Disruption of the foxe1 gene in zebrafish reveals conserved functions in development of the craniofacial skeleton and the thyroid.

Introduction: Mutations in the FOXE1 gene are implicated in cleft palate and thyroid dysgenesis in humans. Methods: To investigate whether zebrafish could provide meaningful insights into the etiology of developmental defects in humans related to FOXE1, we generated a zebrafish mutant that has a disruption in the nuclear localization signal in the foxe1 gene, thereby restraining nuclear access of the transcription factor. We characterized skeletal development and thyroidogenesis in these mutants, focusing on embryonic and larval stages. Results: Mutant larvae showed aberrant skeletal phenotypes in the ceratohyal cartilage and had reduced whole body levels of Ca, Mg and P, indicating a critical role for foxe1 in early skeletal development. Markers of bone and cartilage (precursor) cells were differentially expressed in mutants in post-migratory cranial neural crest cells in the pharyngeal arch at 1 dpf, at induction of chondrogenesis at 3 dpf and at the start of endochondral bone formation at 6 dpf. Foxe1 protein was detected in differentiated thyroid follicles, suggesting a role for the transcription factor in thyroidogenesis, but thyroid follicle morphology or differentiation were unaffected in mutants. Discussion: Taken together, our findings highlight the conserved role of Foxe1 in skeletal development and thyroidogenesis, and show differential signaling of osteogenic and chondrogenic genes related to foxe1 mutation.

Targeting fibroblast growth factor receptors causes severe craniofacial malformations in zebrafish larvae.

Background and Objective: A key pathway controlling skeletal development is fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling. Major regulatory functions of FGF signaling are chondrogenesis, endochondral and intramembranous bone development. In this study we focus on fgfr2, as mutations in this gene are found in patients with craniofacial malformations. The high degree of conservation between FGF signaling of human and zebrafish (Danio rerio) tempted us to investigate effects of the mutated fgfr2 sa10729 allele in zebrafish on cartilage and bone formation. Methods: We stained cartilage and bone in 5 days post fertilization (dpf) zebrafish larvae and compared mutants with wildtypes. We also determined the expression of genes related to these processes. We further investigated whether pharmacological blocking of all FGFRs with the inhibitor BGJ398, during 0-12 and 24-36 h post fertilization (hpf), affected craniofacial structure development at 5 dpf. Results: We found only subtle differences in craniofacial morphology between wildtypes and mutants, likely because of receptor redundancy. After exposure to BGJ398, we found dose-dependent cartilage and bone malformations, with more severe defects in fish exposed during 0-12 hpf. These results suggest impairment of cranial neural crest cell survival and/or differentiation by FGFR inhibition. Compensatory reactions by upregulation of fgfr1a, fgfr1b, fgfr4, sp7 and dlx2a were found in the 0-12 hpf group, while in the 24-36 hpf group only upregulation of fgf3 was found together with downregulation of fgfr1a and fgfr2. Conclusions: Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings enhance our understanding of the role of FGFRs in the etiology of craniofacial malformations.

Regenerating zebrafish scales express a subset of evolutionary conserved genes involved in human skeletal disease.

BACKGROUND: Scales are mineralised exoskeletal structures that are part of the dermal skeleton. Scales have been mostly lost during evolution of terrestrial vertebrates whilst bony fish have retained a mineralised dermal skeleton in the form of fin rays and scales. Each scale is a mineralised collagen plate that is decorated with both matrix-building and resorbing cells. When removed, an ontogenetic scale is quickly replaced following differentiation of the scale pocket-lining cells that regenerate a scale. Processes promoting de novo matrix formation and mineralisation initiated during scale regeneration are poorly understood. Therefore, we performed transcriptomic analysis to determine gene networks and their pathways involved in dermal scale regeneration. RESULTS: We defined the transcriptomic profiles of ontogenetic and regenerating scales of zebrafish and identified 604 differentially expressed genes (DEGs). These were enriched for extracellular matrix, ossification, and cell adhesion pathways, but not in enamel or dentin formation processes indicating that scales are reminiscent to bone. Hypergeometric tests involving monogenetic skeletal disorders showed that DEGs were strongly enriched for human orthologues that are mutated in low bone mass and abnormal bone mineralisation diseases (P< 2× 10-3). The DEGs were also enriched for human orthologues associated with polygenetic skeletal traits, including height (P< 6× 10-4), and estimated bone mineral density (eBMD, P< 2× 10-5). Zebrafish mutants of two human orthologues that were robustly associated with height (COL11A2, P=6× 10-24) or eBMD (SPP1, P=6× 10-20) showed both exo- and endo- skeletal abnormalities as predicted by our genetic association analyses; col11a2Y228X/Y228X mutants showed exoskeletal and endoskeletal features consistent with abnormal growth, whereas spp1P160X/P160X mutants predominantly showed mineralisation defects. CONCLUSION: We show that scales have a strong osteogenic expression profile comparable to other elements of the dermal skeleton, enriched in genes that favour collagen matrix growth. Despite the many differences between scale and endoskeletal developmental processes, we also show that zebrafish scales express an evolutionarily conserved sub-population of genes that are relevant to human skeletal disease.

Loss of sdhb in zebrafish larvae recapitulates human paraganglioma characteristics.

Pheochromocytomas and paragangliomas (PPGLs) caused by mutations in the B-subunit of the succinate dehydrogenase (SDHB) have the highest metastatic rate among PPGLs, and effective systemic therapy is lacking. To unravel underlying pathogenic mechanisms, and to evaluate therapeutic strategies, suitable in vivo models are needed. The available systemic Sdhb knock-out mice cannot model the human PPGL phenotype: heterozygous Sdhb mice lack a disease phenotype, and homozygous Sdhb mice are embryonically lethal. Using CRISPR/cas9 technology, we introduced a protein-truncating germline lesion into the zebrafish sdhb gene. Heterozygous sdhb mutants were viable and displayed no obvious morphological or developmental defects. Homozygous sdhb larvae were viable, but exhibited a decreased lifespan. Morphological analysis revealed incompletely or non-inflated swim bladders in homozygous sdhb mutants at day 6. Although no differences in number and ultrastructure of the mitochondria were observed. Clear defects in energy metabolism and swimming behavior were observed in homozygous sdhb mutant larvae. Functional and metabolomic analyses revealed decreased mitochondrial complex 2 activity and significant succinate accumulation in the homozygous sdhb mutant larvae, mimicking the metabolic effects observed in SDHB-associated PPGLs. This is the first study to present a vertebrate animal model that mimics metabolic effects of SDHB-associated PPGLs. This model will be useful in unraveling pathomechanisms behind SDHB-associated PPGLs. We can now study the metabolic effects of sdhb disruption during different developmental stages and develop screening assays to identify novel therapeutic targets in vivo. Besides oncological syndromes, our model might also be useful for pediatric mitochondrial disease caused by loss of the SDHB gene.

Early Life Glucocorticoid Exposure Modulates Immune Function in Zebrafish (Danio rerio) Larvae.

In this study we have assessed the effects of increased cortisol levels during early embryonic development on immune function in zebrafish (Danio rerio) larvae. Fertilized eggs were exposed to either a cortisol-containing, a dexamethasone-containing (to stimulate the glucocorticoid receptor selectively) or a control medium for 6 h post-fertilization (0-6 hpf). First, we measured baseline expression of a number of immune-related genes (socs3a, mpeg1.1, mpeg1.2, and irg1l) 5 days post-fertilization (dpf) in larvae of the AB and TL strain to assess the effectiveness of our exposure procedure and potential strain differences. Cortisol and dexamethasone strongly up-regulated baseline expression of these genes independent of strain. The next series of experiments were therefore carried out in larvae of the AB strain only. We measured neutrophil/macrophage recruitment following tail fin amputation (performed at 3 dpf) and phenotypical changes as well as survival following LPS-induced sepsis (150 µg/ml; 4-5 dpf). Dexamethasone, but not cortisol, exposure at 0-6 hpf enhanced neutrophil recruitment 4 h post tail fin amputation. Cortisol and dexamethasone exposure at 0-6 hpf led to a milder phenotype (e.g., less tail fin damage) and enhanced survival following LPS challenge compared to control exposure. Gene-expression analysis showed accompanying differences in transcript abundance of tlr4bb, cxcr4a, myd88, il1ß, and il10. These data show that early-life exposure to cortisol, which may be considered to be a model or proxy of maternal stress, induces an adaptive response to immune challenges, which seems mediated via the glucocorticoid receptor.

Divergent Functional Diversification Patterns in the SEP/AGL6/AP1 MADS-Box Transcription Factor Superclade.

Members of SEPALLATA (SEP) and APETALA1 (AP1)/SQUAMOSA (SQUA) MADS-box transcription factor subfamilies play key roles in floral organ identity determination and floral meristem determinacy in the rosid species Arabidopsis (Arabidopsis thaliana). Here, we present a functional characterization of the seven SEP/AGL6 and four AP1/SQUA genes in the distant asterid species petunia (Petunia × hybrida). Based on the analysis of single and higher order mutants, we report that the petunia SEP1/SEP2/SEP3 orthologs together with AGL6 encode classical SEP floral organ identity and floral termination functions, with a master role for the petunia SEP3 ortholog FLORAL BINDING PROTEIN2 (FBP2). By contrast, the FBP9 subclade members FBP9 and FBP23, for which no clear ortholog is present in Arabidopsis, play a major role in determining floral meristem identity together with FBP4, while contributing only moderately to floral organ identity. In turn, the four members of the petunia AP1/SQUA subfamily redundantly are required for inflorescence meristem identity and act as B-function repressors in the first floral whorl, together with BEN/ROB genes. Overall, these data together with studies in other species suggest major differences in the functional diversification of the SEP/AGL6 and AP1/SQUA MADS-box subfamilies during angiosperm evolution.plantcell;31/12/3033/FX1F1fx1.

Early life exposure to cortisol in zebrafish (Danio rerio): similarities and differences in behaviour and physiology between larvae of the AB and TL strains.

Maternal stress and early life stress affect development. Zebrafish (Danio rerio) are ideally suited to study this, as embryos develop externally into free-feeding larvae. The objective of this study was therefore to assess the effects of increased levels of cortisol, mimicking thereby maternal stress, on larval physiology and behaviour. We studied the effects in two common zebrafish strains, that is, AB and Tupfel long-fin (TL), to assess strain dependency of effects. Fertilized eggs were exposed to a cortisol-containing medium (1.1 µmol/l) or control medium from 0 to 6 h following fertilization, after which at 5-day following fertilization, larval behaviour and baseline hypothalamus-pituitary-interrenal cells axis functioning were measured. The data confirmed earlier observed differences between AB larvae and TL larvae: a lower hypothalamus-pituitary-interrenal axis activity in TL larvae than AB larvae, and slower habituation to repeated acoustic/vibrational stimuli in TL larvae than AB larvae. Following cortisol treatment, increased baseline levels of cortisol were found in AB larvae but not TL larvae. At the behavioural level, increased thigmotaxis or 'wall hugging' was found in AB larvae, but decreased thigmotaxis in TL larvae; however, both AB larvae and TL larvae showed decreased habituation to repeated acoustic/vibrational stimuli. The data emphasize that strain is a critical factor in zebrafish research. The habituation data suggest a robust effect of cortisol exposure, which is likely an adaptive response to increase the likelihood of detecting or responding to potentially threatening stimuli. This may enhance early life survival. Along with other studies, our study underlines the notion that zebrafish may be a powerful model animal to study the effects of maternal and early life stress on life history.

Allostatic Load and Stress Physiology in European Seabass (Dicentrarchus labrax L.) and Gilthead Seabream (Sparus aurata L.).

The present study aimed to compare effects of increasing chronic stress load on the stress response of European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) to identify neuroendocrine functions that regulate this response. Fish were left undisturbed (controls) or exposed to three levels of chronic stress for 3 weeks and then subjected to an acute stress test (ACT). Chronic stress impeded growth and decreased feed consumption in seabass, not in seabream. In seabass basal cortisol levels are high and increase with stress load; the response to a subsequent ACT decreases with increasing (earlier) load. Basal cortisol levels in seabream increase with the stress load, whereas the ACT induced a similar response in all groups. In seabass and seabream plasma α-MSH levels and brain stem serotonergic activity and turnover were similar and not affected by chronic stress. Species-specific molecular neuro-regional differences were seen. In-situ hybridization analysis of the early immediate gene cfos in the preoptic area showed ACT-activation in seabream; in seabass the expression level was not affected by ACT and seems constitutively high. In seabream, expression levels of telencephalic crf, crfbp, gr1, and mr were downregulated; the seabass hypothalamic preoptic area showed increased expression of crf and gr1, and decreased expression of mr, and this increased the gr1/mr ratio considerably. We substantiate species-specific physiological differences to stress coping between seabream and seabass at an endocrine and neuroendocrine molecular level. Seabass appear less resilient to stress, which we conclude from high basal activities of stress-related parameters and poor, or absent, responses to ACT. This comparative study reveals important aquaculture, husbandry, and welfare implications for the rearing of these species.

Light regimes differentially affect baseline transcript abundance of stress-axis and (neuro)development-related genes in zebrafish (Danio rerio, Hamilton 1822) AB and TL larvae.

Many strains of zebrafish (Danio rerio) are readily available. Earlier we observed differences between AB and Tupfel long-fin (TL) larvae regarding baseline hypothalamus-pituitary-interrenal (HPI) axis activity and (neuro)development. Light regimes, i.e. 14 h light:10 h dark and 24 h continuous dark or light, affect hatching rate and larval growth. Here, we assessed baseline transcript abundance of HPI-axis-related genes and (neuro)development-related genes of AB and TL larvae (5 days post fertilisation) using these light regimes. A principal component analysis revealed that in AB larvae the baseline expression of HPI-axis-related genes was higher the more hours of light, while the expression of (neuro)development-related genes was higher under 14 h light:10 h dark than under both continuous light or dark. In TL larvae, a complex pattern emerged regarding baseline expression of HPI-axis-related and (neuro)development-related genes. These data extend data of earlier studies by showing that light regimes affect gene-expression in larvae, and more importantly so, strengthen the notion of differences between larvae of the AB and TL strain. The latter finding adds to the growing database of phenotypical differences between zebrafish of the AB and TL strain.

Divergence of the Floral A-Function between an Asterid and a Rosid Species.

The ABC model is widely used as a genetic framework for understanding floral development and evolution. In this model, the A-function is required for the development of sepals and petals and to antagonize the C-function in the outer floral whorls. In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor represents a major A-function protein, but how the A-function is encoded in other species is not well understood. Here, we show that in the asterid species petunia (Petunia hybrida), AP2B/BLIND ENHANCER (BEN) confines the C-function to the inner petunia floral whorls, in parallel with the microRNA BLINDBEN belongs to the TOE-type AP2 gene family, members of which control flowering time in Arabidopsis. In turn, we demonstrate that the petunia AP2-type REPRESSOR OF B-FUNCTION (ROB) genes repress the B-function (but not the C-function) in the first floral whorl, together with BEN We propose a combinatorial model for patterning the B- and C-functions, leading to the homeotic conversion of sepals into petals, carpels, or stamens, depending on the genetic context. Combined with earlier results, our findings suggest that the molecular mechanisms controlling the spatial restriction of the floral organ identity genes are more diverse than the well-conserved B and C floral organ identity functions.

Further characterisation of differences between TL and AB zebrafish (Danio rerio): Gene expression, physiology and behaviour at day 5 of the larval stage.

Zebrafish (Danio rerio) have become popular as model organism in research. Many strains are readily available, which not only differ morphologically, but also genetically, physiologically and behaviourally. Here, we focus on the AB and Tupfel long-fin (TL) strain for which we have previously shown that adults differ in baseline hypothalamus-pituitary-interrenal (HPI)-axis activity (AB higher than TL) affecting inhibitory avoidance behaviour (absent in AB). To assess whether strain differences are already present in early life stages, we compared baseline HPI-axis related gene expression as well as cortisol levels, (neuro)development related as well as (innate) immune system related gene expression, and light-dark as well as startle behaviour in larvae 5 days post fertilisation. The data show that AB and TL larvae differ in baseline HPI-axis activity (AB higher than TL), expression of (neuro)development and immune system related genes (AB higher than TL), habituation to acoustic/vibrational stimuli (AB habituate faster than TL) and light-dark induced changes in motor behaviour (AB stronger than TL). Our data show that already in larval stages differences exist between zebrafish of the AB and TL strain confirming and extending data of earlier studies. To what extent the mutation in connexin 41.8, leading to spots rather than stripes in TL, but also (possibly) affecting eye, heart and brain function, is involved in the expression of (some of) these differences needs to be studied. These results emphasise that differences between strains need to be taken into account to enhance reproducibility both within, and between, laboratories.

Uptake of benzo[a]pyrene, but not of phenanthrene, is inhibited by fatty acids in intestinal brush border membrane vesicles of rainbow trout (Oncorhynchus mykiss).

Partial replacement of fish ingredients with vegetable ingredients has elevated levels of polycyclic aromatic hydrocarbons (PAHs) in Atlantic salmon reared on these feeds. PAH uptake in the intestinal tract is postulated to occur in association with lipid absorption and could well be affected by fatty acid composition. We therefore investigated the effects of a fish oil and vegetable oil fatty acid, eicosapentaenoic acid (EPA; 20:5n-3) and oleic acid (18:1n-9) respectively, on the uptake of benzo[a]pyrene (BaP) and phenanthrene (PHE) across the intestinal brush border membrane in the salmonid species rainbow trout (Oncorhynchus mykiss). BaP and PHE were solubilized in mixed micelles composed of either EPA or oleic acid and administrated to isolated brush border membrane vesicles (BBMV) derived from the pyloric caeca, proximal intestine and distal intestine. In the absence of free fatty acids (FFA) trans-membrane uptake of BaP and PHE was 2-7 times lower than the fraction associated to or in the membrane. In the presence of FFA, trans-membrane BaP uptake had decreased by 80 and 40% at the highest EPA and oleic acid concentration, respectively, whereas PHE uptake was virtually unaffected. In the presence of BaP, but not PHE, trans-membrane EPA uptake in BBMV had decreased. This study obtained evidence for PAH-dependent interactions with FFA uptake. We conclude that intestinal BaP uptake is reduced by luminal FFA contents whereas PHE uptake is not. A large fraction of the administrated BaP and PHE remains associated with the cellular membrane of enterocytes and may interfere with uptake of nutrients.

Providing a food reward reduces inhibitory avoidance learning in zebrafish.

As shown in male rats, prior history of subjects changes behavioural and stress-responses to challenges: a two-week history of exposure to rewards at fixed intervals led to slightly, but consistently, lower physiological stress-responses and anxiety-like behaviour. Here, we tested whether similar effects are present in zebrafish (Danio rerio). After two weeks of providing Artemia (brine shrimp; Artemia salina) as food reward or flake food (Tetramin) as control at fixed intervals, zebrafish were exposed to a fear-avoidance learning task using an inhibitory avoidance protocol. Half the number of fish received a 3V shock on day 1 and were tested and sacrificed on day 2; the other half received a second 3V shock on day 2 and were tested and sacrificed on day 3. The latter was done to assess whether effects are robust, as effects in rats have been shown to be modest. Zebrafish that were given Artemia showed less inhibitory avoidance after one shock, but not after two shocks, than zebrafish that were given flake-food. Reduced avoidance behaviour was associated with lower telencepahalic gene expression levels of cannabinoid receptor 1 (cnr1) and higher gene expression levels of corticotropin releasing factor (crf). These results suggest that providing rewards at fixed intervals alters fear avoidance behaviour, albeit modestly, in zebrafish. We discuss the data in the context of similar underlying brain structures in mammals and fish.

The effects of environmental enrichment and age-related differences on inhibitory avoidance in zebrafish (Danio rerio Hamilton).

The inhibitory avoidance paradigm allows the study of mechanisms underlying learning and memory formation in zebrafish (Danio rerio Hamilton). For zebrafish, the physiology and behavior associated with this paradigm are as yet poorly understood. We therefore assessed the effects of environmental enrichment and fish age on inhibitory avoidance learning. Fish raised in an environmentally enriched tank showed decreased anxiety-like behavior and increased exploration. Enrichment greatly reduced inhibitory avoidance in 6-month (6M)- and 12-month (12 M)-old fish. Following inhibitory avoidance, telencephalic mRNA levels of proliferating cell nuclear antigen (pcna), neurogenic differentiation (neurod), cocaine- and amphetamine-regulated transcript 4 (cart4), and cannabinoid receptor 1 (cnr1) were lower in enriched-housed fish, while the ratios of mineralocorticoid receptor (nr3c2)/glucocorticoid receptor α [nr3c1(α)] and glucocorticoid receptor ß [nr3c1(ß)]/glucocorticoid receptor α [nr3c1(α)] were higher. This was observed for 6M-old fish only, not for 24-month (24 M) old fish. Instead, 24 M-old fish showed delayed inhibitory avoidance, no effects of enrichment, and reduced expression of neuroplasticity genes. Overall, our data show strong differences in inhibitory avoidance behavior between zebrafish of different ages and a clear reduction in avoidance behavior following housing under environmental enrichment.

Identification of novel osteogenic compounds by an ex-vivo sp7:luciferase zebrafish scale assay.

Tight interactions among different cell types contributing to bone formation are of key importance in the maintenance of bone homeostasis. Based on the high similarity in responses to (anti)osteogenic signals between zebrafish scales and mammalian bone, we developed and validated a model to screen large numbers of compounds using ex-vivo cultured scales of a sp7:luciferase transgenic zebrafish. This model combines the high predictive value of explant cultures with quick, sensitive, and quantifiable readout converging the effects via various pathways including WNT-signaling, to SP7/osterix promoter activity. Sp7 is pivotal in osteoblast differentiation and activity and its promoter activity provides an excellent surrogate for sp7 expression. Bmp-2a was shown to dose-dependently increase sp7-driven luciferase activity ex vivo. Next, we identified novel effects on bone for 51.7% of the compounds from a small library of WNT-signaling modulators, including a strong osteogenic effect for niclosamide. From all previously characterized compounds, the effect on bone was correctly predicted for 70% of compounds, resulting in a 7% false positive- and 21% false negative rate. The proposed sp7:luciferase zebrafish scale model is unique, powerful and efficient new tool to assess compounds with osteogenic effects, prior to further testing in rodents.

Unpredictable chronic stress decreases inhibitory avoidance learning in Tuebingen long-fin zebrafish: stronger effects in the resting phase than in the active phase.

Zebrafish (Danio rerio Hamilton) are increasingly used as a model to study the effects of chronic stress on brain and behaviour. In rodents, unpredictable chronic stress (UCS) has a stronger effect on physiology and behaviour during the active phase than during the resting phase. Here, we applied UCS during the daytime (active phase) for 7 and 14 days or during the night-time (resting phase) for 7 nights in an in-house-reared Tuebingen long-fin (TLF) zebrafish strain. Following UCS, inhibitory avoidance learning was assessed using a 3 day protocol where fish learn to avoid swimming from a white to a black compartment where they will receive a 3 V shock. Latencies of entering the black compartment were recorded before training (day 1; first shock) and after training on day 2 (second shock) and day 3 (no shock, tissue sampling). Fish whole-body cortisol content and expression levels of genes related to stress, fear and anxiety in the telencephalon were quantified. Following 14 days of UCS during the day, inhibitory avoidance learning decreased (lower latencies on days 2 and 3); minor effects were found following 7 days of UCS. Following 7 nights of UCS, inhibitory avoidance learning decreased (lower latency on day 3). Whole-body cortisol levels showed a steady increase compared with controls (100%) from 7 days of UCS (139%), to 14 days of UCS (174%) to 7 nights of UCS (231%), suggestive of an increasing stress load. Only in the 7 nights of UCS group did expression levels of corticoid receptor genes (mr, grα, grß) and of bdnf increase. These changes are discussed as adaptive mechanisms to maintain neuronal integrity and prevent overload, and as being indicative of a state of high stress load. Overall, our data suggest that stressors during the resting phase have a stronger impact than during the active phase. Our data warrant further studies on the effect of UCS on stress axis-related genes, especially grß; in mammals this receptor has been implicated in glucocorticoid resistance and depression.

Inhibitory avoidance learning in zebrafish (Danio rerio): effects of shock intensity and unraveling differences in task performance.

The zebrafish (Danio rerio) is increasingly used as a model in neurobehavioral and neuroendocrine studies. The inhibitory avoidance paradigm has been proposed as tool to study mechanisms underlying learning and memory in zebrafish. In this paradigm subjects receive a shock after entering the black compartment of a black-white box. On the next day, latency to enter the black compartment is assessed; higher latencies are indicative of increased avoidance learning. Here, we aimed to understand the effects of different shock intensities (0, 1, 3, and 9 V) and to unravel variation in inhibitory avoidance learning in an in-house reared Tuebingen Long-Fin zebrafish (D. rerio) strain. While median latencies had increased in the 1, 3, and 9 V groups, no increase in median latency was found in the 0 V group. In addition, higher shock intensities resulted in a higher number of avoiders (latency ≥180 s) over nonavoiders (latency <60 s). Both changes are indicative of increased avoidance learning. We assessed whole-body cortisol content and the expression levels of genes relevant to stress, anxiety, fear, and learning 2 h after testing. Shock intensity was associated with whole-body cortisol content and the expression of glucocorticoid receptor alpha [nr3c1(alpha)], cocaine- and amphetamine-regulated transcript (cart4), and mineralocorticoid receptor (nr3c2), while avoidance behavior was associated with whole-body cortisol content only. The inhibitory avoidance paradigm in combination with measuring whole-body cortisol content and gene expression is suitable to unravel (genetic) mechanisms of fear avoidance learning. Our data further show differences in brain-behavior relationships underlying fear avoidance learning and memory in zebrafish. These findings serve as starting point for further unraveling differences in brain-behavior relationships underlying (fear avoidance) learning and memory in zebrafish.

Knockdown of monocarboxylate transporter 8 (mct8) disturbs brain development and locomotion in zebrafish.

Allan-Herndon-Dudley syndrome (AHDS) is an inherited disorder of brain development characterized by severe psychomotor retardation. This X-linked disease is caused by mutations in the monocarboxylate transporter 8 (MCT8), an important thyroid hormone transporter in brain neurons. MCT8-knockout mice lack the 2 major neurological symptoms of AHDS, namely locomotor problems and cognitive impairment. The pathological mechanism explaining the symptoms is still obscure, and no cure for this condition is known. The development of an animal model that carries MCT8-related neurological symptoms is warranted. We have employed morpholino-based gene knockdown to create zebrafish deficient for mct8. Knockdown of mct8 results in specific symptoms in the thyroid axis and brain. The mct8-morphants showed impaired locomotor behavior and brain development. More specifically, we observed maldevelopment of the cerebellum and mid-hindbrain boundary and apoptotic clusters in the zebrafish brain. The mRNA expression of zebrafish orthologs of mammalian TSH, thyroid hormone transporters, and deiodinases was altered in mct8 morphants. In particular, deiodinase type 3 gene expression was consistently up-regulated in zebrafish mct8 morphants. The thyroid hormone metabolite tetrac, but not T3, partly ameliorated the affected phenotype and locomotion disability of morphant larvae. Our results show that mct8 knockdown in zebrafish larvae results in disturbances in the thyroid axis, brain, and locomotion behavior, which is congruent with the clinical aspect of impaired locomotion and cognition in patients with AHDS. Taken together, the zebrafish is a suitable animal model for the study of the pathophysiology of AHDS.

Effects of Pro-Tex on zebrafish (Danio rerio) larvae, adult common carp (Cyprinus carpio) and adult yellowtail kingfish (Seriola lalandi).

Aquaculture practices bring several stressful events to fish. Stressors not only activate the hypothalamus-pituitary-interrenal-axis, but also evoke cellular stress responses. Up-regulation of heat shock proteins (HSPs) is among the best studied mechanisms of the cellular stress response. An extract of the prickly pear cactus (Opuntia ficus indica), Pro-Tex, a soluble variant of TEX-OE(®), may induce expression of HSPs and reduce negative effects of cellular stress. Pro-Tex therefore is used to ameliorate conditions during stressful aquaculture-related practices. We tested Pro-Tex in zebrafish (Danio rerio), common carp (Cyprinus carpio L.) and yellowtail kingfish (Seriola lalandi) exposed to aquaculture-relevant stressors (thermal stress, net confinement, transport) and assessed its effects on stress physiology. Heat shock produced a mild increase in hsp70 mRNA expression in 5-day-old zebrafish larvae. Pro-Tex increased basal hsp70 mRNA expression, but decreased heat-shock-induced expression of hsp70 mRNA. In carp, Pro-Tex increased plasma cortisol and glucose levels, while it did not affect the mild stress response (increased plasma cortisol and glucose) to net confinement. In gills, and proximal and distal intestine, stress increased hsp70 mRNA expression; in the distal intestine, an additive enhancement of hsp70 mRNA expression by Pro-Tex was seen under stress. In yellowtail kingfish, Pro-Tex reduced the negative physiological effects of transport more efficiently than when fish were sedated with AQUI-S(®). Overall, our data indicate that Pro-Tex has protective effects under high levels of stress only. As Pro-Tex has potential for use in aquaculture, its functioning and impact on health and welfare of fish should be further studied.

Identification and applications of the Petunia class II Act1/dTph1 transposable element system.

Transposable genetic elements are considered to be ubiquitous. Despite this, their mutagenic capacity has been exploited in only a few species. The main plant species are maize, Antirrhinum, and Petunia. Representatives of all three major groups of class II elements, viz., hAT-, CACTA- and Mutator-like elements, have been identified in Petunia. Here we focus on the research "history" of the Petunia two-element Act1-dTph1 system and the development of its application in forward- and reverse-genetics studies.