Zebrafish as a model to study the neuroendocrine system and toxicity of endocrine disruptors

Zebrafish is a popular vertebrate model system to study development and perform genetic analysis. It offers numerous advantages such as small size, short generation time, high fecundity, rapid and ex utero development of embryos and optically transparent embryos. Zebrafish is genetically closely related to humans and share high similarity in developmental processes, physiology and behavior. In addition, recent advances in forward and reverse genetics coupled with the availability of a large number of mutants makes zebrafish a useful model for genetic analysis of development. Furthermore, the above mentioned advantages offered by this model organism will be a valuable resource for toxicologists not only to assess toxicity of endocrine disrupting chemicals (EDCs) but also to dissect the mechanisms of toxicity of EDCs. The development and function of the neuroendocrine system in zebrafish is to a great extent similar to other vertebrates. Thus, in an aim to understand the development of the neuroendocrine stress axis we have characterized the expression of corticotropin-releasing hormone (crh) in the embryonic brain of zebrafish. Transcripts of crh were detected in a broad range of adult tissues and also during embryonic and larval stages. Whole-mount in situ hybridization analysis revealed distribution of crh transcripts in various regions of the developing brain including telencephalon, preoptic area, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, hindbrain and retina. Expression of crh in the preoptic area and in extra-hypothalamic regions is consistent with its roles as a hypophysiotropin and a neuromodulator. Estrogen receptors are ligand activated transcription factors involved in regulating the neuroendocrine axis of reproduction. We have analyzed the mRNA levels of the esr genes in the absence and presence of exogenous ligands. The three esr genes, esr1 (ERalpha), esr2b (ERbeta1) and esr2a (ERbeta2) were expressed in all adult tissues tested in the absence of exogenous ligands. Expressions of esr1 and esr2a were altered in the liver, brain, testis and intestine following ligand treatment (17β- estradiol (E2) or diethylstilbestrol (DES) or 4-nonylphenol, (4-NP)). During embryogenesis, only esr1 and esr2b were predominantly expressed and both were regulated by ligands. Our results demonstrate that the esr genes in zebrafish are regulated in a sex- and tissue specific manner and that 4-NP, a well-known endocrine disruptor possess both agonist and antagonist properties in adult tissues. We further extended our study to assess the toxic effects of 4-NP on developing embryos and larvae of zebrafish. A sub-lethal dose of 4-NP not only perturbed the neuroendocrine axis but also induced distortions/kinks and herniations in the notochord. The differentiation of the notochordal cells and the formation of the perinotochordal basement membrane were disrupted by 4-NP. Early disturbances induced by 4-NP in the notochord resulted in deformaties in the vertebral column at late larval stages. The notochord phenotype was accompanied by impaired swimming pattern. Repeated electrical stimulation of the larval muscles of 4-NP treated embryos showed impairment in the relaxation between stimuli which might be a possible reason for the defective swimming observed in 4-NP treated embryos

Changing Paradigms of Indian Foodtech Landscape - Impact of Online Food Delivery Aggregators

The purpose of this study is to depict the perception of consumers on the discount factor, future of food tech applications, tampering of family values, dine-out culture, technological anxiety and their impact on satisfaction behaviour. Millennials are the precedence audience for the online food aggregators. Generation X is different from other generations in perceiving discount factors. Future of food applications rely on the Discounts. Restaurant operators can focus on giving their customers an increased level of convenience and more discounts since these tend to be the reason for them to choose ordering food online. Optimisation is vital as increase in Technological anxiety increases the perceived future of food application and decreases the satisfaction behaviour

Levels of 17β-Estradiol Receptors Expressed in Embryonic and Adult Zebrafish Following In Vivo Treatment of Natural or Synthetic Ligands

The nuclear receptors encompass a group of regulatory proteins involved in a number of physiological processes. The estrogen receptors (ERs), of which one alpha and one beta form exist in mammals function as transcription factors in response to 17β-estradiol (E2). In zebrafish there are three gene products of estrogen receptors and they are denoted esr1 (ERalpha), esr2a (ERbeta2) and esr2b (ERbeta1). Total RNA of zebrafish early life stages (<3, 6, 12, 24, 48, 72, 96 and 120 hours post fertilization) and of adult fish (liver, intestine, eye, heart, brain, ovary, testis, gill, swim bladder and kidney) were isolated following in vivo exposures. Using specific primers for each of the three zebrafish ERs the expression levels were quantified using real time PCR methodology. It was shown that in absence of exposure all three estrogen receptors were expressed in adult fish. The levels of expression of two of these three ER genes, the esr1 and esr2a were altered in organs such as liver, intestine, brain and testis in response to ligand (E2, diethylstilbestrol or 4-nonylphenol). During embryogenesis two of the three receptor genes, esr1 and esr2b were expressed, and in presence of ligand the mRNA levels of these two genes increased. The conclusions are i) estrogen receptor genes are expressed during early development ii) altered expression of esr genes in response to ligand is dependent on the cellular context; iii) the estrogenic ligand 4-nonylphenol, a manufactured compound commonly found in sewage of water treatment plants, acts as an agonist of the estrogen receptor during development and has both agonist and antagonist properties in tissues of adult fish. This knowledge of esr gene function in development and in adult life will help to understand mechanisms of interfering mimicking endocrine chemicals in vivo

Peripheral ameloblastoma of the gingiva

Peripheral Ameloblastoma (PA) is a benign odontogenic tumor, arising from the cell rest of Serres, reduced enamel epithelium and basal cells of the surface epithelium. Peripheral ameloblastoma is a rare odontogenic neoplasm occurring commonly in the mandibular gingiva. PA clinically resembles other peripherally occurring lesions like pyogenic granuloma, peripheral ossifying fibroma, peripheral giant cell granuloma, and squamous papilloma. The recurrence rate of PA is 16-19% which demands a straight follow up. We report a case of recurrent peripheral ameloblastoma occurring in a 72-year old male located in the mandibular lingual gingiva in relation to the 44, 45 element’s regions. The patient had a primary lesion excised from the same site 6 years before which was diagnosed as ameloblastoma

The Zebrafish Orthologue of the Dyslexia Candidate Gene DYX1C1 Is Essential for Cilia Growth and Function

Peer reviewe

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10(-136)) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to themacroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.Peer reviewe

Characterization of brn1.2 and corticotropin-releasing hormone genes in zebrafish

The zebrafish (Danio rerio), a tropical fresh water fish originally found in the rivers of India and Bangladesh has become a popular vertebrate model system over the last decade. The rapid sequencing of the zebrafish genome together with the latest advances in forward and reverse genetics has made this model organism more fascinating as it can be used to decipher the genetic mechanisms involved in the vertebrate development. Corticotropin-releasing hormone (CRH) regulates the hypothalamic-pituitaryadrenal (HPA) axis in response to stress. Alteration in the production of CRH can lead to Cushing s syndrome, anxiety-related disorders and other neurodegenerative disorders such as Alzheimers s disease, Parkinson s dementia and Huntington s disease. In mammals it was reported that a POU class III gene by name Brn-2 is essential for the development of specific neuroendocrine cell types. Brn-2 can also regulate the transcription of corticotropinreleasing hormone by binding to its promoter. Corroborating the above fact, knockout studies in mice have shown that deletion of Brn-2 affects the development of corticotropinreleasing hormone producing neurons in addition to oxytocin and vasopressin synthesizing neurons. In order to understand the genetic mechanisms involved in the development of zebrafish neuroendocrine hypothalamus, we attempted to characterize the developmental expression profile of brn1.2 and crh in zebrafish. Zebrafish crh gene has not been characterized before and only a partial sequence of brn 1.2 was reported earlier. We screened the zebrafish EST database for brn1.2 and crh through a blast search and identified EST s for crh and brn1.2 that contained their full ORF. We used non-quantitative RT-PCR to analyze embryonic and post-embryonic expression pattern of crh. Expression of crh mRNA was detected from 1dpf and it remained stable through 5 dpf. RT-PCR analysis of the tissues revealed strong expression of crh mRNA in the brain, eye, gills and weak expression in the testis. Spatial expression pattern of both the genes were analyzed by whole mount in situ hybridization. Expression of crh was detected in different parts of the embryonic brain including telencephalon, hypothalamus, posterior tuberculum, thalamus, preoptic area, epiphysis, midbrain tegmentum, hindbrain and in the retina. Localization of CRH transcripts in the preoptic area is consistent with its role as a hypophysiotropin in zebrafish. Zebrafish POU class III genes include brn1.2, zp47, zp12, zp23 and zp50. zp47 and brn1.2 are found to be the direct homologs of the mammalian Brn-2 gene. Expression of brn1.2 begins at tailbud stage. From 5-somite stage on, brn1.2 is detected in the midbrain, hindbrain and weakly in the spinal cord. At 24hpf brn1.2 is widely expressed in the diencephalon, midbrain and hindbrain but not detected in the telencephalon. At later stages of development brn 1.2 transcripts were found in the hypothalamus, preoptic area, ventral thalamus, dorsal thalamus, pretectum, tectum, cerebellum and hindbrain. Additionally, brn 1.2 was also detected in the otic vesicle at 48hpf. In this study we have described the developmental expression pattern of brn1.2 and crh. Future studies will include a complete analysis of the function of brn1.2 in the specification of the hypothalamic neurons and also identification of other candidate genes that are required for the development these neurons

Air Pollution Micrometeorology Study in the Neighbourhood Of Coastal Super Power Thermal Plant-A Case Study

Abstract:-Air pollution Micrometrology in the Coastal site other than General Micrometrology will be essential now a days in association with Climate Change. Now a Chennai City had been faced recent trend in the Climate never faced in the last 100 years not yet be cleared. It will need further study which has to be needed in the future of the Chennai City. Chennaities and several researches both in water resources engineers and Environmentalist faced the last December Month

Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2

BackgroundDYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and DCDC2 interact on the genetic and the protein level.ResultsHere, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b in zebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among DYX1C1 and DCDC2 in a cellular model.ConclusionsIn summary, we describe the physical and functional interaction between the two genes DYX1C1 and DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set the stage for future functional studies.Peer reviewe

Knockdown of <i>dyx1c1</i> showed typical cilia phenotypes.

<p>Injection of ATGMO or SPMO at 200 µM concentration produced ventrally curved body axis, hydrocephalus and kidney cysts (A). Arrow in panel A denotes kidney cyst in ATG morphant. RT-PCR showed aberrant splice transcripts in SPMO injected embryos at 1 and 2 dpf (B). Histological sections of 2 day old embryos injected with both ATGMO and SPMO (100 µM each) showed hydrocephalus (D; yellow arrows) compared to normal size brain ventricles in WT (C). Transverse histological sections across the pronephros at 3.5 dpf showed normal pronephros in WT embryos (E). Section of <i>dyx1c1</i> morphant (ATGMO+SPMO) showed severe pronephric distention and a thin glomerulus in the center (F). Yellow arrowheads in panel E and F point out normal pronephros in WT and dilated pronephros in morphant embryo, respectively. Quantitative analysis of the rescue of <i>dyx1c1</i> morphant phenotype to WT phenotype with different combinations of MOs and <i>dyx1c1</i> mRNA (G). Scale bars indicate 100 µm. Abbreviation: gm, glomerulus.</p