be one of mechanisms of branchial arch abnormalities induced by RA and PDGF-C signaling is required for branchial arch morphogenesis.

Background, Aims and Scope: Triazole-derivatives are potent antifungal agents used as systemic agricultural fungicides and against fungal diseases in humans and domestic animals. They act by inhibiting the cytochrome P-450 conversion of lanosterol to ergosterol, thus resulting in faulty fungal cell wall synthesis. Some data have been published about the teratogenic activity of triazoles on rodent embryos: Hypoplasias, abnormal shape, agenesis of the branchial arches, for example, were reported as typical induced malformations. Unfortunately, no data are available on the embryotoxicity of these compounds in amphibians, despite the increasing concern among the scientific community about the phenomenon of global amphibian population declines. The aim of the present work is to evaluate the embryo-lethal and teratogenic potentials of Triadimefon (FON), a triazole-derivative widely used as an antimycotic in agriculture, by the test FETAX (Frog Embryos Teratogenic Assay, Xenopus) with particular attention being paid to the analysis of branchial arch malformations., Methods: Xenopus laevis embryos were exposed continuously from stage 9 to increasing concentrations of FON and analyzed at stage 47 for mortality and teratogenicity (group I) to determine the median lethal (LC50) and teratogenic (TC50) concentrations. Another two pools of larvae were exposed to FON for a 2 hour period at early gastrula (Group II) or neurula (Group III) stages to verify which period of development is the most sensitive to FON. The malformations observed were further investigated by histological section and cartilage staining with Alcian blue., Results and Discussion: The assay has estimated LC50 and TC50 values of 63.8 microM and 2.73 microM, respectively; the resulting TI (Teratogenic Index = LC50/TC50) value of 23.4 has underlined the very high teratogenic risk associated with this compound. Neurulation was more sensitive to FON exposure than gastrulation, since the TC50 estimated values for group III (neurula exposed) specimens was 7.6 times lower than those of group II (gastrula exposed). Interestingly, for each group analyzed, 100% of malformed embryos showed alterations at branchial arch derived cartilages: Anterior cartilages were reduced, missing, fused or incorrectly positioned while gill cartilages were altered only in the most severely affected specimens. In some cases these malformations were associated with hyperpigmentation. Our results support the hypothesis that FON can interfere with Neural Crest Cell (NCC) migration, since craniofacial components and melanophores are derived from neural crest material., Conclusion: In conclusion, our data show Triadimefon to be a potent teratogen able to induce specific craniofacial malformation in Xenopus laevis embryos, probably interfering with the NCC migration into the branchial mesenchyme. These results are also interesting for ecotoxicological reasons as FON, as well as other pesticides, are likely to be present in water systems near agricultural or urban areas which may serve as habitats for developing amphibians and fishes., Recommendation and Outlook: Our results are in agreement with the data obtained on in vitro cultured rat embryos suggesting that the FON mechanism of action involves strongly conserved molecules. The choice of Xenopus laevis as the model organism allows us to extend the toxicological and teratological observations to a molecular level, in order to search for novel genes regulated by FON exposure.

Both retinoic acid (RA) and Tbx1 are definitively indispensable for the development of the pharyngeal arches. The defects produced by a loss of Tbx1 highly resemble those induced by hyper- and hypo-RA. Based on these similarities, the effects of RA on Tbx1 expression pattern were explored during pharyngeal arch development in zebrafish. Whole-mount in situ hybridization and real-time quantitative PCR were used. Zebrafish embryos were treated with 5 x 10(-8)mol/L and 10(-7)mol/L RA at 12.5 hours post fertilization for 1.5 hours, respectively. Whole-mount in situ hybridization showed that Tbx1 was expressed in the cardiac region, pharyngeal arch and otic vesicle between 24 hpf and 72 hpf in zebrafish. Tbx1 expression was obviously reduced, even lost, in the pharyngeal arch and outflow tract in RA treated groups. Real-time quantitative PCR analysis showed that Tbx1 expression rose to a peak level at 36 hpf in wild type group. Repression of Tbx1 expression was most evident at 36 hpf, 24 hours after RA treatment. 10(-7 )mol/L RA caused a more severe effect on the Tbx1 expression level than 5 x 10(-8)mol/L RA. The results suggested that RA could produce an altered Tbx1 expression pattern in zebrafish. In addition, RA could repress Tbx1 expression in a dose-dependent manner.

Triadimefon and triadimenol, fungicides used in agriculture, are suspected of producing craniofacial malformations. As the results of FETAX analysis showed that Triadimefon was highly teratogenic, we studied the action of these triazoles on the development of the branchial apparatus in Xenopus, using early molecular markers and analysis of the cartilaginous-muscular elements of tadpoles. Teratogenic effects were observed, after exposure at the neurula stage, at the level of cartilages and muscles of the 1st and 2nd branchial arches. By in situ hybridization, we observed that in exposed specimens the territories of the branchial arches are perturbed. From these results, we considered the craniofacial malformations related to the influence of triazoles on the differentiation of branchial arches. Comparing the anomalies caused by triazoles and by retinoic acid (RA) led us to the conclusion that triazoles can affect endogenous RA content, as has been shown for mammals.

The effects of ovine prolactin (oPRL) and striped bass prolactin (sbPRL; Morone saxatilis) on plasma osmolality, electrolyte balance, and gill Na(+),K(+)-ATPase activity were investigated in hypophysectomized (Hx), freshwater (FW)-acclimated, hybrid striped bass (M. saxatilisxMorone chrysops). They were kept in dilute (isoosmotic) seawater for about 10 days after surgery. Seven days after transfer to FW, Hx fish had lower plasma osmolality and lower levels of Na(+), Cl(-), and Ca(2+) than sham-operated and intact fish. Fish were injected four times with oPRL (1, 5, or 20 microg/g body mass), sbPRL (10 or 100 ng/g), or hormone vehicle (0.9% NaCl) at 48-h intervals (days 0, 2, 4, and 6) in FW and then sampled for blood plasma 24 h after the fourth injection (day 7). In Hx fish, oPRL (5 and 20 microg/g) and sbPRL (10 and 100 ng/g) were effective in maintaining plasma osmolality and levels of Na(+), Cl(-), and Ca(2+) above values seen in saline-injected controls. Hypophysectomy did not affect branchial Na(+),K(+)-ATPase activity, but enzyme activity was significantly reduced in Hx fish receiving oPRL (20 mug/g) or sbPRL (10 or 100 ng/g). These results indicate that PRL acts to maintain plasma osmotic and ionic balance in FW-adapted hybrid striped bass, and that this may involve downregulation of branchial Na(+),K(+)-ATPase activity.

Homocysteine affects the migration and differentiation of neural crest cells in vitro and can result in neural tube defects in vivo. Furthermore, homocysteine has been described as an important determinant in vascular disease in human adults. However, little is known about the effects of homocysteine on the development of embryonic vessels. In this study, we injected homocysteine (30 micromol/L) into the neural tube lumen of chick embryos at the time point of neural crest cell emigration, and analyzed the effects on the neural crest-derived pharyngeal arch arteries, like the brachiocephalic arteries, and the mesoderm-derived arteries, such as the dorsal aorta. By stage HH35, we observed detachment of the endothelium, decreased expression of the extracellular matrix proteins fibrillin-2, and fibronectin in the pharyngeal arch arteries, whereas the dorsal aorta was identical in homocysteine-neural tube-injected and control embryos. No effect of homocysteine on endothelin-1 mRNA expression was observed. By stage HH40, the brachiocephalic arteries of homocysteine-neural tube-injected embryos displayed a decreased lumen diameter, an increased intima- and media-thickness, and an increased number of actin layers compared with the brachiocephalic arteries in control embryos. We propose that homocysteine affects the neural crest-derived smooth muscle cells and their extracellular matrix proteins in the pharyngeal arch arteries, resulting in an abnormal smooth muscle to endothelial cell interaction, leading to endothelial cell detachment. We suggest that, as in adult life, increased homocysteine concentrations lead to vascular damage in the embryo. This prenatal damage might increase the susceptibility to develop vessel pathology later in life.

The pharyngeal arches are separated by endodermal outpocketings, the pharyngeal pouches. These are structures of considerable importance; they are required to segregate the mesenchymal populations of each arch and to induce the formation of arch components, and they generate specific derivatives, including the parathyroid and the thymus. The pharyngeal pouches are first evident as localised sites at which the endoderm contacts the ectoderm, and they then expand along the proximodistal axis to generate the narrow, tight morphology of the mature pouch. We currently have no knowledge of the morphogenetic mechanisms that direct formation of the pharyngeal pouches. Here, in chick, we show that cells within the pharyngeal pouch endoderm have an abundance of apically located actin fibres that are networked within the endodermal sheet, via their insertion into N-cadherin adherens junctions, to form a web of supra-cellular actin cables. Cytochalasin D disruption of these actin structures results in the formation of aberrant pouches that fail to generate their normal slit-like morphology. This suggests that the process of pharyngeal pouch morphogenesis involves the constraining influence of these actin cables that direct expansion, within the pouch, along the proximodistal axis. These results, importantly, provide us with vital insights into how the pharyngeal pouches form their normal morphology. They also give evidence, for the first time, of actin cables functioning as constraints during complex vertebrate morphogenetic episodes.

The water-soluble fraction (WSF) of crude oil is a complex highly volatile and toxic mixture of hydrocarbon chains (polyaromatics, heterocyclics), phenols, and heterocyclic compounds containing nitrogen and sulfur. To evaluate the toxic effects of WSF in tropical freshwater teleosts and to develop methodologies that could investigate the toxic mechanisms of WSF in tropical organisms, an acute toxicity experiment was conducted with Astyanax sp. Three dilutions (15%, 33%, and 50%) of WSF obtained from Campos Bay's crude oil (Brazil) were used to study morphological and biochemical responses of the fish. Prior to exposure, the distribution and rate of volatilization of the WSF into each aquarium for the same exposure period was quantified by spectrofluorimetry. Five individuals of Astyvanax sp. were exposed to duplicate WSF of 0, 15, 33, and 50% for each of 12-, 24-, and 96-h exposures for a total of 120 individuals. Liver and gills were sampled from five fish from each treatment and were analyzed by histology, scanning and transmission electron microscopy. A fragment of muscle was also collected from each fish to measure acetylcholinesterase activity. Water analysis showed that only 4 h after dilution, an important loss of hydrocarbons in 33% and 50% of WSF was observed. In addition, 50% of hydrocarbon mass was lost in all tested dilutions after 24 h with significant difference for the 50% WSF at all measured times, demonstrating the high volatility of WSF in freshwater. Damage in the liver and the gills included the presence of necrosis, loss of hepatocytes limit, inflammation areas, cellular proliferation, aneurysms, and disorganization of the second lamellae. The 33% WSF significantly reduced acetylcholinesterase activity in fish. Our study demonstrated that the WSF of crude oil caused damage in organs and tissues of tropical freshwater Astyanax sp. and provided also the basis for a better understanding of the toxic mechanisms of WSF in freshwater fishes.

Targeted inactivation of the mouse retinaldehyde dehydrogenase 2 (RALDH2/ALDH1a2), the enzyme responsible for early embryonic retinoic acid synthesis, is embryonic lethal because of defects in early heart morphogenesis. Transient maternal RA supplementation from E7.5 to (at least) E8.5 rescues most of these defects, but the supplemented Raldh2(-/-) mutants die prenatally, from a lack of septation of the heart outflow tract (Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P. and Dollé, P. (2001). Development 128, 1019-1031). We have investigated the developmental basis for this defect, and found that the RA-supplemented Raldh2(-/-) embryos exhibit impaired development of their posterior (3rd-6th) branchial arch region. While the development of the first and second arches and their derivatives, as well as the formation of the first branchial pouch, appear to proceed normally, more posterior pharyngeal pouches fail to form and the pharyngeal endoderm develops a rudimentary, pouch-like structure. All derivatives of the posterior branchial arches are affected. These include the aortic arches, pouch-derived organs (thymus, parathyroid gland) and post-otic neural crest cells, which fail to establish segmental migratory pathways and are misrouted caudally. Patterning and axonal outgrowth of the posterior (9th-12th) cranial nerves is also altered. Vagal crest deficiency in Raldh2(-/-) mutants leads to agenesis of the enteric ganglia, a condition reminiscent of human Hirschprung's disease. In addition, we provide evidence that: (i) wildtype Raldh2 expression is restricted to the posteriormost pharyngeal mesoderm; (ii) endogenous RA response occurs in both the pharyngeal endoderm and mesoderm, and extends more rostrally than Raldh2 expression up to the 2nd arch; (iii) RA target genes (Hoxa1, Hoxb1) are downregulated in both the pharyngeal endoderm and mesoderm of mutant embryos. Thus, RALDH2 plays a crucial role in producing RA required for pharyngeal development, and RA is one of the diffusible mesodermal signals that pattern the pharyngeal endoderm.

Background: A widely-used antimycotic agent, bis-triazole fluconazole (FLUCO), is able to produce abnormalities to the branchial apparatus (hypoplasia, agenesis, and fusion) in postimplantation rodent embryos cultured in vitro. The branchial apparatus is a complex and transient structure in vertebrate embryos and is essential for the development of the face skeleton. Branchial arch mesenchyme is formed by two different cellular populations: paraxial mesenchyme and ectomesenchyme, which originate from rhombencephalic neural crest cell (NCC) migration. We investigated the possible pathogenic pathways involved in FLUCO-related branchial arch abnormalities. Perturbations in physiological apoptosis, cell proliferation, NCC migration and branchial mesenchyme induction have been considered., Methods: Rat embryos (9.5-day postcoitum; 1-3 somites) were exposed in vitro to 0 or 500 microM FLUCO. After 24, 36, or 48 hr of culture, embryos were examined for apoptosis (acridine orange method) and cell proliferation (BrdU incorporation and detection method). Rhombencephalic NCC migration was analyzed using immunostaining of NCC (using anti-CRABP antibodies) and the extracellular matrix (using anti-fibronectin antibodies). The differentiative capability of the branchial mesenchymes was investigated using anti-endothelin and anti-endothelin-receptor antibodies., Results: During the whole culture period, no alterations in physiological apoptosis, cell proliferation, and mesenchymal cell induction were observed in FLUCO-exposed embryos in comparison to controls. On the contrary, severe alterations in NCC migration pathways were observed in FLUCO-exposed embryos., Conclusions: The findings suggest that FLUCO produces teratogenic effects by interfering with the cellular and molecular mechanisms that control NCC migration.

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.

The rapid action of cortisol and corticosterone on branchial Na(+)-K(+) ATPase, Ca(2+) ATPase activity and Na(+), K(+) and Ca(2+) ion contents was studied both in vivo and in vitro employing transcription inhibitor actinomycin D in Oreochromis mossambicus. Cortisol and corticosterone administration had significantly increased the activity of branchial Na(+)-K(+) ATPase and Ca(2+) ATPase in vivo after 30 min of injection, and the trend continued for 60 and 120 min for cortisol. The ionic contents were also significantly increased after 30 min in vivo. Na(+)-K(+) ATPase activity was significantly increased 5 min after hormone application in the in vitro system. Actinomycin D did not inhibit the effect of glucocorticoids on ATPase activity both in vivo and in vitro. It is concluded from the present study that cortisol and corticosterone produced a rapid stimulatory effect on branchial ATPase activity and ions in O. mossambicus both in vivo and in vitro. This effect could be due to a non-genomic action of these hormones since the enzyme activity was insensitive to actinomycin D.

Triazole-derivatives are antimycotics used in agriculture as well as in clinical and veterinary therapy. The aim of the present work is the in vitro comparative study of the teratogenic activity of triazole (the parental compound), flusilazole (an agricultural triazole mono-derivative fungicide), and fluconazole (a clinically used bis-triazole derivative). Rat embryos, 9.5 days old (1 to 3 somites) were exposed in vitro to triazole 500 to 5000 microM, flusilazole 3.125 to 250 microM, or fluconazole 62.5 to 500 microM. After 48 h in culture, the embryos were morphologically examined and processed for histologic and biochemical analysis. Flusilazole and fluconazole showed similar teratogenic effects (abnormalities at the branchial apparatus level and cell death at the level of the branchial mesenchyme) at concentration levels of 6.25 microM and higher for flusilazole and of 125 microM and higher for fluconazole. By contrast, only slight developmental retardation and blood discoloration were observed at the highest concentrations of triazole, suggesting no teratogenic activity for the triazole group.

Alcohol abuse by pregnant women can result in fetal alcohol effects (FAE) and fetal alcohol syndrome (FAS). Both ethanol itself and its main metabolite, acetaldehyde (Ach), are able to produce specific FAS-related malformations. In previous in vitro studies, we documented that 10-day-old rat embryos exposed to Ach show a characteristic embryonic Ach syndrome, histologically characterized by marked cellular death. As both necrosis and pathological apoptosis are teratological mechanisms, the aim of this work was to evaluate if cellular death, observed in Ach-exposed embryos, can be related to necrotic or apoptotic events. Ten-day-old rat embryos were cultured in the presence of Ach 30-60 microg/ml and stained with the vital dye acridine orange to visualize apoptotic areas. After fixation, the TUNEL [3' terminal deoxynucleotide transferase (TdT)-mediated dUTP-biotin nick end labeling] method was used to histologically identify apoptosis. Both acridine orange and TUNEL staining showed signs of physiological apoptosis in controls and abnormal apoptotic regions in Ach-exposed embryos. Our results show a clear correlation between malformed organs and apoptotic embryonic districts, suggesting the role of apoptosis in Ach-induced abnormalities.

The fates of cranial neural crest cells are unique compared to trunk neural crest. Cranial neural crest cells form bone and cartilage and ultimately these cells make up the entire facial skeleton. Previous studies had established that exogenous retinoic acid has effects on neurogenic derivatives of cranial neural crest cells and on segmentation of the hindbrain. In the present study we investigated the role of retinoic acid on the skeletal derivatives of migrating cranial neural crest cells. We wanted to test whether low doses of locally applied retinoic acid could respecify the neural crest-derived, skeletal components of the beak in a reproducible manner. Retinoic acid-soaked beads were positioned at the presumptive mid-hindbrain junction in stage 9 chicken embryos. Two ectopic cartilage elements were induced, the first a sheet of cartilage ventral and lateral to the quadrate and the second an accessory cartilage rod branching from Meckel's cartilage. The accessory rod resembled a retroarticular process that had formed within the first branchial arch domain. In addition the quadrate was often displaced laterally and fused to the retroarticular process. The next day following bead implantation, expression domains of Hoxa2 and Hoxb1 were shifted in an anterior direction up to the mesencephalon and Msx-2 was slightly down-regulated in the hindbrain. Despite down-regulation in neural crest cells, the onset of Msx-2 expression in the facial prominences at stage 18-20 was normal. This correlates with normal distal beak morphology. Focal labeling of neural crest with DiI showed that instead of migrating in a neat group toward the second branchial arch, a cohort of labeled cells from r4 spread anteriorly toward the proximal first arch region. AP-2 expression data confirmed the uninterrupted presence of AP-2-expressing cells from the anterior mesencephalon to r4. The morphological changes can be explained by mismigration of r4 neural crest into the first arch, but at the same time maintenance of their identity. Up-regulation of the Hoxa2 gene in the first branchial arch may have encouraged r4 cells to move in the anterior direction. This combination of events leads to the first branchial arch assuming some of the characteristics of the second branchial arch.

The requirement of retinoic acid (RA) in the initial formation of the pharyngeal arches was investigated by treating headfold-stage mouse embryos with a pan-RAR antagonist in vitro and in vivo. This results in a complete absence of mesenchyme, arteries, nerves and epibranchial placodes of the 3rd and 4th pharyngeal arches, complete agenesis of the 3rd and 4th pouches and consistent lack of the 6th arch artery. Mesodermally derived endothelial cells are absent from the 3rd and 4th pharyngeal arch region and the distribution domain of EphA2 transcripts in mesodermal cells is shifted caudally. In situ hybridization with CRABPI, kreisler and EphA4 probes and the pattern of expression of a Wnt1-lacZ transgene show that neural crest cells (NCC) normally destined to the 3rd and 4th arches migrate ectopically. Most interestingly, the appearance of the 3rd and 4th arches is prevented by the antagonist only during a very narrow window of time, which does not correspond to the period of post-otic NCC migration. Both the timing of appearance and the nature of the defects in RAR antagonist-treated embryos indicate that migrating NCC and mesodermal cells destined to the caudal pharyngeal arches do not represent primary targets of RA action. Alterations in the endodermal expression pattern of Hoxa1, Hoxb1, Pax1, Pax9, Fgf3 and Fgf8 in response to the antagonist-induced block in RA signal transduction demonstrate for the first time that RA signaling is indispensable for the specification of the pharyngeal endoderm and suggest that this signaling is necessary to provide a permissive environment locally for the migration of NCC and mesodermal cells. Our study also indicates that the formation of the 2nd pharyngeal arch and that of the 3rd and 4th pharyngeal arches probably involve distinct RA-dependent developmental processes.

This study was undertaken to examine the normal and abnormal epithelial alterations of secondary palate in rats. Control and dexamethasone-treated embryos and fetuses of Wistar rats were evaluated by macroscopic and scanning electron microscopic analysis prior to, during, and after fusion of palatal processes. Normal alterations of the surface topography included growth and disorganization of medial edge epithelial cells followed by fusion and posterior migration to both the oral and nasal aspects of the palate. No evidence of epithelial cell death or transformation was observed. Dexamethasone-treated fetuses showed epithelial cells increased in size with a large amount of desquamation, followed by deposition of a disorganized cell layer with keratin-like characteristics. This allowed no fusion of palatal processes.

All-trans retinoid acid (RA) induces a stereotypic spectrum of stage-specific malformations in vertebrate conceptuses. The present work evaluated the anatomic and biochemical effects of exposure to RA in mouse embryos at a peri-implantational stage of development - gestational day (GD) 5. The RA receptors (RARs) beta and gamma, the retinoid X receptors (RXRs) alpha and beta, and the cellular retinoid acid binding proteins (CRABPs) I and II were detected by RT-PCR in both control and treated individual GD 5 decidua/embryo complexes 3 h after RA injection, indicating the presence of the mRNAs coding for the proteins that mediate the effects of RA. In contrast, the RAR alpha mRNA was detected in some but not all decidua/embryo complexes, both control and treated, suggesting that its expression is initiated at approximately GD 5, while RXR gamma mRNA was not detected. Examination of the control and RA-exposed embryos on GD 10, 12, or 17 showed that greater than 50% of the RA-exposed embryos were adversely affected, many with defects found only after serial histopathological examination. The malformations were localized primarily in the central nervous system, the branchial arches, and their derivatives. These terata included excessive folding and elevation of the neural tube floor plate, exencephaly (with detachment of the cephalic neuroepithelium and rarefied cephalic mesenchyme), persistent patency of Rathke's pouch, small trigeminal ganglia, neural diverticula (chiefly from the spinal cord), and/or various optic and otic defects. Unexpectedly, limb reduplications were not apparent in RA-exposed fetuses. Those litters examined on GD 17 had a high percentage of resorbed or malformed implantations, and the few apparently normal fetuses were developmentally delayed with respect to bone ossification. These data confirm that the development of neural- and neural crest-derived structures are severely disrupted by RA exposure prior to initial specification of the neural plate and suggest that many of the proteins that regulate RA signaling are available in early vertebrate embryos at this developmental stage.

This study investigates hindbrain and associated neural crest (NCC), otocyst, and pharyngeal arch development in monkey embryos following teratogenic exposure to 13-cis-retinoic acid (cRA). cRA was orally administered (5 mg/kg) to pregnant long-tailed macaques (Macaca fascicularis) between gestational days (GD) 12 and 27. Embryos were surgically collected at desired stages during treatment, analyzed for external morphological changes, and processed for immunohistochemistry. Two transiently expressed nuclear proteins, Krox-20 and Pax-2, were used as markers for the target cellular and anatomical structures. Rhombomere (r) expression patterns of Pax-2 (r4/r6) and Krox-20 (r3/r5) were maintained after cRA treatment, but r4 and r5 were substantially reduced in size. In untreated embryos, Krox-20 immunoreactive NCC derived from r5 migrated caudally around the developing otocyst to contribute to the third pharyngeal arch mesenchyme. In cRA-treated embryos, a subpopulation of NCC rostral to the otocyst also showed Krox-20 immunoreactivity, but there was a substantial reduction in Krox-20 post-otic NCC. Pax-2 immunoreactive NCC migrating from r4 to the second pharyngeal arch were substantially reduced in numbers in treated embryos. Alteration in the otic anlage included delayed invagination, abnormal relationship with the adjacent hindbrain epithelium, and altered expression boundaries for Pax-2. cRA-associated changes in the pharyngeal arch region due to cRA included truncation of the distal portion of the first arch and reduction in the size of the second arch. These alterations in hindbrain, neural crest, otic anlage, and pharyngeal arch morphogenesis could contribute to some of the craniofacial malformations in the macaque fetus associated with exposure to cRA.

The effect of hormone treatment on the abundance of Na+-K+-ATPase alpha- and beta-subunit mRNA in Sparus sarba branchial tissue was investigated. Groups of seawater (33/1000) and hypo-osmotic (6/1000) acclimated fish were injected daily, with either saline, cortisol, recombinant bream growth hormone (rbGH) or ovine prolactin (oPRL). Total RNA from branchial tissue was analyzed by Northern blotting using PCR amplified Na+-K+-ATPase alpha- and beta-subunit cDNA clones. Na+-K+-ATPase alpha- and beta- subunit transcripts of 3.3kb and 2.4kb respectively, were detected and their abundance, after hormone treatment was assessed using RNA dot blots. The abundance of subunit mRNAs increased 1.4-1.9 fold, relative to controls, after cortisol treatment. The alpha:beta mRNA ratio also increased in cortisol treated seawater acclimated fish. Growth hormone treatment did not cause any significant changes in Na+-K+-ATPase subunit mRNA, whereas prolactin significantly reduced alpha-subunit mRNA levels by approximately 0.5 fold in both seawater and hypo-osmotic conditions. The data from this study add further support to the generally accepted roles that cortisol and prolactin have in the modulation of Na+-K+-ATPase activity. It can be concluded from this study that S. sarba branchial Na+-K+-ATPase subunit expression is multihormonally regulated.

In the present study, we have applied an antagonist treatment to the chick embryo in ovo in order to demonstrate and dissect the essential roles of the endothelin type A (ETA) receptor in the embryonic development. We have cloned, sequenced and expressed the cDNA of the chick ETA receptor and shown that its affinity for endothelin antagonists is very similar to that shown by its mammalian counterparts. We have studied the spatio-temporal expression pattern of this receptor by in situ hybridization and shown that there is a high level of its mRNA within the mesenchyme of the branchial arches at E3-E5, in keeping with the direct effect of endothelin-1 (ET-1) on the fate of this region of the embryo. Unlike the endothelin type B (ETB) receptor mRNA, ETA mRNA is not expressed in neural crest cells during emigration from the neural tube, but is detected in neural crest-derived ectomesenchyme of the branchial arches. Finally, the functional involvement of this receptor in craniofacial and cardiovascular organogenesis was assessed by selectively inactivating the ETA receptor with specific antagonists applied during the time period corresponding to the expression of the ETA receptor and colonisation of the branchial arches. Embryos treated by these antagonists show a severe reduction and dysmorphogenesis of the hypobranchial skeleton, as well as heart and aortic arch derivative defects. This phenotype is very similar to that obtained in mice by gene inactivations of ET-1 and ETA. These results are observed with ETA antagonists but not with an ETB antagonist, and are dependent on the dose of the antagonists used and on the time of application to the embryo. Altogether, these data strongly show that the ET-1/ETA pathway, in chicken as in mammals, is a major factor involved directly and functionally in morphogenesis of the face and heart. This experimental model of pharmacological inactivation of a gene product described in this study offers a simple and rapid alternative to gene inactivation in mouse. This strategy can be applied to other ligand-receptor systems and extended to compounds of various chemical and functional natures.