Osmoregulation in zebrafish: ion transport mechanisms and functional regulation

Fish, like mammals, have to maintain their body fluid ionic and osmotic homeostasis through sophisticated iono-/osmoregulation mechanisms, which are conducted mainly by ionocytes of the gill (the skin in embryonic stages), instead of the renal tubular cells in mammals. Given the advantages in terms of genetic database availability and manipulation, zebrafish is an emerging model for research into regulatory and integrative physiology. At least five types of ionocytes, HR, NaR, NCC, SLC26, and KS cells, have been identified to carry out Na+ uptake/H+ secretion/NH4+ excretion, Ca2+ uptake, Na+/Cl- uptake, K+ secretion, and Cl- uptake/HCO3- secretion, respectively, through distinct sets of transporters. Several hormones, namely isotocin, prolactin, cortisol, stanniocalcin-1, calcitonin, endothelin-1, vitamin D, parathyorid hormone 1, catecholamines, and the renin-angiotensin-system, have been demonstrated to positively or negatively regulate ion transport through specific receptors at different ionocytes stages, at either the transcriptional/translational or posttranslational level. The knowledge obtained using zebrafish answered many long-term contentious or unknown issues in the field of fish iono-/osmoregulation. The homology of ion transport pathways and hormone systems also means that the zebrafish model informs studies on mammals or other animal species, thereby providing insights into related fields

Perfused Gills Reveal Fundamental Principles of pH Regulation and Ammonia Homeostasis in the Cephalopod Octopus vulgaris

In contrast to terrestrial animals most aquatic species can be characterized by relatively higher blood [Formula: see text] concentrations despite its potential toxicity to the central nervous system. Although many aquatic species excrete [Formula: see text] via specialized epithelia little information is available regarding the mechanistic basis for NH3/[Formula: see text] homeostasis in molluscs. Using perfused gills of Octopus vulgaris we studied acid-base regulation and ammonia excretion pathways in this cephalopod species. The octopus gill is capable of regulating ammonia (NH3/[Formula: see text]) homeostasis by the accumulation of ammonia at low blood levels (<260 μM) and secretion at blood ammonia concentrations exceeding in vivo levels of 300 μM. [Formula: see text] transport is sensitive to the adenylyl cyclase inhibitor KH7 indicating that this process is mediated through cAMP-dependent pathways. The perfused octopus gill has substantial pH regulatory abilities during an acidosis, accompanied by an increased secretion of [Formula: see text]. Immunohistochemical and qPCR analyses revealed tissue specific expression and localization of Na+/K+-ATPase, V-type H+-ATPase, Na+/H+-exchanger 3, and Rhesus protein in the gill. Using the octopus gill as a molluscan model, our results highlight the coupling of acid-base regulation and nitrogen excretion, which may represent a conserved pH regulatory mechanism across many marine taxa

A Positive Regulatory Loop between foxi3a and foxi3b Is Essential for Specification and Differentiation of Zebrafish Epidermal Ionocytes

BACKGROUND: Epidermal ionocytes play essential roles in the transepithelial transportation of ions, water, and acid-base balance in fish embryos before their branchial counterparts are fully functional. However, the mechanism controlling epidermal ionocyte specification and differentiation remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In zebrafish, we demonstrated that Delta-Notch-mediated lateral inhibition plays a vital role in singling out epidermal ionocyte progenitors from epidermal stem cells. The entire epidermal ionocyte domain of genetic mutants and morphants, which failed to transmit the DeltaC-Notch1a/Notch3 signal from sending cells (epidermal ionocytes) to receiving cells (epidermal stem cells), differentiates into epidermal ionocytes. The low Notch activity in epidermal ionocyte progenitors is permissive for activating winged helix/forkhead box transcription factors of foxi3a and foxi3b. Through gain- and loss-of-function assays, we show that the foxi3a-foxi3b regulatory loop functions as a master regulator to mediate a dual role of specifying epidermal ionocyte progenitors as well as of subsequently promoting differentiation of Na(+),K(+)-ATPase-rich cells and H(+)-ATPase-rich cells in a concentration-dependent manner. CONCLUSIONS/SIGNIFICANCE: This study provides a framework to show the molecular mechanism controlling epidermal ionocyte specification and differentiation in a low vertebrate for the first time. We propose that the positive regulatory loop between foxi3a and foxi3b not only drives early ionocyte differentiation but also prevents the complete blockage of ionocyte differentiation when the master regulator of foxi3 function is unilaterally compromised

<b>Knock-down </b><i>foxi3a</i> Expression Severely Reduces Epidermal Ionocyte Progenitor Number and Abolishes the Later Differentiation Program.

<p>(A–G) Interfering with <i>foxi3a</i> and <i>foxi3b</i> functions of epidermal ionocyte differentiation by a morpholino (0.5 mM/embryo) injection. Morphants were fixed at 24 hours post fertilization (hpf) and stained with <i>atp1b1b</i> (green), <i>ca2a</i> (red), and P63 (blue) to detect Na<i>⁺</i>,K<i>⁺</i>-ATPase rich cells (NaRCs), H<i>⁺</i>-ATPase rich cells (HRCs), and epidermal stem cells, respectively. (H–I) A rescue experiment to show the specificity of <i>foxi3a</i> morpholinos. The <i>foxi3a</i> mRNA used for the rescue experiment does not contain a binding site for MO2. (J–L) Comparison of the vital dye uptake ability between the wild type (wt), <i>foxi3a</i> morphants, and <i>foxi3b</i> morphants. NaRCs and HRCs in either wild-types or <i>foxi3b</i> morphants can absorb MitoTracker (red) and Con-A (green) through to their apical openings. For <i>foxi3a</i> morphants, no MitoTracker or Con-A staining was detected due to blockage of the entire differentiation program. (M–O) Detection of the apical opening of epidermal ionocytes in wild-types, <i>foxi3a</i> morphants, and <i>foxi3b</i> morphants by scanning electron microscopy. The apical openings of NaRCs and HRCs in wild-types are shaped as deep holes (green box) or a mesh (red box), respectively. The apical openings were totally undetected in <i>foxi3a</i> morphants due to blockage of the entire differentiation program. For <i>foxi3b</i> morphants, the apical openings for both NaRCs and HRCs were reduced. Embryos in (A–I) were scored at 24 hpf, while in (J–O), they were scored at 72 hpf. In I, embryos are orientated in a dorsal-up and anterior-top position.</p

Prostaglandin E2 synchronizes lunar-regulated beach spawning in grass puffers

Many organisms living along the coastlines synchronize their reproduction with the lunar cycle. At the time of spring tide, thousands of grass puffers (Takifugu alboplumbeus) aggregate and vigorously tremble their bodies at the water’s edge to spawn. To understand the mechanisms underlying this spectacular semilunar beach spawning, we collected the hypothalamus and pituitary from male grass puffers every week for 2 months. RNA sequencing (RNA-seq) analysis identified 125 semilunar genes, including genes crucial for reproduction (e.g., gonadotropin-releasing hormone 1 [gnrh1], luteinizing hormone β subunit [lhb]) and receptors for pheromone prostaglandin E (PGE). PGE2 is secreted into the seawater during the spawning, and its administration activates olfactory sensory neurons and triggers trembling behavior of surrounding individuals. These results suggest that PGE2 synchronizes lunar-regulated beach-spawning behavior in grass puffers. To further explore the mechanism that regulates the lunar-synchronized transcription of semilunar genes, we searched for semilunar transcription factors. Spatial transcriptomics and multiplex fluorescent in situ hybridization showed co-localization of the semilunar transcription factor CCAAT/enhancer-binding protein δ (cebpd) and gnrh1, and cebpd induced the promoter activity of gnrh1. Taken together, our study demonstrates semilunar genes that mediate lunar-synchronized beach-spawning behavior

Raw datasets from Cellular mechanisms underlying extraordinary sulphide tolerance in a crustacean holobiont from hydrothermal vents

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulphide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulphur-rich vent systems. Despite the toxicity of hydrogen sulphide, X. testudinatus occupies an ecological niche in a sulphide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulphide detoxification. Here sulphide is oxidized to thiosulphate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulphate and hypotaurine is mediated by the sodium-independent sulphate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulphide-oxidizing endo-symbiotic bacteria, creating an effective sulphide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet

Supplemental Figures from Cellular mechanisms underlying extraordinary sulphide tolerance in a crustacean holobiont from hydrothermal vents

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulphide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulphur-rich vent systems. Despite the toxicity of hydrogen sulphide, X. testudinatus occupies an ecological niche in a sulphide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulphide detoxification. Here sulphide is oxidized to thiosulphate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulphate and hypotaurine is mediated by the sodium-independent sulphate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulphide-oxidizing endo-symbiotic bacteria, creating an effective sulphide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet

Supplemental Information for Materials and Methods from Cellular mechanisms underlying extraordinary sulphide tolerance in a crustacean holobiont from hydrothermal vents

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulphide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulphur-rich vent systems. Despite the toxicity of hydrogen sulphide, X. testudinatus occupies an ecological niche in a sulphide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulphide detoxification. Here sulphide is oxidized to thiosulphate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulphate and hypotaurine is mediated by the sodium-independent sulphate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulphide-oxidizing endo-symbiotic bacteria, creating an effective sulphide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet