Pharyngula Stage Research Articles

Dehorionization of <i>DANIO RERIO</i> embryos to assess the acute toxicity of organomineral chelate complexes

One of the main issues when working with fish embryos is the throughput of the chorion. It is believed that heavy metals can also be blocked by chorion. In the context of studying the acute toxicity of an organomineral chelate complex containing metals such as iron, manganese, copper, zinc, and cobalt, modification of standard tests is required. A possible approach to studying the effect of chorion on the results of acute toxicity may be the removal of chorion (dehorionization). The aim of the study was to compare the sensitivity of dehorionized embryos in assessing the acute toxicity of organomineral chelate complexes. The research was conducted at the Department of Biochemistry and Physiology of the St. Petersburg State University of Veterinary Medicine. The object of the study was the embryos of Danio rerio. To obtain caviar, the parent pairs were planted in spawning jigs. Dehorionization was performed mechanically at 24 hpf (pharyngula stage). After dehorionization, the embryos were transferred to agarose-coated wells of 24-well tablets filled with 2 ml of solution per well. During the study, 15 concentrations of the studied dietary supplement were produced, varying by 2.0 times (64...0.0039 g/l). Working concentrations were prepared by diluting dietary supplements with E3 medium. The results of comparing the sensitivity of de- and nondehorionized embryos in assessing the acute toxicity of organomineral chelate complexes were obtained. It was shown that the LC50 of the studied drug for non-dehorionized embryos is 3.754 g/l, for dehorionized embryos the LC50 is 2.326 g/l. The data indicate that the removal of the chorion leads to an increase in the toxicity of the drug for Danio rerio embryos.

Sturgeon gut development: a unique yolk utilization strategy among vertebrates.

In vertebrates, maternally supplied yolk is typically used in one of two ways: either intracellularly by endodermal cells or extracellularly via the yolk sac. This study delves into the distinctive gut development in sturgeons, which are among the most ancient extant fish groups, contrasting it with that of other vertebrates. Our observations indicate that while sturgeon endodermal cells form the archenteron (i.e., the primitive gut) dorsally, the floor of the archenteron is uniquely composed of extraembryonic yolk cells (YCs). As development progresses, during neurulation, the archenteric cavity inflates, expands laterally, and roofs a semicircle of YCs. By the pharyngula stage, the cavity fully encompasses the YC mass, which begins to be digested at the hatching stage. This suggests a notable deviation in sturgeon gut development from that in other vertebrates, as their digestive tract initiates its function by processing endogenous nutrition even before external feeding begins. Our findings highlight the evolutionary diversity of gut development strategies among vertebrates and provide new insights into the developmental biology of sturgeons.

Year-Round Spawning, Filial Cannibalism, and Embryonic and Larval Development of the Coral Reef Fish Orchid Dottyback, Pseudochromis fridmani

Aquaculture of marine ornamental fish could potentially reduce the fishing pressure on wild stocks by the global aquarium trade, but its expansion is often constrained by the limited understanding on the biology and early life history of candidate species. The orchid dottyback Pseudochromis fridmani is a valuable and popular marine ornamental fish, but scientific reports on its baseline biology, especially the egg filial cannibalistic behaviour, are scarce. The present study documented key aspects of reproductive biology and early ontogeny of P. fridmani in captivity, including reproductive behaviour, patterns of spawning and filial cannibalism throughout a 12-month period by seven pairs of brood fish, as well as the embryonic and larval development. The results showed that the captive spawning of the broodstock pairs generally occurred every 5–11 days, most commonly every 6–8 days, throughout a year. Despite feeding the brood fish to satiation throughout the data collection period, the average monthly rate of egg filial cannibalism reached 55 ± 37%, but the cannibalism frequency appeared to be pair-specific. The egg incubation duration was approximately 96 h at 27 ± 1 °C, and the development of embryos from cleavage, blastula, gastrula, and segmentation to the pharyngula stage is herein described in detail. The newly hatched P. fridmani larvae possessed important structures and organs for first feeding, including pigmented eyes, developed jaws, and a straight-tube gut. The 0 to 14 days post-hatching (DPH) period appeared to be an important larval stage, as P. fridmani larvae were observed to complete major changes in morphology, gut development, and phototactic behaviour by 14 DPH. Under the culture conditions of this study, the earliest transition to the juvenile stage was observed on 31 DPH, and the majority of fish became juveniles by 56 DPH. The results of this study inform baseline aquaculture production protocols and direct future research, particularly to reduce filial cannibalism through broodstock management and to improve larval culture through supporting the early ontogenetic development of P. fridmani. Additionally, these findings form a foundation for further studying the biology and ecology of P. fridmani in the wild.

Assessment of Embryonic and Larval Development of Nile Tilapia under the Traditional and Re-Circulatory Thermostatic System in Relation to Climatic and Water Quality Variations

Embryonic and larval development of tilapia (Oreochromis niloticus) is very vulnerable to climate change. This study was conducted for an assessment of the embryonic and larval development of Nile tilapia in traditional hatchery and re-circulatory thermostatic systems. Daily changes in embryonic and larval development were measured through microscopic observation and image analysis in the laboratory. Climatic data and water quality parameters were measured every day using appropriate devices. Water temperature was varied with room temperature at the traditional hatchery system while it was maintained at 28.50 °C in the re-circulatory thermostatic system. A total of 200 unhatched eggs were stocked in every three trays of both systems. The egg diameters of the gastrula, segmentation, and pharyngula stages were measured at higher (2261.47 ± 81.66 µm, 2646.24 ± 17.98 µm, and 2710.90 ± 16.60 µm) in the re-circulatory thermostatic system than in the traditional hatchery system (2261.07 ± 81.52 µm, 2645.47 ± 18.24 µm, and 2710.01 ± 16.45 µm), respectively. For both systems, egg colors, egg size, black pigments, germinal ring, eye shape, tail, and heartbeat were determined through microscopic observation. Higher hatching and survival rates were found under the re-circulatory thermostatic system (95% and 97%) than under the traditional hatchery system (85% and 81%). About 6 h less hatching time was required under the re-circulatory thermostatic system than under the traditional system. At the end of 30 DAH (Days After Hatching), larval length and weight under the re-circulatory thermostatic system were found to be higher (15.736 ± 0.424 mm and 0.0528 ± 0.004 g) than under the traditional hatchery system (15.518 ± 0.415 mm and 0.050 ± 0.004 g), respectively. Larval growth patterns for both systems were found to have an exponential trend. PCA analysis revealed that two components were identified, one primarily associated with morphometric characteristics and the other with climatic and water quality parameters. These components showed that there were several interrelationships between the morphometric changes and the climatic and water quality parameters. The characteristic changes of larval development under the re-circulatory thermostatic system and the traditional hatchery system were found to be remarkably similar except for some deformities denoted under the traditional hatchery system. The changes of yolk sac, body pigmentation, dorsal and caudal fin shape, eye size, and head length and width were determined from 1 DAH to 30 DAH. After absorbing the yolk sac, ready-made feed was provided. The water temperature was varied from 30.50 °C to 35.50 °C in the traditional hatchery system. The highest air temperature and humidity were 33.87 °C and 69.94% while the lowest were 29.63 °C and 45.62%, respectively, in the traditional hatchery system. There has been no such comprehensive comparative study on hatchery production in Bangladesh, and therefore, further research might be carried out on broader aspects. This research would be highly beneficial for improving seed production at the tilapia fish hatchery level in the country.

Embryonic Development of Black Neon Tetra Hyphessobrycon herbertaxelrodi Géry, 1961

The current study described the embryonic development stages of black neon tetra (Hyphessobrycon herbertaxelrodi), an economic ornamental fish. We characterized the stages of the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching occurring during embryogenesis, which emphasizes changing spectrum of the main development processes from fertilization to incubation. The findings were put forth and photographed by examining live embryos under microscopy. Embryonic development of black neon tetra concluded at 24 ± 0.5°C water temperature at 20-21 hours. The first embryonic division occurs within the first 43 minutes after fertilization, and the process goes on to blastula at 02 hours and 28 minutes. The gastrula stage began at 02.57 hours, while 6 somite segmentation stages were observed to occur at 08.14 hours. Following the pharyngula stage seen between 17 to 20 hours, the hatching occurred at 20-21hours. The results of this study can provide significant benefits to professional breeders in the aquaculture of black neon tetra and other ornamental fish species.

Temporal Dynamic Analysis of Alternative Splicing During Embryonic Development in Zebrafish.

Alternative splicing is pervasive in mammalian genomes and involved in embryo development, whereas research on crosstalk of alternative splicing and embryo development was largely restricted to mouse and human and the alternative splicing regulation during embryogenesis in zebrafish remained unclear. We constructed the alternative splicing atlas at 18 time-course stages covering maternal-to-zygotic transition, gastrulation, somitogenesis, pharyngula stages, and post-fertilization in zebrafish. The differential alternative splicing events between different developmental stages were detected. The results indicated that abundance alternative splicing and differential alternative splicing events are dynamically changed and remarkably abundant during the maternal-to-zygotic transition process. Based on gene expression profiles, we found splicing factors are expressed with specificity of developmental stage and largely expressed during the maternal-to-zygotic transition process. The better performance of cluster analysis was achieved based on the inclusion level of alternative splicing. The biological function analysis uncovered the important roles of alternative splicing during embryogenesis. The identification of isoform switches of alternative splicing provided a new insight into mining the regulated mechanism of transcript isoforms, which always is hidden by gene expression. In conclusion, we inferred that alternative splicing activation is synchronized with zygotic genome activation and discovered that alternative splicing is coupled with transcription during embryo development in zebrafish. We also unveiled that the temporal expression dynamics of splicing factors during embryo development, especially co-orthologous splicing factors. Furthermore, we proposed that the inclusion level of alternative splicing events can be employed for cluster analysis as a novel parameter. This work will provide a deeper insight into the regulation of alternative splicing during embryogenesis in zebrafish.

Comprehensive expression analysis for the core cell cycle regulators in the chicken embryo reveals novel tissue-specific synexpression groups and similarities and differences with expression in mouse, frog and zebrafish.

The core cell cycle machinery is conserved from yeast to humans, and hence it is assumed that all vertebrates share the same set of players. Yet during vertebrate evolution, the genome was duplicated twice, followed by a further genome duplication in teleost fish. Thereafter, distinct genes were retained in different vertebrate lineages; some individual gene duplications also occurred. To which extent these diversifying tendencies were compensated by retaining the same expression patterns across homologous genes is not known. This study for the first time undertook a comprehensive expression analysis for the core cell cycle regulators in the chicken, focusing in on early neurula and pharyngula stages of development, with the latter representing the vertebrate phylotypic stage. We also compared our data with published data for the mouse, Xenopus and zebrafish, the other established vertebrate models. Our work shows that, while many genes are expressed widely, some are upregulated or specifically expressed in defined tissues of the chicken embryo, forming novel synexpression groups with markers for distinct developmental pathways. Moreover, we found that in the neural tube and in the somite, mRNAs of some of the genes investigated accumulate in a specific subcellular localisation, pointing at a novel link between the site of mRNA translation, cell cycle control and interkinetic nuclear movements. Finally, we show that expression patterns of orthologous genes may differ in the four vertebrate models. Thus, for any study investigating cell proliferation, cell differentiation, tissue regeneration, stem cell behaviour and cancer/cancer therapy, it has to be carefully examined which of the observed effects are due to the specific model organism used, and which can be generalised.

Thermal stress induces a positive phenotypic and molecular feedback loop in zebrafish embryos.

Aquatic organisms must cope with both rising and rapidly changing temperatures. These thermal changes can affect numerous traits, from molecular to ecological scales. Biotic stressors are already known to induce the release of chemical cues which trigger behavioural responses in other individuals. In this study, we infer whether fluctuating temperature, as an abiotic stressor, may similarly induce stress-like responses in individuals not directly exposed to the stressor. To test this hypothesis, zebrafish (Danio rerio) embryos were exposed for 24h to fluctuating thermal stress, to medium in which another embryo was thermally stressed before ("stress medium"), and to a combination of these. Growth, behaviour, expression of molecular markers, and of whole-embryo cortisol were used to characterise the thermal stress response and its propagation between embryos. Both fluctuating high temperature and stress medium significantly accelerated development, by shifting stressed embryos from segmentation to pharyngula stages, and altered embryonic activity. Importantly, we found that the expression of sulfide:quinone oxidoreductase (SQOR), the antioxidant gene SOD1, and of interleukin-1β (IL-1β) were significantly altered by stress medium. This study illustrates the existence of positive thermal stress feedback loops in zebrafish embryos where heat stress can induce stress-like responses in conspecifics, but which might operate via different molecular pathways. If similar effects also occur under less severe heat stress regimes, this mechanism may be relevant in natural settings as well.

Congenital Craniofacial Abnormalities in Olive Ridley Sea Turtle Hatchlings

The Olive Ridley (Lepidochelys olivacea), is the most abundant species of sea turtles in the world, but is still threatened according to the red list of endangered species. Apparently, the Olive Ridley turtle seems more prone to congenital malformations compared to other species of sea turtles. In northwestern Mexico, an incidence of congenital malformations of 2% has been reported for this species, most of which occur in the craniofacial region (49%). The objective of this study was to deduce the etiology of the malformations based on the pattern of the affected structures. During the 2019 nesting season at El Caimanero nesting beach, Sinaloa, Mexico, 11 specimens with external craniofacial malformations and 10 that did not show external malformation were collected. The normal animals hatched, but died from other natural causes within the nests. Specimens were divided between histological analysis or µCT with phosphotungstic acid contrast enhancement. Specimens with malformations consistent with a Holoprosencephaly (HPE) phenotype were detected (Table 1). Analysis PTA staining and µCT showed there was a single external naris. There was variability in the morphology of the nasal passages. Other midline structures were also deficient including absent interorbital septum and small premaxilla. However, two other cardinal signs of severe HPE were not seen. The brain was divided into right and left hemispheres and the eyes were separated. There were additional defects in the mandible affecting mesodermal derivatives of the first arch. The tongue was severely hypoplastic and the musculature in the floor of the oral cavity was deficient. The tracheal opening was also displaced posteriorly in the tongue compared to the usual position in the center of the tongue. The nasal defects may be caused by a hypoplastic frontonasal process which allows merging of the nasal pits. The muscular deficiencies in the tongue are likely due to incomplete formation of lingual swellings in the first pharyngeal arch. Nasal development was normal in the majority of animals and the most common defect was mandibular deviation combined with hypoplasia. The defects observed are consistent with a mild form of HPE but also with ciliopathies that include tongue agenesis. Further histological analysis will be carried out to assess cellular and molecular phenotypes. This work suggest pleotropic environmental effects cause the embryonic defects after the eggs are oviposited in the nest. This turtle species lays eggs with embryos in the gastrula stage of development, reaching pharyngula stage, 3-4 days after laying. Therefore, we postulate an exposure to an environmental teratogen in the first 4 days is responsible for the congenital abnormalities.

Post-Ovipositional Developmental Stages of the Japanese Grass Lizard, Takydromus tachydromoides (Squamata: Lacertidae)

The Japanese grass lizard, Takydromus tachydromoides, is a species endemic to Japan. While several studies have investigated the reproductive ecology of T. tachydromoides, the embryogenesis of this species has not been reported in detail. In the current study, we observed the external morphological characteristics of 247 embryo specimens that developed under a constant temperature (28°C) throughout the duration from oviposition to hatching. We identified 17 consecutive developmental stages for the post-ovipositional developmental process of this species based on the staging criteria that have been widely used for the description of embryogenesis of lizards. The youngest embryos on the day of oviposition corresponded to stage 26, which is a relatively early period in pharyngula stages, and juveniles hatched at stage 42, approximately 30 days after oviposition. The entire developmental sequence of key morphological features was shared with other species of Lacertidae, except for the timing of the beginning of the first body pigmentation. This is the first description of the complete sequence of post-ovipositional developmental stages for the oviparous species of Lacertidae, providing valuable information for further evolutionary developmental studies.

Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 ​h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b−/− and maternal zygotic (MZ)cdx1b−/− mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b−/− mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b−/− mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b−/− mutant embryos at 72 hpf. On the other hand, Kupffer’s vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b−/− mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 ​mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23–24 ​s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Inhibition of ecto-5\u2032-nucleotidase and adenosine deaminase is able to reverse long-term behavioural effects of early ethanol exposure in zebrafish (Danio rerio)

The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5′-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5′-nucleotidase and adenosine deaminase activities.

Effect of nano-encapsulation of β-carotene on Xenopus laevis embryos development (FETAX)

Vitamin A plays a vital role during embryo development as most precursor of embryonic retinoic acid, a key morphogen during embryogenesis. Carotenoids, including β-carotene, are important vegetal source of Vitamin A and in contrast to teratogenic potential of animal-derived retinoids, β‐carotene is usually considered freed from embryotoxic effects and supplements in pregnancy with β-carotene are suggested. The aim of the present work is to evaluate the effect of bulk and nano-encapsulated β-carotene on embryo development, by using the animal model Frog Embryo Teratogenesis Assay: Xenopus- FETAX. Xenopus laevis embryos were exposed from late gastrula till pharyngula (the phylotypic stage for vertebrates) to the concentrations of BULK β-carotene 150-3000 ng/mL and NANO β-carotene 0.75-30 ng/mL. At pharyngula stage, some embryos were processed for whole mount neural crest cell immunostaining, while others embryos were allowed to develop till tadpole for morphological and histological evaluation of neural crest cells-derived structures. In this model, the nano-encapsulated β-carotene induced specific malformations at craniofacial and eye level, while the bulk formulation only induced developmental delays. Finally, the applied alternative animal model resulted a rapid and sensitive screening method able to re-evaluate the teratogenic profile of nano-encapsulated micronutrients.

Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action

In (eco)toxicology, there is a critical need for efficient methods to evaluate the neurotoxic potential of environmental chemicals. Recent studies proposed analysis of early coiling activity in zebrafish embryos as a powerful tool for the identification of neurotoxic compounds. In order to demonstrate that the analysis of early tail movements of zebrafish embryos allows for the discrimination of neurotoxicants acting via different mechanisms, the present study investigated the effects of four different neurotoxicants on the embryogenesis (fish embryo toxicity test) and early tail coiling movements of zebrafish embryos. Cadmium predominantly increased the frequency of tail coiling at the late pharyngula stage. Dichlorvos delayed embryonic development and caused convulsive tail movements resulting in prolonged duration of tail coils. Embryos exposed to teratogenic concentrations of fluoxetine and citalopram displayed absence of spontaneous tail movements at 24 h post-fertilization. In contrast, a non-teratogenic test concentration of citalopram decreased coiling frequency at multiple time points. Results demonstrated that the analysis of tail coiling movements of zebrafish embryos has the potential to discriminate neurotoxic compounds with different primary modes of action. In addition, chemical-induced effects on coiling activity were shown to potentially overlap with effects on embryogenesis. Further studies are needed to clarify the interplay of unspecific developmental toxicity of neurotoxic chemicals and effects resulting from specific neurotoxic mechanisms.

The effects of salinity on reproductive development and egg and larvae survival in the spotted scat Scatophagus argus under controlled conditions

In the present study, we report the first successful instance of controlled reproduction in Scatophagus argus, which has recently emerged as a new aquaculture resource. The controlled reproduction process for S. argus was optimized with regard to salinity acclimation. Gonadal maturation was affected by salinity in both sexes. Levels of plasma 17β-estradiol (E2) and 11-ketotestosterone (11-kT) were salinity dependent and increased significantly with the duration of acclimation. Plasma levels of gonadal steroids were higher in fish held at 25‰ salinity. The highest gonadosomatic indices (GSI), 15.1 ± 1.6 in the female and 6.4 ± 1.2 in the male, were also observed at 25‰ salinity. Nevertheless, the optimal salinity for S. argus embryonic development and larval culture was 15‰. Thus, the salinity requirement for gonadal maturation and early development are quite different. The use of advanced reproductive technologies combining salinity acclimation and stimulation of luteinizing hormone-releasing hormone analog (LHRH-A2) resulted in a fertilization rate of 83.2%–91.3% and embryonic survival rates of over 90%. Embryos of S. argus at the 2-cell, blastula, gastrula and pharyngula stages were observed. Most embryos hatched after 21.0 hr of incubation at 28.0 ± 1.0°C. The development of larvae into juveniles was completed at 40–45 days posthatch (dph). In this study, we provide information about the controlled reproduction of S. argus and identify the optimal environmental parameters for S. argus embryonic and larval culture, with the aim of developing reliable reproductive techniques for its mass production.

Comparative analysis of embryonic muscle development in wildtype zebrafish and its intermuscular bone deficiency mutant

PDF HTML阅读 XML下载 导出引用 引用提醒 肌间刺缺失突变对斑马鱼胚胎发育过程中肌肉发育的影响 DOI: 作者: 作者单位: 1. 上海海洋大学, 水产科学国家级实验教学示范中心, 上海 201306;2. 中国水产科学研究院黑龙江水产研究所, 黑龙江 哈尔滨 150070 作者简介: 杨建(1987-),男,博士研究生,主要从事鱼类遗传育种的研究.E-mail:last0templar@126.com 通讯作者: 中图分类号: S917 基金项目: 中国水产科学研究院基本科研业务费项目（2016HY-ZD0301）. Comparative analysis of embryonic muscle development in wildtype zebrafish and its intermuscular bone deficiency mutant Author: Affiliation: 1. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;2. Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为阐明肌间刺缺失对斑马鱼（、）在囊胚期（3 hpf，hours post fertilization）、原肠胚期（6 hpf）、体节期（12 hpf）、咽囊期（24 hpf）和孵化期（72 hpf）5个发育时期的表达情况，对比分析了肌间刺缺失对斑马鱼胚胎发育过程中肌肉发育特异性基因的时序表达差异和肌肉发育的影响，以及对受精率、孵化率和畸形率的影响。结果显示，5个特异性基因在肌间刺缺失突变型与野生型斑马鱼各发育时期的表达量无显著性差异（、基因在囊胚期和原肠胚期表达量很低，在体节期和咽囊期表达量迅速升高，并在孵化期开始下降。此外，受精率、孵化率和畸形率的统计结果显示，肌间刺突变型与野生型间亦无显著性差异（>0.05）。综上，推测肌间刺的缺失对斑马鱼胚胎发育及胚胎发育过程中肌肉的发育没有影响。 Abstract:In order to explore the difference in embryonic muscle development between an intermuscular bone (Imb) deficiency mutant and wildtype zebrafish, we used a qRT-PCR method to analyze the expression of 5 muscle-specific genes, including , in the development stages blastula (3 hpf, hours post fertilization), gastrula (6 hpf), segmentation (12 hpf), pharyngula (24 hpf), and hatching period (72 hpf), and discussed the impact of Imb's deficiency on muscle development during embryonic development. Furthermore, the differences in fertilization rate, hatching, and abnormal rate of larvae were assessed. The results showed that the expression of genes were elevated in the gastrula stage, and downregulated in the segmentation and pharyngula stages; the expression levels of in the blastula and gastrula stages were extremely low, and were upregulated significantly in the segmentation and pharyngula stages before being downregulated in the hatching period. The results also showed that there were no significant differences in 5 muscle-specific genes' expression between the wildtype zebrafish and its Imb deficiency mutant. Additionally, no significant difference in fertilization rate, hatching, and abnormal rate occurred between the two Imb phenotypes. In summary, the deletion of Imb do not impact muscle development or the survival of embryos during embryonic development of zebrafish. 参考文献 相似文献 引证文献

Persistent increase in ecto‑5′‑nucleotidase activity from encephala of adult zebrafish exposed to ethanol during early development

Prenatal alcohol exposure causes alterations to the brain and can lead to numerous cognitive and behavioral outcomes. Long-lasting effects of early ethanol exposure have been registered in glutamatergic and dopaminergic systems. The purinergic system has been registered as an additional target of ethanol exposure. The objective of this research was to evaluate if the ecto‑5′‑nucleotidase and adenosine deaminase activities and gene expression of adult zebrafish exposed to 1% ethanol during early development could be part of the long-lasting targets of ethanol. Zebrafish embryos were exposed to 1% ethanol in two distinct developmental phases: gastrula/segmentation (5–24 h post-fertilization) or pharyngula (24–48 h post-fertilization). At the end of three months, after checking for morphological outcomes, the evaluation of enzymatic activity and gene expression was performed. Exposure to ethanol did not promote gross morphological defects; however, a significant decrease in the body length was observed (17% in the gastrula and 22% in the pharyngula stage, p < 0.0001). Ethanol exposure during the gastrula/segmentation stage promoted an increase in ecto‑5′‑nucleotidase activity (39.5%) when compared to the control/saline group (p < 0.0001). The ecto‑5′‑nucleotidase gene expression and the deamination of adenosine exerted by ecto and cytosolic adenosine deaminase were not affected by exposure to ethanol in both developmental stages. HPLC experiments did not identify differences in adenosine concentration on the whole encephala of adult animals exposed to ethanol during the gastrula stage or on control animals (p > 0.05). Although the mechanism underlying these findings requires further investigation, these results indicate that ethanol exposure during restricted periods of brain development can have long-term consequences on ecto‑5′‑nucleotidase activity, which could have an impact on subtle sequels of ethanol early exposure.

Segmentation pattern of zebrafish caudal fin is affected by developmental temperature and defined by multiple fusions between segments.

Caudal-fin lepidotrichia is composed of numerous segments, which are linked to each other by intersegmental joints. During fish growth, lepidotrichia elongate by the addition of new segments at their distal margin, whereas the length of each segment remains constant after it is formed. In the present paper, we examined whether the water temperature affects the segmentation pattern of the juvenile and adult caudal fin. For this purpose, zebrafish (Danio rerio) embryos and larvae were exposed to three different temperature conditions (24°C, 28°C, and 32°C) from the pharyngula stage (1 day postfertilization [dpf]) to metamorphosis, whereas the control temperature (28°C) was applied to all the groups before and after this period. Results demonstrated that water temperature had a significant effect on the length of the segments of each lepidotrichium, at both the juvenile and adult stages. Moreover, at higher temperatures, there was a significant proximal shift of the position of the first bifurcation of the second lepidotrichium of the dorsal lobe. At all the experimental conditions, the length of proximal segment was not constant during fish growth, but it followed a discontinuous saltatory growth. Histological analysis of the proximal lepidotrichia segments revealed that the observed apparent growth of segments is the result of fusions between segments. Fusion occurs not by mineralization of the intersegmental joints, but by bone deposition around the joints.

Early exposure to ethanol is able to affect the memory of adult zebrafish: Possible role of adenosine

Ethanol is one of the most widely consumed drugs in the world, and the effects of ethanol during early development include morphological and cognitive problems. The regulation of adenosine levels is essential for the proper function of major neurotransmitter systems in the brain, particularly glutamate and dopamine; thus, the investigation of the relation of adenosine and memory after early ethanol exposure becomes relevant. Embryos of zebrafish were exposed to 1% ethanol during two distinct developmental stages: gastrula/segmentation or pharyngula. The evaluation of memory, morphology, and locomotor parameters was performed when fish were 3 months old. The effect of ecto-5′-nucleotidase and adenosine deaminase inhibition on the consequences of ethanol exposure with regard to memory formation was observed. Morphological evaluation showed decreases in body length and the relative telencephalic and cerebellar areas in ethanol exposed animals. The locomotor parameters evaluated were not affected by ethanol. In the inhibitory avoidance paradigm, ethanol exposure during the gastrula/segmentation and pharyngula stages decreased zebrafish memory retention. When ethanol was given in the pharyngula stage, the inhibition of ecto-5′-nucleotidase in the acquisition phase of memory tests was able to revert the effects of ethanol on the memory of adults. These findings suggest that the increased adenosine levels caused by ethanol could alter the neuromodulation of important components of memory formation, such as neurotransmitters. The adjustment of adenosine levels through ecto-5′-nucleotidase inhibition appears to be effective at restoring normal adenosine levels and the acquisition of memory in animals exposed to ethanol during the pharyngula stage.

Evaluation of the influence of arsenic on embryonic development of zebrafish (Danio rerio)

Arsenic is one of heavy metal elements which is dangerous to human and aquatic animals. Accumulating in the body,arsenic causes serious damage to organ systems such as the nervous system, circulatory system, digestive system, reproductive system, ect and even cancer, skin cancer is the most common type. This study aims to evaluate the influences of Arsenic (As) concentrations on different stages in embryonic development of zebrafish (Danio rerio): blastura, gastrula, segmentation, pharyngula and hatching. After mating, embryos were exposed to As in the examined concentrations: 0, 20, 50, 80, 110, 140, 170, 200, 230, 260 µg/L in the Hank’s embryonic medium. It was observed that the survival rate of embryonic zebrafish decreased gradually corresponding to the increasing the concentration of As and the development stages of the embryo. However, examined concentrations of As in this study did not reach the threshold lethal concentration (LCt50) of embryos. With the increasing of examined As concentrations, the heartbeat increased linearly, the body turning beat of each stage decreased linearly. The highest heartbeat was 237.73±1.87 beat/min in 260 µg/L concentration while it was 197.60±2.20 beat/min in the control group (p &lt; 0.05) in the hatching stage. The lowest body turning was 2.53 beat/min while it was 5.50 beat/min (p &lt; 0.05) in the control group in the pharyngula stage. Moreover, the As prolonged the hatching duration and reduced the hatching rate of embryonic zebrafish, after 72 hour fertilization, the hatching rate was 77.78% in 260 µg/L concentration while it was 98.86% in the control group (p &lt; 0.05).