Head-trunk Angle Research Articles

Prednisolone Accelerates Embryonic Development of Zebrafish via Glucocorticoid Receptor Signaling at Low Concentrations.

Synthetic glucocorticoids have been widely detected in aquatic ecosystems and may pose a toxicological risk to fish. In the present study, we described multiple end point responses of zebrafish to a commonly prescribed glucocorticoid, prednisolone (PREL), at concentrations between 0.001 and 9.26 μg/L. Of 23 end points monitored, 7 were affected significantly. Significant increases in the frequency of yolk extension formation, spontaneous contraction, heart rate, and ocular melanin density and significant decreases of ear-eye distance at PREL concentrations of 0.001 μg/L and above clearly pointed to the acceleration of embryonic development of zebrafish by PREL. Further confirmation came from the alterations in somite numbers, head-trunk angle, and yolk sac size, as well as outcomes obtained via RNA sequencing, in which signaling pathways involved in tissue/organ growth and development were highly enriched in embryos upon PREL exposure. In addition, the crucial role of glucocorticoid receptor (GR) for PREL-induced effects was confirmed by both, the coexposure to antagonist mifepristone (RU486) and GR-/- mutant zebrafish experiments. We further demonstrated similar accelerations of embryonic development of zebrafish upon exposure to 11 additional glucocorticoids, indicating generic adverse effect characteristics. Overall, our results revealed developmental alterations of PREL in fish embryos at low concentrations and thus provided novel insights into the understanding of the potential environmental risks of glucocorticoids.

Toxicity and teratogenicity investigation of methanolic bark extract of Terminalia arjuna (Roxb.) wight and arn in zebrafish (Danio rerio) embryo model

The present research work aims to establish the teratogenic effect of the Terminalia arjuna (T.arjuna) bark extract using fish embryos through zebrafish embryo model. In the present study, selected zebrafish embryos were exposed to a range of methanolic extract of T.arjuna bark and the changes the embryos undergo were identified by comparing them with a control group. The embryo toxicity was assessed by observing morphological abnormalities after 24, 48, 72 and 96 h exposure and end teratogenicity points were assessed after 48 h exposure. All the embryos were imaged and analyzed for various developmental anomalies such as coagulated embryos, lack of somite formation, non-detachment of the tail, and lack of heartbeat, using imageJ software. From the results obtained, it was found that the embryos exposed with 10 mg/L of T.arjuna showed regular developmental patterns similar to control up to 72 h 5 % mortality was observed post 72 h period. 50 and 100 mg/L showed 100 % mortality after 96 h and 72 h respectively. At 48 h, Embryos treated with 50 mg/L of T.arjuna showed 5.58 % decreased heart beat rate, 7.89 % of decrease in eye diameter decreased head trunk angle of 3.56 % and increased yolk sac width of 0.79 %. After 48 h exposure to 50 and 100 mg/L of T.arjuna extract, 45 % and 85 % of the embryos showed the teratogenic endpoint of coagulation respectively with bent tail. Increased yolk sac width was observed in 100 mg/L treated embryos with 1.01 % after 24 h, also at 48 h decreased heart beat rate of 4.02 %,16.8 % of increased eye diameter, with decreased head trunk angle of 2.35 %, decreased yolk sac width of 5.13 % were observed. After 48 h exposure of T.arjuna bark extract, the morphological, physiological changes and teratogenic end points were eminent in 50 and 100 mg/L treated embryos, thus the results signify that methanolic bark extract of T. arjuna possess teratogenic effects at this concentration in zebrafish embryos.

Developmental abnormalities and epigenetic alterations in medaka (Oryzias latipes) embryos induced by triclosan exposure

Triclosan (TCS), an antibacterial and antifungal agent present in some consumer products, has been detected in the environment at varying concentrations. TCS exposure has been found to cause developmental abnormalities and endocrine disruption in various species of fish. It is not clearly understood whether TCS exposure causes epigenetic alterations in developing embryos and their germ cells. In the present study, we examined the effects of TCS exposure (0, 50, 100 and, 200 μg/L) on embryonic development and primordial germ cells (PGCs), which are precursors of sperm and eggs, in medaka (Oyzias latipes). Developmental TCS exposure from 8 h post-fertilization through 15 days post-fertilization (dpf) resulted in several developmental abnormalities, including enlarged yolk sac, decreased head trunk angle (HTA), and severe edema in the pericardial region. The male ratio increased in the 100 μg/L TCS exposure group, which was negatively correlated with the expression of cyp19ala (a gene encoding aromatase) and arα (androgen receptor alpha). Developmental 50 μg/L TCS exposure resulted in global hypomethylation in the whole body but not in the isolated PGCs. Expression of the gene encoding DNA methyltransferases (dnmt1 and dnmt3aa) was decreased by 50 μg/L TCS exposure both in the whole body and PGCs. TCS altered the expression of genes encoding enzymes involved in DNA methylation and demethylation in PGCs, suggesting epigenetic effects on germ cells. The present results demonstrate that the embryos exposed to the tested concentrations of TCS develop deformities during the early life stages and that the TCS within this range possesses endocrine disrupting properties potential enough to alter sex ratios of developing embryos.

Teratogenic effects of aqueous extract of Ficus glomerata leaf during embryonic development in zebrafish (Danio rerio)

Over past few decades, many reports published in scientific journals describing medicinal properties of Ficus glomerata. However, its effects on embryonic development and its safety characteristics have not been studied. The purpose of this investigation was to determine LC50 value and study the effect of aqueous extract of Ficus glomerata leaf on developmental abnormalities in zebrafish embryos. LC50 value of aqueous extract of Ficus glomerata leaf was calculated by using probit analysis. Effect on percentage hatchability, heartbeat rate, total body length and developmental morphological abnormalities i.e delayed growth, abnormal movement, tail detachment, abnormal head trunk angle, scoliosis/ flexure, yolk sac edema were recorded. Aqueous extract of Ficus glomerata leaf revealed LC50 of 239.88 ppm. The result showed a significant reduction in percent hatchability (p

Assay for the developmental toxicity of safflower (Carthamus tinctorius L.) to zebrafish embryos/larvae

ObjectiveTo explore the developmental toxicity and the potential toxicological mechanism of safflower (Carthamus tinctorius L.) on zebrafish embryos/larvae. MethodsMortality, malformations and increased apoptosis induced by safflower were assessed in zebrafish embryos from 6 to 96 hours post-fertilization. Enzymes and genes in the anti-oxidative and apoptotic pathways were also assayed. ResultsThe lethal concentration 50 of safflower to zebrafish embryos was 345.6 mg/L. Hatching inhibition, abnormal spontaneous movement, depressed heart rate, pericardial edema, yolk sac edema, abnormal head-trunk angle, inhibition of melanin release, enlarged yolk, and short body length were observed in safflower-treated zebrafish. Additional apoptotic cells mainly appeared around the heart. Safflower exposure changed the activities of defense enzymes (SOD↑, CAT↑, MDA↑, GPX without trend), increased MDA content, decreased caspase-3 activity, and altered mRNA levels of related genes (ogg1↓, p53↓, Cu/Zn-sod↑, Mn-sod↓, cat↓, gpx↑). ConclusionSafflower exhibits developmental toxicity for zebrafish embryos/larvae. The developing heart was speculated as the target organ of toxicity. Oxidative stress and increased apoptosis have roles in the developmental toxicity of safflower. This article provides a novel method to research the teratogenicity and possible mechanisms of toxicity of traditional Chinese medicines that are prohibited or contraindicated in pregnant women.

Persistent hydrocephalus due to postural activation of a ventricular shunt anti-gravity device

The ever present need to balance over drainage with under drainage in hydrocephalus has required innovations including adjustable valves with antigravity devices. These are activated in the vertical position to prevent siphoning. We describe a group of bedridden patients who presented with unexplained under drainage caused by activation of antigravity shunt components produced by peculiar head/body position.Retrospective single centre case series of hydrocephalus patients, treated with ventriculo-peritoneal (VP) shunt insertion between April 2014 – February 2016. These patients presented with clinical and radiological under drainage syndrome. Medical notes were reviewed for clinical picture and outcome. Radiological studies were reviewed assessing shunt placement and ventricular size.Seven patients presented with clinical and radiological under drainage syndrome. A consistent posturing of long term hyper-flexion of the neck whilst lying supine was observed. All patients had similar shunt construct (adjustable Miethke ProGAV valve and shunt assistant anti-gravity component). In each of those patients a hypothesis was formulated that neck flexion was activating the shunt assistance anti-gravity component in supine position. Five patients underwent shunt revision surgery removing the shunt assistant device from the cranium and adding an anti-gravity component to the shunt system at the chest. One had the shunt assistant completely removed and one patient was managed conservatively with mobilisation. All patients had clinical and radiological improvement.Antigravity shunt components implanted cranially in bedridden hydrocephalus patients will produce underdrainage due to head flexion induced anti-gravity device activation. In these patients, anti-gravity devices should be placed at the chest. Alternatively, special nursing attention should be paid to head-trunk angle.

Early-life glucocorticoids programme behaviour and metabolism in adulthood in zebrafish.

Glucocorticoids (GCs) in utero influence embryonic development with consequent programmed effects on adult physiology and pathophysiology and altered susceptibility to cardiovascular disease. However, in viviparous species, studies of these processes are compromised by secondary maternal influences. The zebrafish, being fertilised externally, avoids this problem and has been used here to investigate the effects of transient alterations in GC activity during early development. Embryonic fish were treated either with dexamethasone (a synthetic GC), an antisense GC receptor (GR) morpholino (GR Mo), or hypoxia for the first 120h post fertilisation (hpf); responses were measured during embryonic treatment or later, post treatment, in adults. All treatments reduced cortisol levels in embryonic fish to similar levels. However, morpholino- and hypoxia-treated embryos showed delayed physical development (slower hatching and straightening of head–trunk angle, shorter body length), less locomotor activity, reduced tactile responses and anxiogenic activity. In contrast, dexamethasone-treated embryos showed advanced development and thigmotaxis but no change in locomotor activity or tactile responses. Gene expression changes were consistent with increased (dexamethasone) and decreased (hypoxia, GR Mo) GC activity. In adults, stressed cortisol values were increased with dexamethasone and decreased by GR Mo and hypoxia pre-treatments. Other responses were similarly differentially affected. In three separate tests of behaviour, dexamethasone-programmed fish appeared ‘bolder’ than matched controls, whereas Mo and hypoxia pre-treated fish were unaffected or more reserved. Similarly, the dexamethasone group but not the Mo or hypoxia groups were heavier, longer and had a greater girth than controls. Hyperglycaemia and expression of GC responsive gene (pepck) were also increased in the dexamethasone group. We conclude that GC activity controls many aspects of early-life growth and development in the zebrafish and that, like other species, manipulating GC status pharmacologically, physiologically or genetically in early life leads to programmable metabolic and behavioural traits in adulthood.

Developmental toxicity of the PBDE metabolite 6-OH-BDE-47 in zebrafish and the potential role of thyroid receptor β

6-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47) is both a polybrominated diphenyl ether (PBDE) flame retardant metabolite and a marine natural product. It has been identified both as a neurotoxicant in cell-based studies and as a developmental toxicant in zebrafish. However, hydroxylated PBDE metabolites are also considered thyroid hormone disruptors due to their structural similarity to endogenous thyroid hormones. The purpose of this study was to evaluate the effects of 6-OH-BDE-47 on a developmental pathway regulated by thyroid hormones in zebrafish. Morphological measurements of development (head trunk angle, otic vesicle length, and eye pigmentation) were recorded in embryos at 30h post fertilization (hpf) and detailed craniofacial morphology was examined in 4 day old larvae using cartilage staining. Exposure to 6-OH-BDE-47 resulted in severe developmental delays. A 100nM concentration resulted in a 26% decrease in head trunk angle, a 54% increase in otic vesicle length, and a 42% decrease in eye pigmentation. Similarly, altered developmental morphology was observed following thyroid receptor β morpholino knockdown, exposure to the thyroid hormone triiodothyronine (T3) or to thyroid disrupting chemicals (TDC; iopanoic acid and propylthiouracil). The threshold for lower jaw deformities and craniofacial cartilage malformations was at doses greater than 50nM. Of interest, these developmental delays and effects were rescued by microinjection of TRβ mRNA during the 1–2 cell stage. These data indicate that OH-BDEs can adversely affect early life development of zebrafish and suggest they may be impacting thyroid hormone regulation in vivo through downregulation of the thyroid hormone receptor.

Assessment of developmental delay in the zebrafish embryo teratogenicity assay

In this study we analyzed some aspects of the assessment of developmental delay in the zebrafish embryotoxicity/teratogenicity test and explored the suitability of acetylcholinesterase (AChE) activity as a biochemical marker and as a higher throughput alternative to morphological endpoints such as head–trunk angle, tail length and morphological score. Embryos were exposed from 4 to 52h post-fertilization (hpf) to a selection of known embryotoxic/teratogen compounds (valproic acid, retinoic acid, caffeine, sodium salicylate, glucose, hydroxyurea, methoxyacetic acid, boric acid and paraoxon-methyl) over a concentration range. They were evaluated for AChE activity, head–trunk angle, tail length and several qualitative parameters integrated in a morphological score. In general, the different patterns of the concentration–response curves allowed distinguishing between chemicals that produced growth retardation (valproic and methoxyacetic acid) and chemicals that produced non-growth-delay related malformations. An acceptable correlation between the morphological score, AChE activity and head–trunk angle as markers of developmental delay was observed, being AChE activity particularly sensitive to detect delay in the absence of malformations.

Early morphological variation and induction of phenotypic plasticity in Patagonian pejerrey

The aim of this work was to study two aspects of phenotypic plasticity in the Patagonian pejerrey Odontesthes hatcheri (Teleostei: Atherinopsidae) the dependence of the early morphology on developmental time and temperature, and the induction of morphological changes by controlled feeding in juveniles. Newly hatched free embryos, incubated at two different temperatures (13 and 18oC), and juveniles were used for the study and induction of phenotypic plasticity. Body and head shapes were analyzed with geometric morphometrics and linear measurements. Our results showed that shape variation at hatching was related to the bending of the embryo head on the yolk sac, increasing the head-trunk angle due to progressive straightening of the embryo. The head-trunk angle was related with temperature at incubation, with embryos incubated at higher temperature being more bent. Embryos that hatched earlier had bigger yolk sacs than those that hatched later. In juveniles, controlled feeding experiments added new morphological variation to that of wild juveniles. In all comparisons, the slenderness of the head, the size of premaxilla and jaw, and the position of the eye showed an enlarged variation due to controlled feeding. These results will contribute to comprehending the complexity of the morphological variation of O. hatcheri.

Combined antisense knockdown of type 1 and type 2 iodothyronine deiodinases disrupts embryonic development in zebrafish ( Danio rerio)

Thyroid hormones (THs) are important regulators of gene expression during vertebrate development. In teleosts, early embryos rely on the maternal TH deposit in the egg yolk, consisting predominantly of T 4. Activation of T 4 to T 3 by iodothyronine deiodinases (Ds) may therefore be an important factor in determining T 3-dependent development. In zebrafish, both Ds capable of T 3 production, D1 and D2, are first expressed very early during embryonic development. We sought to determine their relative importance for zebrafish embryonic development by inhibiting their expression via antisense oligonucleotides against D1 and D2, and by a combined knockdown of both deiodinases. The impact of these treatments on the rate of embryonic development was estimated via three morphological indices: otic vesicle length, head-trunk angle and pigmentation index. Knockdown of D1 alone seemed not to affect developmental progression. In contrast, D2 knockdown resulted in a clear developmental delay in all parameters scored, suggesting that D2 is the major contributor to TH activation in developing zebrafish embryos. Importantly, combined knockdown of D1 and D2 caused not only a more pronounced developmental delay than D2 knockdown alone but also the appearance of dysmorphologies in a substantial minority of treated embryos. This shows that although D1 may not be essential in euthyroid conditions, it may be crucial under depleted thyroid status as is the case when T 3 production by D2 is inhibited. These results indicate that zebrafish embryos are dependent on T 4 uptake and its subsequent activation to T 3, and suggest that substantial inhibition of embryonic T 4 to T 3 conversion reduces intracellular T 3 availability below the threshold level necessary for normal development.

Type 2 Iodothyronine Deiodinase Is Essential for Thyroid Hormone-Dependent Embryonic Development and Pigmentation in Zebrafish

Despite the known importance of thyroid hormones (THs) in vertebrate growth and development, the role of tissue-specific TH activation in early embryogenesis remains unclear. We therefore examined the function of type 2 iodothyronine deiodinase (D2), one of the two tissue-specific enzymes catalyzing the conversion of T4 to T3, in developing zebrafish embryos (Danio rerio). Microinjection of early embryos with antisense oligonucleotides targeting either the D2 translation start site or the splice junction between the first exon and intron induced delays in development and pigmentation, as determined through the measurement of otic vesicle length, head-trunk angle, and pigmentation index at 31 h after fertilization. The antisense-induced delays in developmental progression and pigmentation were reversible through treatment with T3, suggesting that these phenotypic effects may be due to the depletion of intracellular T3 levels. Additional evidence for this hypothesis was provided by quantitative RT-PCR analysis of TH receptor-beta expression in D2 knockdown embryos, revealing a significant down-regulation of this T3-induced transcript that could be reversed by T3 treatment. Tyrosinase expression was also down-regulated in D2 knockdown embryos to a greater degree than could be predicted by the observed delay in developmental progression, suggesting that reduced D2 activity and resultant low intracellular T3 availability may directly influence pigmentation in zebrafish. These data indicate that TH activation by D2 is essential for embryonic development and pigmentation in zebrafish.

Perceived Head-Trunk Angle During Microgravity Produced by Parabolic Flight

Neck proprioceptors are essential for orienting the head relative to the trunk. However, it has been shown that the available information about the relationship of gravity to different body parts would augment the clues about their relative orientation. In weightlessness, the absence of relevant body position signals from the otoliths and other inertial graviceptors requires the substitution of other sensory information. The aim of the present study was to investigate the ability of humans to accurately locate the head relative to the trunk in microgravity. Experiments were conducted during two separate sessions: on Earth and during parabolic flights. Volunteers were asked to adjust a visual rod until it looked parallel to their head or trunk axis in two different segmental configurations: head and trunk aligned or head tilted. There was no effect of microgravity when the head and trunk were aligned. However, when the head was tilted with respect to the trunk, the orientation of the visual rod relative to the head or the trunk (visual egocentric coordinates) was deviated toward the head tilt, although the orientation between the body parts themselves (head-trunk angle) was correctly estimated. These results suggested that, in microgravity, the proprioceptive signals from neck muscles seem sufficient to provide accurate head on trunk information. However, the representation of orientation in visual space was modified. This experiment provides evidence for the role of gravity on the visual perception of head- and trunk-based egocentric coordinates.