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 4dpf
pH4
pH7
pH10
Measurement of body length, mortality (exp. 1) SEM observation and quantification of ionocytes (exp. 3) pH4
pH7
pH10
Calcein stain and measurement of vertebra number (exp. 2)
7dpf Sampling for
exp. 2
Fertilized eggs
Sampling for exp. 1& 3
Exp. 4. Time-course changes of whole body [H+] in larvae subjected pH changes
0h 1h 4dpf
Fertilized eggs pH4
pH7
pH4
pH7
pH4
pH7 pH7
pH4
0m 30m
Acclimation to Transfer to
Acclimation to Transfer to
Sampling for SIET
60m 90m 120m
Measurement of whole body [H+]
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
0h 1h 4dpf
SEM observation and quantification of ionocytes (exp. 5)
Measurement of acid secretion (exp. 6) pH4
pH7
pH4
pH7
pH4
pH7 pH7
pH4
0h 2h 4h 6h
Acclimation to Transfer to
Acclimation to Transfer to Fertilized eggs
Sampling for SEM & SIET
Exp. 7. Short-term changes of the apical surface of the Type II cells
0h 1h 4dpf
Fertilized eggs pH4
pH7
pH4
pH7
pH4
pH7 pH7
0m 10m
Sampling for SEM
Acclimation to Transfer to
Acclimation to Transfer to
30m 60m 120m
SEM observation and quantification of ionocytes
Immunostaining and confocal observation Sampling for Confocal
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
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.
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
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
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.
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
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
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
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
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
(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.
References
Al-Awqati, Q. (1996). Plasticity in epithelial polarity of renal intercalated cells: targeting of the H+-ATPase and band 3. Am. J. Physiol. 270, C1571-1580.
Breton, S. and Brown, D. (2007). New insights into the regulation of V-ATPase-dependent proton secretion. Am. J. Physiol. Renal. Physiol.
292, F1-10.
Breton, S., Wiederhold, T., Marshansky, V., Nsumu, N. N., Ramesh, V.
and Brown, D. (2000). The B1 subunit of the H+-ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells. J. Biol. Chem. 275, 18219-18224.
Brown, D. and Breton, S. (2000). H+-V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas
deferens: vesicle recycling and transcytotic pathways. J. Exp. Biol. 203, 137-145.
Brown, P. (1992). Gill chloride cell surface-area is greater in
fresh-water-adapted adult sea trout (Salmo trutta L) than those adapted to seawater. J. Fish Biol. 40, 481-484.
Chang, I. C., Lee, T. H., Yang, C. H., Wei, Y. Y., Chou, F. I. and Hwang, P. P. (2001). Morphology and function of gill
mitochondria-rich cells in fish acclimated to different environments.
Physiol. Biochem. Zool. 74, 111-119.
Chang, I. C., Wei, Y. Y., Chou, F. I. and Hwang, P. P. (2003).
Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis
mossambicus). Physiol. Biochem. Zool. 76, 544-552.
Choe, K. P., O'Brien, S., Evans, D. H., Toop, T. and Edwards, S. L.
(2004). Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis. J. Exp. Zoolog. A. Comp. Exp. Biol. 301, 654-665.
Claiborne, J. B. (1998). Acid-base regulation. In: Evans DH, editor. The physiology of fishes. Boca Raton: CRC Prese, p 179-200.
Claiborne, J. B., Compton-McCullough, D. and Walton, J. S. (2000).
Branchial acid-base transfers in the euryhaline oyster toadfish (Opsanus tau) during expose to dilute seawater. J. Fish Biol. 56, 1539-1544.
Claiborne, J. B., Edwards, S. L. and Morrison-Shetlar, A. I. (2002).
Acid-base regulation in fishes: cellular and molecular mechanisms. J.
Exp. Zool. 293, 302-319.
Esaki, M., Hoshijima, K., Kobayashi, S., Fukuda, H., Kawakami, K.
and Hirose, S. (2007). Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R470-480.
Evans, D. H., Piermarini, P. M. and Choe, K. P. (2005). The multifunctional fish gill: dominant site of gas exchange,
osmoregulation, acid-base regulation and excretion of nitrogenous waste. Physiol. Rev. 85, 97-177.
Galvez, F., Wong, D. and Wood, C. M. (2006). Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 291, R170-176.
Goss, G. G., Laurent, P. and Perry, S. F. (1992). Evidence for a morphological component in the regulation of acid-base balance in hypercapnic catfish (Ictalurus nebulosus). Cell Tissue Res. 268, 539-552.
Goss, G. G., Laurent, P. and Perry, S. F. (1994a). Gill morphology during hypercapnia in brown bullhead (I. nebulosus): role of chloride cells and pavement cells in acid-base regulation. J. Fish Biol. 45, 705-718.
Goss, G. G. and Perry, S. F. (1993). Physiological and morphological regulation of acid-base status during hypercapnia in rainbow trout (Oncorhynchus mykiss). Can. J. Zool. 71, 1673-1680.
Goss, G. G. and Perry, S. F. (1994b). Different mechanisms of acid-base regulation in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) during NaHCO3 infusion. Physiol. Zool. 67, 381-406.
Goss, G. G., Perry, S. F. and Laurent, P. (1995). Gill morphology and
Goss, G. G., Perry, S. F. and Laurent, P. (1995). Gill morphology and