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In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

ABSTRACT: Molecular and physiological analyses in ionoregulatory organs (e.g., adult gills and embryonic skin) are essential for studying fish ion regulation. Recent progress in the molecular physiology of fish ion regulation was mostly obtained in embryonic skin; however, studies of ion regulation in adult gills are still elusive and limited because there are no direct methods for in vivo functional assays in the gills. The present study applied the scanning ion-selective electrode technique (SIET) in adult gills to investigate branchial H⁺-excreting functions in vivo. We removed the opercula from zebrafish and then performed long-term acid acclimation experiments. The results of Western blot and immunofluorescence showed that the protein expression of H⁺-ATPase (HA) and the number of H⁺-ATPase-rich ionocytes were increased under acidic situations. The SIET results proved that the H⁺ excretion capacity is indeed enhanced in the gills acclimated to acidic water. In addition, both HA and Na⁺/H⁺ exchanger (Nhe) inhibitors suppressed the branchial H⁺ excretion capacity, suggesting that H⁺ is excreted in association with HA and Nhe in zebrafish gills. These results demonstrate that SIET is effective for in vivo detection in fish gills, representing a breakthrough approach for studying the molecular physiology of fish ion regulation.

SUBMITTER: Shih SW

PROVIDER: S-EPMC9031880 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

Shih Shang-Wu SW Yan Jia-Jiun JJ Tsou Yi-Ling YL Lu Shao-Wei SW Wang Min-Chen MC Chou Ming-Yi MY Hwang Pung-Pung PP

International journal of molecular sciences 20220416 8

Molecular and physiological analyses in ionoregulatory organs (e.g., adult gills and embryonic skin) are essential for studying fish ion regulation. Recent progress in the molecular physiology of fish ion regulation was mostly obtained in embryonic skin; however, studies of ion regulation in adult gills are still elusive and limited because there are no direct methods for in vivo functional assays in the gills. The present study applied the scanning ion-selective electrode technique (SIET) in ad ...[more]

PMID: 35457237

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Project description:BackgroundZinc deficiency is detrimental to organisms, highlighting its role as an essential micronutrient contributing to numerous biological processes. To investigate the underlying molecular events invoked by zinc depletion we performed a temporal analysis of transcriptome changes observed within the zebrafish gill. This tissue represents a model system for studying ion absorption across polarised epithelial cells as it provides a major pathway for fish to acquire zinc directly from water whilst sharing a conserved zinc transporting system with mammals.ResultsZebrafish were treated with either zinc-depleted (water = 2.61 μg L-1; diet = 26 mg kg-1) or zinc-adequate (water = 16.3 μg L-1; diet = 233 mg kg-1) conditions for two weeks. Gill samples were collected at five time points and transcriptome changes analysed in quintuplicate using a 16K oligonucleotide array. Of the genes represented the expression of a total of 333 transcripts showed differential regulation by zinc depletion (having a fold-change greater than 1.8 and an adjusted P-value less than 0.1, controlling for a 10% False Discovery Rate). Down-regulation was dominant at most time points and distinct sets of genes were regulated at different stages. Annotation enrichment analysis revealed that 'Developmental Process' was the most significantly overrepresented Biological Process GO term (P = 0.0006), involving 26% of all regulated genes. There was also significant bias for annotations relating to development, cell cycle, cell differentiation, gene regulation, butanoate metabolism, lysine degradation, protein tyrosin phosphatases, nucleobase, nucleoside and nucleotide metabolism, and cellular metabolic processes. Within these groupings genes associated with diabetes, bone/cartilage development, and ionocyte proliferation were especially notable. Network analysis of the temporal expression profile indicated that transcription factors foxl1, wt1, nr5a1, nr6a1, and especially, hnf4a may be key coordinators of the homeostatic response to zinc depletion.ConclusionsThe study revealed the complex regulatory pathways that allow the organism to subtly respond to the low-zinc condition. Many of the processes affected reflected a fundamental restructuring of the gill epithelium through reactivation of developmental programs leading to stem cell differentiation. The specific regulation of genes known to be involved in development of diabetes provides new molecular links between zinc deficiency and this disease. The present study demonstrates the importance of including the time-dimension in microarray studies.

Project description:BackgroundDietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. We exploited the zebrafish (Danio rerio) gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. This organ forms a conduit for zinc uptake whilst exhibiting conservation of zinc trafficking components.ResultsFish were maintained with either zinc supplemented water (4.0 μM) and diet (2023 mg zinc kg-1) or water and diet containing Zn2+ at 0.25 μM and 233 mg zinc kg-1, respectively. Gill tissues were harvested at five time points (8 hours to 14 days) and transcriptome changes analysed in quintuplicate using a 16 K microarray with results anchored to gill Zn2+ influx and whole body nutrient composition (protein, carbohydrate, lipid, elements). The number of regulated genes increased up to day 7 but declined as the fish acclimated. In total 525 genes were regulated (having a fold-change more than 1.8 fold change and an adjusted P-value less than 0.1 which is controlling a 10% False discovery rate, FDR) by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. Many genes displayed cyclic expression, typical for homeostatic control mechanisms. Annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". A suite of genes linked to "development" were also statistically overrepresented. More specifically, early regulation of genes was linked to a few key transcription factors (e.g. Mtf1, Jun, Stat1, Ppara, Gata3) and was followed by hedgehog and bone morphogenic protein signalling.ConclusionsThe results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell differentiation, a response likely reflecting gill remodelling in response to its altered environment. This provides insight to the role of zinc during cell differentiation and illustrates the critical nature of maintaining zinc status. The study also highlights the importance of temporal transcriptomics analysis in order resolve the discrete elements of biological processes, such as zinc acclimation.

Dataset Information

In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

Publications

In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

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