斑馬魚仔魚富含氫幫浦細胞對環境酸鹼值改變之短期調節機制

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(1)國立台灣師範大學生命科學系碩士論文. 斑馬魚仔魚富含氫幫浦細胞對環境酸鹼值改變之短期調節機制 Short-term regulation of H+-ATPase-Rich Cells in zebrafish larvae subjected to environmental pH changes. 研究生：陳鶴文 Ho-Wen Chen 指導教授：林豊益博士 Li-Yih Lin. 中華民國九十八年一月.

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(6) Table of Contents Abstract…………………………………..…….1 摘要………………………………….....……....2 Introduction…………………………………….3 Materials and Methods……….………….……10 Flowcharts of acclimation…….…………..…..15 Results……………………….……………..…19 Discussions………………….……………..….24 References………………….………………....30 Tables and Figures………….…………………39. 6.

(7) Abstract Acid-base regulation is crucial for fish to cope with environmental pH changes particularly during embryonic and larval development. H+-ATPase-rich cells (HRCs) in the skin of zebrafish larvae are the major sites for H+ secretion. In this study, I examined the short-term (in hours) regulation of H+ secretion in zebrafish subjected to ambient pH changes. Scanning electron microscopy (SEM) and confocal microscopy were used to observe and quantify the apical surface (membrane) of HRCs. Whole body pH and H+ secreting by the skin of larvae were measured with scanning ion-selective electrode technique (SIET). Results showed that whole body H+ concentration significantly increased in the larvae transferred from pH 7 to pH 4 for 30 min. The apical surface area and density of HRCs were higher in pH 4 than in pH 7 acclimated larvae. As early as 10 min after being transferred from pH 4 to pH 7, the apical surface area dramatically decreased, meanwhile morphological change and internalization of the apical surface were observed. Down-regulation of H+ secretion was also found after the transfer. In contrast, when the larvae were transferred from pH 7 to pH 4, the apical surface gradually increased after 4 h.. 1.

(8) 摘要魚類胚胎或仔魚必須進行酸鹼調節以應付水中酸鹼變動。斑馬魚仔魚體表上的富含氫幫浦細胞(HRCs)是主要魚體排酸的細胞。本實驗目的是探討斑馬魚仔魚排酸的短期調節機制。利用掃瞄式電子顯微鏡和共軛焦顯微鏡來觀察和量化 HRCs 的頂膜開口型態和密度。並利用掃瞄離子電極技術(SIET)來測量魚體內酸鹼值和魚體排出氫離子的能力。結果顯示當仔魚由 pH 7 轉移到 pH 4 環境 30 分鐘後，魚體內氫離子濃度有顯著增加。仔魚馴養在 pH 4 環境中，HRCs 的頂膜開口面積和密度顯著大於馴養在 pH 7 環境下的仔魚。當仔魚由 pH 4 轉移到 pH 7 環境 10 分鐘之內，HRCs 頂膜開口面積顯著下降，同時可以觀察到頂膜型態皺縮和內吞現象。然而，當仔魚由 pH 7 轉移到 pH 4 環境 4 小時後，HRCs 頂膜開口面積才會逐漸增加。本研究證實斑馬魚仔魚具有短期調節排酸的機制。. 2.

(9) Introduction Acid-base regulation in fish Fish, like other vertebrate, have to maintain homeostasis of intracellular and extracellular pH. Fish utilize the parallel strategies of buffering and excretion to maintain intracellular and extracellular pH. Cellular metabolic processes generate acid compounds directly with the generation of free H+ and indirectly via the production of CO2. CO2 could convert to H+ and HCO3- as described by the Henderson-Hasselbalch equation (Heisler, 1993 ; Claiborne, 1998). This can be simplified to the equilibrium reaction : CO2 + H2O ←→ H+ + HCO3In intracellular fluids (ICF) of fishes, amino acid residues, proteins, inorganic and organic phosphates can act as buffers to limit the levels of excess free H+. These buffers could significantly reduce the acid and base variations in ICF pH (pHi, a “typical” fish pHi is ~7.0-7.4, normally ~0.6 pH unit lower than the plasma pH; Heisler, 1986a). The extracellular fluids (ECF) and blood are also protected by the predominant buffers like erythrocyte hemoglobin and plasma HCO3-. A primary mammalian response to metabolic acidosis is to increase lung respiratory exchanges in order to reduce the plasma partial pressure of CO2 (PCO2). This in turn drives a shift in the equation to the left and increases pH once again. A high initial PCO2 (in humans, typically about 40 mmHg) allows the respiratory adjustments necessary to excrete more CO2. In contrast, fishes cannot utilize this strategy to the same extent because of the much lower PCO2 normally found in these animals. When 3.

(10) compared to air breathers, the requirements for extracting sufficient oxygen from the water necessitate a 10- to 20-fold higher ventilatory water flow over the gills (normalized for blood oxygen capacity and flow rates). This water flow, combined with a high water CO2 solubility, allows the rapid transfer of CO2 across the gills into the water, and reduces the PCO2 of the plasma to only 1-4mmHg in most species (Heisler, 1986a). At these low levels, additional respiratory reductions in PCO2 are possible (Claiborne et al. 2000), but small. Thus, fishes must utilize another mechanism to excrete excess H+.. Gills in acid-base regulation The primary compensation of pH imbalance is due to the direct transepithelial transfer of acid-base relevant molecules between the animal and the external media. The gills, intestine (Wilson et al. 1996 ; Grosell et al. 2001), and kidneys (Wood et al, 1999) have all been shown to be involved in acid-base transfers under some conditions. However, branchial epithelium is generally thought to account for the majority of acid-base movements (90% or higher) in most fishes studied (Heisler, 1986b). The gill epithelium is composed of several distinct cell types like pavement cells (PVCs), mitochondria-rich cells (MR cells), mucous cells, neuro-epithelial cells, etc., but primarily consists of PVCs and MR cells, which compose >90% and <10% of the epithelial surface area, respectively (Evan et al. 2005). MR cell features an abundance of energy-providing mitochondria, display extraordinary development of cytoplasmic tubular system, and contain an abundance of Na+-K+-ATPase. Plenty of literature have been accumulated on the study of the functions 4.

(11) of MR cells in fish gills, and several models for gill ion regulation have been proposed. However, there are still many issues that are unclear and require convincing evidence to answer. In general, MR cells and probably pavement cells are both involved in fish acid-base regulation (Evans et al. 2005; Claiborne et al. 2002).. Morphology of MR cells and acid-base regulation SEM observations have been widely used to examine structure/function correlation in MR cells of various fishes. A series of ultrastructural SEM studies were conducted to determine if morphological changes in PVCs or MR cells occur in response to acid-base disturbances (Goss, 1994a, 1995; Laurent, 1995). Goss and colleagues demonstrated that 30% and 85% decreases in the apical surface of MR cells during hypercapnia acidosis in rainbow trout and catfish, respectively (Goss, 1993, 1992). They also reported a 50% increase in the MR cell apical surface during metabolic alkalosis in rainbow trout (Goss, 1993, 1994b, c). Another ultrastructural observation contradicting these findings was reported; metabolic acidosis was correlated with a 135% increase in the MR cell apical surface of rainbow trout (Goss, 1994c). Morphological changes in MR cells were also reported in several species of FW fishes in response to ambient ionic compositions. Enlargement of the apical surface of MR cells and increases in cell density were found in salmon acclimated to low-NaCl water (Laurent et al. 1985; Perry and Laurent 1989; Greco et al. 1996).. 5.

(12) Transporters and acid-base regulation In early literatures, a variety of mechanisms have been proposed to drive the transfer of acid-base relevant ions across the gill epithelium of fishes. The dominant model is Na+/H+ and Cl-/HCO3- exchanges, in which electroneutral exchanges of ions permit the excretion of acid or base for the uptake of Na+ or Cl- (Evans et al. 2005). Na+/H+ exchange has been proposed in early literatures as a possible mechanism of Na+ uptake and H+ excretion in fish gills. The molecular evidences for Na+/H+ exchanger (NHE) in fish gills have been reported (Wilson et al. 2000; Hirata et al. 2003; Yan et al. 2007; Hiroi et al. 2008), although this mechanism has been questioned about the driving force for Na+ uptake in freshwater (Parks et al. 2008). Another model for acid section in FW fish includes an apical H+-ATPase in gill epithelium has been reported in some species including lamprey (Geotria australis) (Choe et al. 2004), tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss) (Wilson et al. 2000). Recently, Lin et al. (2006) used molecular and electrophysiological approaches to demonstrate the acid secretion by H+-ATPase-rich cells (HRCs, a subtype of ionocytes) in the skin of zebrafish (Denio rerio) larvae. The H+-ATPase droved Na+ uptake was also suggested to occur in the HRCs of zebrafish (Horng et al. 2007; Esaki et al. 2007). Since H+-ATPase has been linked to acid secretion of fish, plenty of studies have been conducted to investigate the regulatory of H+-ATPase in fishes during acid-base disturbance. The protein and mRNA amount of branchial H+-ATPase was increased in gills of rainbow trout (O.mykiss) in response to acidosis (Sullivan et al. 1995; Perry et al. 2000; 2003). 6.

(13) However, no increase and a decrease in H+-ATPase mRNA levels were respectively reported in Japanese dace (Tribolodon hakonensis) exposed to pH 3.5 water (Hirata et al. 2003) and in Atlantic salmon (Salmo salar) exposed to hypercapnia (Seidelin et al. 2001). Most studies investigated the regulation of H+-ATPase in fishes subjected to acid-base disturbance for several days. However, little is known about the short-term mechanisms of acid-base regulation in fish. In mammalian H+-ATPase-rich cells, proton secretion is regulated via recycling of H+-ATPase-containing vesicles to and from the plasma membrane (Al-Awqati, Q. 1996; Breton et al. 2000; Brown and Breton 2000). In kidney intercalated cells, systemic acidosis causes the accumulation of H+-ATPase in the apical membrane of type A intercalated cells, and conversely, alkalosis induces the retrieval of H+-ATPase molecules from the membrane via endocytosis. This short-term regulation of H+-ATPase recycling usually takes place in minutes and followed by de novo H+-ATPase synthesis which takes place in hours to days.. Zebrafish as a model for ion and acid-base regulation During fish embryonic stages, the skin is believed to regulate body fluid pH and ionic composition before the development and functioning of gills, kidneys, and intestine (Hiroi et al. 1999 ; Hwang et al. 1999 ; Kaneko et al. 2002 ; Lin and Hwang 2001, 2004). Skin of zebrafish embryos or larvae may be an excellent model to study the functions of these ionocytes, because the embryonic skin is comparatively easy to use for morphological observations and functional assays in vivo. Comparing to salmon, trout, tilapia, etc., zebrafish has became a new and powerful 7.

(14) model specie because of its plentiful genetic database and mutants, as well as the various established molecular physiological approaches (gain-of-function and loss-of-function). Lin et al. (2006) first reported two subtypes of ionocytes, HRCs and NaRCs, in zebrafish embryos. Pan et al. (2005) showed that a portion of NaRCs have epithelial Ca2+ channel (zECaC) for Ca2+ uptake. Horng et al. (2007) further provided evidence for the uptake of Na+ via HRCs. Yan et al. (2007) showed that Na+/H+ exchange (NHE3) is located in HRCs and involved in Na+ uptake mechanism. Hwang and Lee (2007) proposed a model of ion regulatory mechanisms in zebrafish gill/skin ionocytes including HRCs, NaRCs and NCC cells. Recently, Shih et al. (2008) demonstrated that H+-ATPase and Rhcg1 are involved in ammonia excretion of zebrafish larvae. Those reports reveal that zebrafish is a good model for studying ion regulation in fish.. Purpose To cope with ambient pH disturbance, it is necessary for fishes and particular fish embryos to regulate the capability of acid or base secretion. In a recent study, zebrafish embryos were shown to be tolerant of extreme acid water (pH 4) and developed a up-regulation of acid secretion by increasing H+-ATPase expression in HRCs and promoting HRCs differentiation in the skin (Horng et al. unpublished data). In this study, I focused on the short-term regulation of the HRCs in zebrafish larvae subjected to external pH changes. Using electron microscopic, immunocytochemical, and electrophysiological approaches, this study reveals an apical membrane modification and fast regulation of proton 8.

(15) secretion by the HRCs of zebrafish larvae.. 9.

(16) Materials and Methods Zebrafish Wild type AB strain zebrafish (Denio rerio) were obtained from the Institute of Cellular and Organismic Biology, Academia Sinica and maintained as described in “The Zebrafish Book”. Zebrafish were raised in aquatic tank at 28°C water temperature under illumination cycle as 14 hours brightness/ 10 hours darkness. Eggs were collected from mating pairs about 1 hour after being laid. Unfertilized eggs were discarded after egg collection.. Preparation of artificial water Fertilized eggs were incubated in normal water containing (in mM) 0.5 NaCl, 0.2 MgSO4, 0.2 CaSO4, 0.16 KH2PO4, 0.16 K2HPO4. pH 4, 7, 10 water were prepared by adding HCl or NaOH to the normal water which containing 0.3 mM appropriate buffers (MES for pH4, MOPS for pH7, and Tricine for pH10).. Calcein stain Calcein immersion solutions (0.2%) were prepared by dissolving 0.2 g of calcein powder (Sigma Chemical, St. Louis, MO) in 100ml of normal water. Zebrafish embryos immersed for 15 min. After the immersions, the embryos were rinsed a number of times in fresh water, and then allowed to stand for 15 min to allow the excess, unbound calcein to diffuse out of the tissues. The embryos were then anesthetized in tricaine-methanesulfonate (MS222) and mounted on glass slides. 10.

(17) Observations were carried out at a magnification of x10 eyepiece and 10x/0.4 object lens by using an Olympus BX60 fluorescent microscope.. SEM Zebrafish larvae were fixed at 4°C in cacodylate-buffered 2.5% glutaraldehyde overnight. After rinsing with 0.1 M cacodylate buffer, the specimens were postfixed with 2% osmium tetroxide in 0.1 M cacodylate buffer for another 2 h. After rinsing with cacodylate buffer and dehydration with ethanol, the specimens were critical-point dried with liquid CO2 in a critical-point drier (Hitachi HCP-2, Tokyo, Japan) and sputter coated for 3 min with a gold-palladium complex in a vacuum evaporator (Cressington, Watford, UK). The coated specimens were examined with an SEM (FEI Quanta 200, Holand). Zebrafish larvae yolk and yolk tube total divided into four areas (110x95 µm2 =10,450 µm2 each) and captured at 2500x magnification with the enclosed software to the SEM (FEI Quanta 200, Holand). Calculation of surface area and density is by using Image J software (Version 1.38).. Whole mount immunocytochemistry For double staining of concanavalin A (Con-A) and H+-ATPase, live larvae were pre-incubated in normal water containing 0.5 mg/ml Alexa 488-conjugated Con-A (Molecular Probes, Eugene, OR) for 10 min. After being washed in normal water for 3 min, the Con-A-labeled larvae were fixed with 4% paraformaldehyde in 0.1 M PB (pH 7.4) for 1 h at 4°C. After being rinsed with PBS, the larvae were postfixed and permeabilized with 70% ethanol at -20°C for 10 min. After being washed 11.

(18) with PBS, samples were incubated with 3% bovine serum albumin for 30 min to block nonspecific binding. The samples were then incubated overnight at 4°C with polyclonal antibody aganst the A subunit of killifish H+-ATPase (Katoh et al. 2003). After being rinsed with PBS for 20 min, the larvae were further incubated in goat anti-rabbit IgG conjugated with Alexa 488 (or Alexa 568, diluted 1:100; Molecular Probes, Eugene, OR), for 2 h at RT (26-28°C). Observations and image acquisitions were made using a Leica TCS-NT confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) equipped with 20x/0.4 and 40x/1.2 lenses, and appropriate filter sets for simultaneous monitoring of various fluorescence signals.. Scanning ion-selective electrode technique (SIET) SIET was used to measure extracellular H+ activity (concentration) at the surface of zebrafish larvae. Microelectrodes with tip diameters of 3-4μm were pulled from glass capillary tubes (World Precision Instruments, Sarasota, FL; no. TW 150-4 with 1.12- and 1.5-mm inner and outer diameters, respectively) with a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA). These were then baked in covered dishes at 200°C overnight and vapor silanized with dimethyl chlorosilane (Fluka, Buchs, Switzerland) for 30 min, and the covers were removed before further baking at 200°C for at least 1 h. The capillaries were backfilled with a 1-cm column of 100 mM KCl-H2PO4 (pH 7.0) and then frontloaded with a 20- to 30-μm column of liquid ion exchanger cocktail (Hydrogen Ionophore I-cocktail B; Fluka). The ion-selective microelectrode was connected to an operational amplifier 12.

(19) (IP Amp ion polarographic amplifier; Applicable Electronics, East Falmouth, MA) via an Ag/AgCl wire electrode holder (World Precision Instruments), and the circuit was completed by placing a salt bridge (3 M potassium acetate, 10 mM KCl in 3% agarose connected to a Ag/AgCl wire). Electrode vibration and positioning were achieved with a stepper motor-driven three-dimensional (3D) positioner (Applicable Electronics). Data acquisition, preliminary processing, and control of the 3D electrode positioner were performed with ASET software (Science Wares, East Falmouth, MA). The vibrating electrode system was attached to an Olympus upright microscope (BX-50WI). A ×10 dry and a ×40 water immersion objective lens (working distance 3.3 mm) were used for differential interference contrast (DIC) imaging. To detect the surface pH of zebrafish embryo, SIET was performed at RT (24-26°C) in a small plastic recording chamber filled with 1 ml of “recording solution” that contained zebrafish solution, 300μM MOPS buffer (Sigma, St. Louis, MO), and 0.1 mg/l ethyl 3-aminobenzoate (Tricaine, Sigma; pH = 7.0). An anesthetized embryo was positioned in the center of the chamber with its lateral side contacting the base of the chamber and the probe was moved to the skin surface of yolk sac. Voltage output signals in millivolts were recorded every 3.0 s and averaged for 3.0 min. The averaged voltages were converted to H+ concentration after three-point calibration (pH 6, 7 and 8). After recording, the probe was moved to about 0.5 cm away from the embryo to record the background values of the medium.. Calibration of ion-selective vibrating probe Before the collection of biological data, the efficiency of the H+ 13.

(20) probe were measured by placing the electrode in a series of standard pH solutions (pH 6, 7 and 8). When the voltage output of the probe was plotted against log H+ activity, linear regression yielded a Nernstian slope of 57.75 ± 2.1) (n = 10).. Statistical analysis Values are presented as the mean ± SD and were compared using Student's t-test or one-way analysis of variance (ANOVA) (Tukey’s pairwise comparison).. 14.

(21) Flowcharts of acclimation. Exp. 1. Development of zebrafish embryos in various pH media Exp. 2. Formation of vertebra in the larva acclimated to pH4, pH7 and pH10 Exp. 3. Apical Morphology of the ionocytes in larval skin. 0h. 1h. Sampling for exp. 1& 3. Sampling for exp. 2. 4dpf. 7dpf. Fertilized eggs. pH4. pH4. pH7. pH7. pH10. pH10. Measurement of body length, mortality (exp. 1) SEM observation and quantification of ionocytes (exp. 3). 15. Calcein stain and measurement of vertebra number (exp. 2).

(22) Exp. 4. Time-course changes of whole body [H+] in larvae subjected pH changes Sampling for SIET. 0m. 0h. 1h. 60m. 30m. 90m 120m. 4dpf. Acclimation to pH4. Transfer to. pH4. pH4 pH7. Fertilized eggs Acclimation to pH7. Transfer to. pH7. pH7. pH4. 16. Measurement of whole body [H+].

(23) Exp. 5. Time-course changes of Type II cells during pH acclimation Exp. 6. Time-course changes of acid secretion by the larvae subjected to different pH. Sampling for SEM & SIET. 0h. 1h. 0h. 2h. 4h. 6h. 4dpf. Acclimation to pH4. Transfer to. pH4. pH4. SEM observation and quantification. pH7. of ionocytes (exp. 5). Fertilized eggs Acclimation to pH7. Measurement of. Transfer to. pH7. pH7. pH4. 17. acid secretion (exp. 6).

(24) Exp. 7. Short-term changes of the apical surface of the Type II cells. Sampling for SEM. 0m. 1h. 0h. 30m. 10m. 60m. 120m. 4dpf Sampling for Confocal. Acclimation to pH4. Transfer to. pH4. pH4 pH7. SEM observation and quantification of ionocytes Immunostaining and confocal observation. Fertilized eggs Acclimation to pH7. Transfer to. pH7. pH7. 18.

(25) Results Exp. 1. Development of zebrafish embryos in various pH media To evaluate if zebrafish embryo can be tolerant wide range of pH water (pH4-pH10), the mortality and body length were measured after 4 days of acclimation. There is no significant difference in mortality between various pH groups (Table 1). However, the length of larvae is slightly shorter in pH4 than in the other groups (Table 2). The larvae seem to be able to be tolerant a wide range of pH very well.. Exp. 2. Calcification of vertebra in the larvae acclimated to pH 4, pH 7 and pH 10 To evaluate the calcification of vertebra in zebrafish larvae acclimated to acidic or basic water, fluorescent calcein was used to stain the calcified vertebra of live zebrafish larvae. Fig. 1 shows the stained vertebrae of 7dpf larvae. The number of calcified vertevrae in pH 4 larvae is significantly less than that in pH 7 or pH 10 larvae (Fig.1. D), indicating that pH 4 larvae may lose Ca2+ due to systematic acidosis.. Exp. 3. Apical Morphology of the ionocytes in the larval skin The skin surface of zebrafish larva was observed with scanning electron microscopy (SEM) to examine the morphology of the ionocytes in the skin. Morphological differences were found in the apical surface (exposed apical membrane) of the ionocytes. These apical surfaces are categorized into 3 major types, type I, II and III (Fig. 2A, B, C). Type I is featured by a convex surface with short microvilli on it. Type II is 19.

(26) featured by an alveolar surface which is usually composed of numerous small pits. Type III is the surface with a hole and is usually smaller than the other two types. The size (area) and density of the 3 types of apical surfaces were quantified and compared between 3 different groups of larvae acclimated to pH 4, 7 or 10 media for 4 days (Fig. 2D, E, F). A significant increase was found in the area and density of type II surface in the larvae acclimated to pH 4 (Fig. 3). Density of type III decreased significantly in the larvae acclimated to pH 4 medium (Fig. 3B). However, the size of the other two types did not change significantly (Fig. 3A). This data implicates that the type II cell is the HR cell which is responsible for proton secretion in the skin of zebrafish larvae (Lin et al. 2006).. Exp. 4. Time-course changes of whole body [H+] in larvae subjected to pH changes The whole body [H+] of zebrafish larvae was determined by measuring the supernatant [H+] of homogenized larvae (10 larvae pooled together for 1 sample). The larvae were measured after being transferred from pH 4 to pH 7 or vice versa to determine the systematic pH regulation of the larvae. Fig. 4 shows that a significant increase of whole body [H+] was found in the larvae transferred from pH 7 to pH 4 for 30 min but no significant difference occurred at other time points. By contrast, being transferred from pH4 to pH7, the larvae seem to be able to maintain the pH very well.. 20.

(27) Exp. 5. Time-course changes of Type II cells in response to pH changes The area and density of the apical surface of Type II cell were measured in the larvae subjected to short-term (0-6 h) pH changes (from pH 4 to pH 7 or vice versa) to examine the modification of HRCs (Type II cells) in response to pH changes. Before the transfer, two groups of larvae were pre-acclimated to pH 4 or pH 7 medium for 4 days. Then the pH 4 group and the pH 7 group were transferred to pH 7 or pH 4 medium respectively. At every time point after the transfer, the size of pH 4 group is about 2-3 fold larger than that of pH 7 group (Fig. 5A). Dramatic decrease in the size was found in the larvae transferred from pH 4 to pH 7 medium (Fig. 5A). At 2h after the transfer, the size already dropped to the same as the pH 7 group and still maintained low at latter time points (Fig. 5A). By contrast, increase in the size was found in the larvae transferred from pH 7 to pH 4 medium after 4 h (Fig. 5A). However, no significant change was found in the density of type II cells (Fig. 5B).. Exp. 6. Time-course changes of acid secretion by the larvae subjected to different pH SIET was used to measure proton gradient (Δ [H+]) at larval surface which reflects the capability of acid secretion by the larval skin. At every time point after the pH transfer, the pH 4 acclimated larvae were always higher than the pH 7 acclimated larvae in proton gradient (Fig. 6). Dramatic decrease in the Δ [H+] was found in the larvae transferred from pH 4 to pH7 medium (Fig. 6). At 2h after the transfer, the Δ [H+] already 21.

(28) decreased to the level of the pH 7 group and still maintained low at latter time points (Fig. 6). By contrast, gradually increase in the Δ [H+] was found in the larvae transferred from pH 7 to pH 4 medium after 4 h (Fig. 6).. Exp. 7. Short-term changes of the apical surface of the Type II cells In this experiment, the change of the type II surface was examined within 1h of transfer from pH 4 to pH 7. As early as 10 min after the transfer, significant decrease in the size of type II surface was found (Fig. 7A). In addition to the size of type II surface, the density also decreased slightly with time (Fig. 7B). It was noticed that the apical surface of type II was shrinking during the short-term pH4 to pH7 acclimation (Fig. 7A, B). Unlike the oval and slightly convex surface in pH 4 group, the surface of type II cell showed a polygonal and flat surface during the first hour of the transfer (Fig. 8A, B). The ratio of these shrinking surfaces is above 90% before first hour and then drop to normal level after 2 h (Fig. 8C). This phenomenon was further examined by the fluorescent staining with Con-A and antibody against H+-ATPase. Con-A has been shown to specifically binding on the apical membrane of HRCs (Lin et al. 2006). Confocal laser scanning images show that the Con-A signal (in green, Fig. 9A, C, E) and H+-ATPase signal (in red, Fig. 9B, D, F) are mostly co-localized in the apical surface of the HRCs. Either Con-A or H+-ATPase signals show larger apical surface of HRCs in pH 4 group (Fig. 9A, B) than in pH7 group (Fig. 9C, D). In addition to the strong signals concentrated in the center of apical surface, we also noticed some little spots surrounding the center (thin arrows). These spotty signals were 22.

(29) shown to be the endocytotic vesicles in most of which H+-ATPase are located. In the pH 4 to pH 7 group, we found that the morphology of the apical surface is quite different from the other two groups. Shrinking apical surfaces occurred predominantly in the HRCs of the larvae transferred from pH 4 to pH 7 for 30 min (arrows in Fig. 9E, F). Fig. 9G, H, I shows the optical sections at the top (G), 2 µm below (H) and 4 µm below (I) the cells pointed in E, F. Arrowheads indicate the internalization of apical membrane which was not found in pH4 or pH7 acclimated larvae.. 23.

(30) Discussion In the previous studies, H+-ATPase-rich cells (HRCs) have been identified as an acid-secreting subtype of MRCs in the skin of zebrafish embryos and larvae (Lin et al. 2006 ; Horng et al. 2007). In this study, I examined the apical morphology of HRCs in response to ambient pH and the short-term regulatory mechanism of acid secretion by the HRCs. The major findings were (1) three different type of apical surface of ionocytes were shown in the skin of the larvae; among them, an alveolar type was identified as the apical surface of HRCs; (2) the apical surface area and density of HRCs change remarkably in response to environmental pH changes; (3) the larvae show down- or up-regulation of acid secretion in response to acute pH changes; as early as 10 min after transferring from pH 4 to pH 7, the apical surface area of HRCs dramatically decrease through shrinking and internalization of the apical membrane. The mortality of the larvae is slightly higher in pH 4 group than in the other groups. However, the shorter body length and less calcified vertebra of pH 4 group indicate a retardation of growth. By contrast, pH 10 group is as normal as pH 7 group. The zebrafish embryos and larvae seem to be more sensitive to acidic water than to basic water. With their development, gradually increase of acid secretion is required for maintaining internal pH balance and normal growth (Horng et al. 2007). Therefore, an acid environment may increase the load of acid secretion by the skin and consequently consume the energy for normal growth. Basic water in contrast may facilitate acid secretion in the larvae. Knockdown of H+-ATPase has been shown to retard growth of zebrafish embryos and 24.

(31) lead to ionic lose (particular Ca2+ ) of the embryos (Horng et al. 2007). The retardation of growth and vertebra calcification in this study might be also due to systematic acidosis and lose of internal Ca2+. The apical membrane of MRCs were documented to be polymorphic and has been examined with SEM/TEM in plenty species of teleosts including brown bullhead (Ictalurus nebulosus) (Perry et al. 1992), American eel (Anguilla rostrata) (Perry et al. 1992), rainbow trout (Oncorhynchus mykiss) (Perry et al. 1992), killifish (Fundulus heteroclitus) (Katoh et al. 2001), brown trout (Salmo trutta) (Brown 1992), tilapia (Oreochromis mossambicus) (Lee et al. 1996c), carp (Cyprinus carpio) (Lee et al. 1996a), and medaka (Oryzias latipes) (Lee et al. 1996b). In SEM micrographs of the gill surface, the FW MRCs generally showed flat or slightly invaginated surface with microvillus or microplicae on it. In the tilapia (Oreochromis mossambicus), the apical membrane was also recessed below the adjacent pavement cells to form a hole. Those morphological differences were also used to discriminate subtype of MRCs, such as α cell/β cell in brown trout (Brown 1992), wavy convex/shallow basin/deep hole or type I/II/III in tilapia (Lee et al. 1996c ; van der Heijden et al. 1999), A cell/B cell in carp (Lee et al. 1996a). The subtypes of MRCs showing different functions were also suggested (Pisam et al. 1995 ; Lee et al. 1996c ; Katoh et al. 2001 ; Chang et al. 2001, 2003). However, those early studies usually lacked molecular evidence to support the functional difference of the subtypes. Recent studies on rainbow trout have provided convincing molecular evidence to support this idea. Goss’s group isolated PNA- and PNA+ cells from branchial cell fractions of rainbow trout and demonstrated them to 25.

(32) exhibit different transporters and ion uptake (Reid et al. 2003 ; Galvez et al. 2006 ; Parks et al. 2007). Hiroi et al. (2005) identified four subtypes of MRCs with different expression pattern of transporters in tilapia larvae. Lin et al. (2006) identified HRCs and NaRCs in zebrafish larvae and demonstrated their functional difference by using molecular and in vivo electrophysiological approaches. Recently, Hwang’s group further found the third subtype of ionocytes (NCC cell) in zebrafish (Hwang and Lee, 2007). Apparently, the functional differentiation of MR cell subtypes has being a critical topic in fish ion-regulation. To further link the morphology and function of the subtypes in zebrafish, I analyzed the apical surface of ionocytes with SEM to see if structure-function relationships exist. I found three major apical structures (type I/II/III) in the skin of zebrafish larvae (Fig. 2). The type I is similar to the wavy-convex in tilapia, and is commonly shown in other freshwater teleosts (Evans et al. 2005). The type III is similar to the deep-hole in FW tilapia and SW teleosts. However, the type II (alveolar type) is not as common as the other two types but has been shown in brown bullhead (Ictalurus nebulosus) (Perry et al. 1992). I acclimated the larvae to various pH media and analyzed the changes of the apical surface. A significant increase was found in the size and density of the type II cell in pH 4 acclimated larvae indicating that type II should be the acid-secreting cells, the HRCs. Increase of size and density in type II of zebrafish seems to be required for compensating acid secretion in pH 4 medium, since a higher acid secretion was measured at the larval surface. Another study on zebrafish by Horng et al. (unpublished data) also directly demonstrated that the HRCs with larger apical surface 26.

(33) secrete more acid than those with smaller apical surface. Similar phenomenon was found in other species subjected to extremely low Na+/Cl- freshwater. Enlargement of the apical surface of MRCs were reported in killifish acclimated to low-NaCl water (katoh et al. 2003). Both the apical surface and cell density of MR cell were increased in tilapia acclimated to low Cl- medium (Chang et al. 2001, 2003 ; Lin and Hwang 2001, 2004). The increase was suggested to compensate for the Cl- lost in low Cl- medium. Horng and Lin (2008) further reported that the apical NKCC (NCC) immunoreactivity of MR cell increases in tilapia acclimated to low Cl- medium, and suggested a molecule involved in Cl- uptake. Moreover, Lin et al. (unpublished data) have recently demonstrated a higher Cl- uptake by MR cells with large (convex) surface by using SIET probing. The identity of type I/III cell is still unclear in this study, if they represent the NaRC/NCC cells needs to be further examined. I have also acclimated zebrafish larvae to low Na+/Cl-/Ca2+ media to see is there a clear structure/function relationship. However, the effect is not as clear as that shown in this study. An alternative way to identify the three subtypes could be via their distributions on the larval skin. With SEM observation, the type II surface was mostly found on yolk-sac and yolk-extension domains and only type III could be found on trunk region. Their distributions are coincident with that of HRCs and NaRCs respectively (Lin et al. 2006). Down-regulation of acid secretion seems to be relatively faster than up-regulation of that in the larvae. From the time-course observation, it takes more than 4h to increase the apical surface of type II cells to a 27.

(34) significant level in the larvae transferred from pH7 to pH4 medium. Coincidently, the acid secretion from the larvae showed a gradually increase with time. Interestingly, when transferred from pH4 to pH7 media, the larvae showed a fast down-regulation of apical surface and acid secretion. I suggest that the increase of apical membrane and H+-ATPase on it may take longer time than just modifying the configuration of apical structure (shrinking and internalization of apical membrane). Based on this phenomenon, I wonder how the HRCs can react to ambient pH changes so fast (shrinking in 10 min). In general, the reaction by the control of hormones cannot be so fast. The reaction of the HRCs should be controlled by nervous signals or probably a sensing system within HRCs themselves. Recent studies have proposed some evidence for physical connection of neural fiber and MR cells in skin/gills of zebrafish, implicating a direct control of MR cells by nervous system (Jonz and Nurse 2006). It would be interesting to further investigate if the function of HRCs is controlled by nervous signals. Mammalian acid-secreting cells of epithelium, such as clear cells of the epididymis, intercalated cells of kidney also contain abundant H+-ATPase in their apical membrane and are responsible for acidifying the lumen (Brown and Breton 2000). Acid secretion by those cells are regulated fast via active recycling (endoctyosis and exocytosis) of H+-ATPase (Breton and Brown 2007). Recently, it has been demonstrated that this recycling is regulated by luminal pH and bicarbonate through a sAC (soluble adenylyl cyclase)-dependent signaling pathway, and it was also suggested that this signaling pathway may be a widespread mechanism that allows cells to sense and regulate acid secretion 28.

(35) (Pastor-Soler et al. 2003 ; Paunescu et al. 2008). In this study, the internalization of apical membrane may be a result of apical membrane recycling as shown in the mammalian cells, since I also observed quick endocytosis of apical membrane with fluorescent Con-A labeling. Further study is needed to investigate the sAC-dependent pH sensing and H+-ATPase recycling system in zebafish.. 29.

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(45) Tables and Figures. Table 1. The mortality of larvae acclimated to different pH freshwater Medium. pH4. pH5. pH6. pH7. pH10. 6.6%. 4.2%. 4.3%. 4.4%. 4.2%. N=150 was indicated.. 39.

(46) Table 2. The body length (mm) of larvae acclimated to different pH freshwater Medium. pH4 3.56±0.19. pH5 a. 3.71±0.15. pH6 b. 3.72±0.12. pH10. pH7 b. 3.75±0.11. b. 3.73±0.14. Mean ± SD (N=40) was indicated. Different letter indicates significant difference among different groups (one-way ANOVA, Tukey’s comparison, p<0.05). 40. b.

(47) A. D b. Number of vertebrae. 6 5. b a. 4 3 2 1. pH4 0. B. pH4. pH7. pH10. Fig. 1. Calcein staining of 7dpf larvae acclimated to different pH media. Arrows indicate the stained vertebrae. The number is shown in D. Mean ± SD (N=40) is indicated. Different letter indicates significant difference among different groups (one-way ANOVA, Tukey’s comparison, p < 0.05). pH7. C. pH10. 41.

(48) Type I. Type II. A. Type III. B. C. 5 µm. 5 µm. D. E. F. 10 µm. pH 4. 5 µm. 10 µm. pH 7. 10 µm. pH 10. Fig. 2. Apical morphologies of 3 main ionocytes (A, B, C) in the skin of zebrafish larvae were observed with SEM. Apical morphologies of Type II cells were different in larvae acclimated to different pH media (D, E, F). Enlargement of Type II cell was found in pH 4 acclimated larvae. Arrows indicate Type II cells.. 42.

(49) A. Area of apical surface (µm2). 50. Type I Type II Type III. a (246). 40 30. a (52) a (91). a (60). 20 b (208) 10 a (82) 0. b (124). a (160). pH 4. a (91) pH 10. pH 7. B a (24). Density of apical surface (mm-2). 1200. b (24) 1000. b (24). Type I Type II Type III b (24). 800. ab (24) a (24). 600 400. a (24). a (24). a (24). 200 0 pH 4. pH 7. pH 10. Fig. 3. Effects of ambient pH on the area (A) and density (B) of apical surface of ionocytes in the yolk-sac skin of zebrafish larvae. Data are means ± SD. The same type of cell was compared between different pH groups. Different letter indicates significant difference (p < 0.05, one-way ANOVA and Tukey’s comparisons).. 43.

(50) pH4 pH4 to pH7 pH7 pH7 to pH4. Whole body [ H+] (10-7 M). 1.5. b 1.25 ab a 1.0. ab. a. a a. a a. a. a. a 0.0 0. 30. 60 Time (min). 90. 120. Fig. 4. Short-term changes of whole body [H+] in zebrafish larvae subjected to acute pH changes (from pH 4 to pH 7 or vice versa). pH 4, pH 7 controlled groups indicate the larvae acclimated to pH 4 or pH 7 for 4 days. Data are mean ± SD (N = 5). Different letter indicates significant difference among different time points (one-way ANOVA, Tukey’s comparison, p < 0.05).. 44.

(51) A. pH4 pH4 to pH7 pH7 pH7 to pH4. Apical Surface (µm2). 50 a (59). a (119). 40. a (100). a (120). 30 20 10. b (111). b (225) b (169). b (343). b (97). c (219) c (70). b (395) b (175) c (84). 0 0. 2. 4. 6. Time (h). B. pH4 pH4 to pH7 pH7 pH7 to pH4 1800 1600. a (12). 1400. a (12) a (12) a (12). Density (mm-2). a (12) 1200 1000. a (12) a (12). a (12) a (12) a (12). 800 600. a (12). a (12). b (12). 400 0 0. 2. 4. a (12) 6. Time (h) Fig. 5. Changes of apical surface area (A) and density (B) of H+-ATPase-rich cell (HRCs) in the larvae subjected to pH changes. Data are mean ± SD. Different letter indicates significant difference among different groups at the same time (one-way ANOVA, Tukey’s comparison, p < 0.05). A significant decrease was found in the larvae transferred from pH4 to pH7.. 45.

(52) pH4 pH4 to pH7 pH7 pH7 to pH4. 0.15. a. Δ [ H+] (µM). a. a. 0.10 b a. b. b. c 0.05 b. bc b. c. c. c. 0.00 0. 2. 4. 6. Time (h) Fig. 6. Changes of Δ [H+] at skin surface of the larvae subjected to pH changes. The Δ [H+] represents acid secretion from the skin. Different letter indicates significant difference among different groups at the same time (N= 10, one-way ANOVA, Tukey’s comparison, p < 0.05).. 46.

(53) A. pH4 pH4 to pH7 pH7. Apical Surface (µm2). 60 50 40. a (132). a (128). 30 b (128). bc (102). 20. c (84) a (57). 10. a (93). 0 0. 10. 20. 30 40 Time (min). B. 60. pH4 pH4 to pH7 pH7. 1400 1200. Density (mm-2). 50. a (12). a (12). 1000 800. ab (12). ab (12). 600. b (12) a (12). a (12). 400 0 0. 10. 20. 30. 40. 50. 60. Time (min) Fig. 7. Short-term changes of apical surface area and density of HRCs in the larvae subjected to pH changes. Data are mean ± SD. Different letter indicates significant difference among different time points at the same group (one-way ANOVA, Turkey’s comparison, p < 0.05). A significant decrease was found in the larvae transferred from pH4 to pH7.. 47.

(54) A. B. 10 µm. 2.5 µm. Ratio of shrinking apical surface (%). C. b (12). b (12). pH4 pH4 to pH7 pH7. b (12). 100 80 60 a (12) 40. a (12) a (12). a (12). 20 0.0 0. a (12). a (12). a (12) 30. 60 Time (min). 90. 120. Fig. 8. Morphological changes in HRCs in larvae transferred from pH4 to pH7. Shrinking of the apical surface was observed in HRCs during short-term pH transfer. The locations of HRCs are indicated by arrows (A, B). The percentage of shrinking HRCs’ apical surface is shown (C). Data are presented as the mean±SD. Different letters indicate a significant difference among different time points at the same group (one-way ANOVA, Turkey’s comparison, p < 0.05). 48.

(55) A. B. 10 µm. pH 4. 10 µm. pH 4. D. C. 10 µm. pH 7. E. 10 µm. pH 7. F. 10 µm. G. 10 µm. I. H. 5 µm. 5 µm. 5 µm. Fig. 9. Confocal images of Con-A (A, C, E) and H+-ATPase (B, D, F) staining in HRCs of the larvae acclimated to pH4 (A, B), pH7 (C, D) for 4 days, and transferred from pH4 to pH7 for 30 min (E, F). G, H, I shows the optical sections of the arrows pointed cells in E, F. The apical surface (G), 2 um (H), and 4 um below (I) are shown. Arrowheads indicate internalization of apical membrane which was not found in pH4 and pH7 acclimated larvae.. 49.

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