JOURNAL OF MORPHOLOGY 275:933–948 (2014)

The Structure of the Caudal Wall of the Zebrafish (Danio rerio) Swim Bladder: Evidence of Localized Lamellar Body Secretion and a Proximate Neural Plexus George N. Robertson,1,2* Roger P. Croll,3 and Frank M. Smith1 1

Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 Department of Biology, Saint Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 2W5 3 Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 2

ABSTRACT In this study, we present a morphological description of the fine structure of the tissues composing the caudal tip of the adult zebrafish swim bladder and an immunochemical survey of the innervation at this site. The internal aspect of the caudal tip is lined by an epithelium specialized to secrete surfactant into the lumen as evinced by the exocytosis of lamellar bodies. The sole innervation to this region consists of a neural plexus, present on the external surface, of nitric oxide synthase-positive (nNOS) neuronal cell bodies that are contacted by axon terminals, some containing neuropeptide Y and vasoactive intestinal polypeptide. As the specialized epithelium and neural plexus are coincident and of common extent, we suggest that the morphological relationship between the two elements allows the nervous system to affect surfactant processing, possibly through a paracrine mechanism. J. Morphol. 275:933–948, 2014. VC 2014 Wiley Periodicals, Inc. KEY WORDS: surfactant; autonomic nervous system; cyprinid; ultrastructure

INTRODUCTION In previous studies, we examined the structure of the zebrafish swim bladder to determine the nature of the innervation to peripheral target tissues that function to maintain buoyancy (Finney et al., 2006; Robertson et al., 2007; Dumbarton et al., 2010). Here, we report the results of a histological investigation and immunocytochemical survey of the caudal tip of the swim bladder wall which, we believe, relates axonal terminals to a small, discrete area of epithelium that is specialized to exocytose surfactant into the swim bladder. Surfactants are vital fluid mixtures of lipid and protein that line gas-filled structures such as swim bladders and lungs and act to lessen the work of reinflation by reducing surface tension and also serve as a lubricant to prevent adjacent tissue surfaces from adhering after exhalation or collapse (Daniels and Orgeig, 2003). There are identifiable morphological structures that are manifestations of surfactant production, maturation, and expression. Surfactants are processed, stored, and secreted from distinctive cellular organelles called lamellar bodies which have been C 2014 WILEY PERIODICALS, INC. V

found within the alveolar type II (AT II) cells in the lung (Rooney et al., 1975; Schmitz and Muller, 1991) and within epithelial cells of the swim bladders of many different fishes including mummychog, cod, toadfish (Copeland, 1969), trout (Brooks, 1970), goldfish (Morris and Albright, 1979), perch, eel (Prem et al., 2000), pirarucu, tarpon, and snapper (see for review, Daniels et al., 2004). The lamellar body appears round or oval in profile, is encapsulated by a surrounding membrane, and is filled with alternating lamellae of electrondense and electron-lucent layers that extend the full width of the organelle. Maturation of this organelle is advanced by fusion with multivesicular bodies, organelles containing many small vesicles (Weaver et al., 2002). The mature lamellar body then fuses with the apical membrane of the surfactant-producing cell and secretes surfactant into the gas-filled lumen. This transposition, from an intracellular to an extracellular location, results in conformational changes of the surfactant from compact lamellae to characteristic expanded whorls or tubules, which may also be registered in meshes, known as tubular myelin. Tubular myelin is a repository of surfactant that provides a single layer of lipids and proteins, in the proper proportions, to the air–liquid interface (Fehrenbach, 2001; Daniels and Orgeig, 2003). In the present study, we used these established ultrastructural

Contract grant sponsor: Canadian Space Agency; Grant number: #046016/001/ST (R.P.C.; F.M.S.); Contract grant sponsor: Natural Sciences and Engineering Council of Canada; Grant number: #38863-02 (Discovery Grant R.P.C.). *Correspondence to: George Robertson, Department of Biology, Saint Francis Xavier University, Antigonish, N.S., Canada B2G 2W5. E-mail: [email protected] Received 16 December 2013; Revised 11 February 2014; Accepted 25 February 2014. Published online 19 March 2014 in Wiley Online Library ( DOI 10.1002/jmor.20274


G.N. ROBERTSON ET AL. TABLE 1. Details of primary antibodies

Antibody zn-12 Choline acetyltransferase (ChAT) Tyrosine hydroxylase (TH) Neuropeptide Y (NPY) Vasoactive intestinal peptide (VIP) Neuronal nitric oxide synthase (nNOS)

Immunogen L2 HNK-1 epitope on zebrafish axons Human placental enzyme TH from rat PC 12 cells Synthetic neuropeptide Y (porcine) coupled to bovine thyroglobulin Synthetic VIP (human, porcine, rat) coupled to bovine thyroglobulin Synthetic peptide corresponding to amino acids 1411–1425 of human nNOS



Specimens examined (n)

Developmental Studies Hybridoma Bank, mouse, monoclonal Millipore/Chemicon (cat. no. AB144P) goat, polyclonal Immunostar (cat. no. 22941) mouse, monoclonal Immunostar (cat. no. 22940) rabbit, polyclonal Immunostar (cat. no. 20077) rabbit, polyclonal Abcam (cat. no. ab5586) rabbit, polyclonal













zn-12 and nNOS

criteria (presence of lamellar bodies, evidence of their exocytosis and occurrence of multivesicular bodies) to demonstrate that the specialized epithelium at the caudal end of the zebrafish swim bladder secretes surfactant. There is evidence that both the quality and quantity of swim bladder surfactant are controlled. The swim bladder of the goldfish, a cyprinid like the zebrafish, is a double-chambered organ of hour-glass shape that is linked at the center, yet the surfactant components of the two chambers differ in composition and rate of formation. This difference may be related to the functions of the two chambers as the anterior chamber aids audition and the posterior aids buoyancy (Doneen and Gutmann, 1981; Daniels and Skinner, 1994). Surfactant release can be affected by the autonomic nervous system as adrenergic and cholinergic agents will stimulate surfactant secretion when applied to the isolated lungs or cultured cells of lung and swim bladder tissue of a wide variety of vertebrates (lung of rat, Brown and Longmore, 1981; fat-tailed dunnart, Wood et al., 2000; bullfrog, Wood et al., 2000; bearded dragon lizard, Wood et al., 1995, 1997, 1999; swim bladder of Australian lungfish, Wood et al., 2000). However, the structural components of the neural systems contributing to surfactant release, and their specific relation to the target surfactant-secreting cells, is not yet understood. Here, we provide a detailed morphological description of the specialized caudal tip of the swim bladder, from the external nerve terminals to the internal epithelium, to define the structural relationships between these different tissue elements. A novel discovery, reported here, is that a discrete neural plexus is situated on the external surface of the swim bladder at a site that is coincident and of common extent to the internal specialized epithelium. As this plexus provides the sole innervation to this location and contains the terminal portions of fibers of the nervous system, we propose that there is a possible functional relaJournal of Morphology


tionship between the plexus and the surfactantsecreting cells based on proximity and that, by this structural configuration, the nervous system may affect the surfactant-secreting cells. MATERIALS AND METHODS Animals Adult zebrafish, Danio rerio (Hamilton, 1822), were purchased from a local pet store (Aqua Creations Tropical Fish, Halifax, Canada) and kept in aerated, dechlorinated tap water at 28 C on a 14:10 h light–dark schedule. Sixty zebrafish were used in this study. Fish were maintained following the procedures outlined in Westerfield (1995); animals of both sexes were kept in 75–80 l glass aquaria and fed Nutrafin staple fish food (Rolf C. Hagen, Montreal, Canada). Institutional approval for these experiments was obtained from the University Committee on Laboratory Animals at Dalhousie University, where experiments were performed between 2004 and 2008.

Immunochemical Labeling All fish were anesthetized in a 0.01% solution of tricane methanesulphonate (MS-222) in tank water until all movement ceased. The swim bladder, pneumatic duct, and an attached segment of the esophagus were removed and immersion-fixed in 4% paraformaldehyde (TAAB, UK) in phosphate-buffered saline (PBS, 50 mmol l21 Na2HPO4, 140 mmol l21 NaCl, pH 7.2) at room temperature for 1–4 h and processed as whole mounts. After fixation, all tissues were rinsed three times with PBS for 10 min each and placed in a PBS solution containing 4.0% Triton X-100 to permeabilize the tissue for 12–16 h at 4 C. Tissues were then incubated in the appropriate primary antibody (see below and Table 1 for antibody sources and dilutions) for 7–10 days. Thereafter, tissues were rinsed in PBS as above, and then incubated in a 1:200 dilution of secondary antibody, in PBS, for 3–7 days (see Table 2). Following three final rinses in PBS, tissues were placed in a solution of three parts glycerol to one part 0.1 mmol l21 Tris, pH 8.0, with 2% n-propyl gallate, overnight, before being mounted on slides for viewing and orientation. The specialized segment of tissue was prepared for a more detailed investigation by cutting the small caudal portion of the posterior chamber free of the rest of the swim bladder. This conical structure was flattened by a series of radial cuts, and then remounted, inspected, and photographed to provide a clear image of the structure reported here.

Antibody Characterization The specificity of each antibody and the pertinent preabsorption controls were related in Finney et al. (2006), with the



TABLE 2. Details of secondary antibodies Antibody (zn-12) goat anti-mouse (ChAT) donkey anti-goat (TH) goat anti-mouse (NPY) goat anti-rabbit (VIP) goat anti-rabbit (nNOS) goat anti-rabbit

Fluorophore Alexa CY5 Alexa FITC Alexa Alexa

Fluor 488/555 Fluor 555 Fluor 488 Fluor 488

exception of nNOS (see below). In general, whole mounts of the swim bladder, pneumatic duct and esophagus were used as positive controls to confirm the known immunochemical staining patterns of the antibodies, as first reported in Finney et al. (2006; see also Robertson et al., 2007), before removing the caudal tip for detailed examination. The overall pattern of innervation to the posterior portion of the swim bladder was revealed by the antibody zn-12, an axonal marker in zebrafish (Trevarrow et al., 1990). The cholinergic neurons associated with the parasympathetic nervous system were identified with an antibody against choline acetyltransferase (ChAT), the enzyme that catalyzes the formation of acetylcholine. Similarly, an antibody that binds to tyrosine hydroxylase (TH), the enzyme that catalyzes the rate-limiting step in the formation of catecholamines, was used as a marker for adrenergic neurons of the sympathetic nervous system. The situations of neuropeptide Y (NPY) and vasoactive intestinal peptide were elucidated using antibodies directed toward these neuroactive peptides. Cells capable of forming nitric oxide (NO) were identified by an antibody directed towards neuronal nitric oxide synthase (nNOS), the enzyme that catalyzes the formation of this chemical within the nervous system. See Tables 1 and 2 for antibody types, sources, dilutions, and the number of animals used in each assay. To test the specificity of the nNOS antibody (Abcam ab5586 rabbit polyclonal antibody to a synthetic peptide, residues 1411–1425 from human nNOS: 93% homology with the zebrafish), we compared the staining of tissue using a 1:400 nNOS dilution with that of an adsorbed antibody preparation. To adsorb the antibody and block staining, we added nNOS peptide (Abcam ab5891, used to generate the antibody) in the ratio of 5 mg peptide to 1 mg nNOS antibody, agitated the solution for 20 min on a rocker then stored it at 4 C overnight. The solution was spun at 5,000 rpm for 10 min and the supernatant used as a probe. The caudal tip was removed from each of four swim bladders; two were incubated in the immunostaining solution and two in the immunoadsorbed solution. In addition, to take advantage of the bilaterally symmetrical distribution of a cluster of nNOS-positive cells situated at the junction of the lateral band and the rete on both sides of the swim bladder, the remainder of the posterior chamber tissue was cut in half along the sagittal plane, separating the corresponding nNOS positive cell clusters, and one half was put into the positive control with the opposite half being placed in the adsorbed solution. All tissue incubated in the positive solution stained cell bodies at the appropriate positions; there was no staining apparent in the tissue from the adsorption control.

Fluorescent Images Digital images of the swim bladder tissue were made using a Zeiss LSM 510 confocal microscope. Confocal z-stacks, with an interval of 1 mm between adjacent optical sections, were projected to create single images with proprietary Zeiss software.

Light Microscopy One micrometer resin sections were stained with a combination of acridine orange and thionin (Sievers, 1971).



1:200 1:200 1:200 1:50 1:200 1:200

Molecular probes A11001/A21422 Jackson 705-175-147 Molecular probes A21422 Jackson 111-095-003 Molecular probes A11008 Molecular probes A11008

Electron Microscopy Dissected tissues were immersion-fixed for 1 h in 3% formaldehyde and 3% glutaraldehyde in 0.1 mmol l21 sodium cacodylate buffer with 340 ll of 0.1mmol l21 CaCl2.2H2O added to 10 ml of fixative solution. After rinsing, tissues were stored at 4 C overnight in 1% tannic acid, and then osmicated (1% OsO4 in distilled water) for 1 h. The tissues were again rinsed and stained en bloc in a saturated aqueous solution of uranyl acetate for 1 h after which they were dehydrated through an ascending series of acetone solutions. The tissues were next infiltrated with epoxy resin, placed in wells and cured at 60 C for 2 days. Ultrathin sections were cut on a LKB Ultratome III and stained for 1 h in uranyl acetate and 4 min in lead citrate. Sections were examined on a JOEL 1230 electron microscope at 80KV and images were captured with a Hamamatsu digital camera. All images were adjusted for consistency of brightness and contrast, labeled and assembled into plates using Photoshop CS5 (Adobe Systems, San Jose, CA).

RESULTS The tip of the caudal extension of the posterior chamber in the zebrafish swim bladder (Fig. 1, arrow in Fig. 1c) was the site of a region specialized for secretion of lamellar bodies. Paired lateral nerves and small blood vessels coursed along the serosal side of the lateral muscle bands (arrowheads, Fig. 1b,c) to converge at this caudal tip. The ultrastructure and the patterns of innervation of the posterior chamber wall in this region are described below. Wall Ultrastructure Overview: Comparison of general and specialized wall structure. Figure 2a shows a low-power, bright-field photomicrograph of a section of the caudal posterior chamber that serves to orient the tissue sections in the other panels. This section, oblique to the frontal plane, included a region of the lateral wall dorsal to the lateral muscle band (box b) and the thickened specialized region of the wall at the caudal tip where secretion of lamellar bodies occurred (box c). Figure 2b shows ultrastructural details of the general wall structure typical of most of the posterior chamber excluding the lateral muscle bands and the posterior tip. The general wall was approximately 6 lm thick, consisting of a simple squamous epithelium on the luminal surface and a discontinuous layer of fibroblasts in the midwall with connective tissue Journal of Morphology



Fig. 1. Location and structure of the zebrafish swim bladder. a) Schematic diagram of the swim bladder and associated structures in left lateral view. The pneumatic duct (pd) links the esophagus (e) to the posterior chamber (pc) of the swim bladder; the anterior chamber (ac) of the swim bladder is linked to the posterior chamber by the ductus communicans (dc). Bar 5 1 cm. b) Schematic diagram of the posterior chamber in left lateral view. Nerve trunks along the swim bladder artery (sa) and pneumatic duct (pd) contribute multiple axons (large arrows) to bilateral nerve trunks (arrowhead indicates left lateral nerve) coursing the length of the posterior chamber. Hatching indicates the extent of the lateral muscle band. c) Photomicrograph of isolated posterior chamber corresponding to schematic shown in panel b; arrowhead indicates position of lateral nerve and arrow indicates caudal region specialization. Scale bar in c represents 1 mm.

(primarily collagen fibers) making up the bulk of the wall. Nerves were, occasionally, observed at the serosal surface. The wall gradually thickened toward the caudal tip of the chamber, attaining a maximal thickness of approximately 40–60 lm at the tip (Fig. 2c). This increase in thickness was largely due to an increased amount of collagen and the addition of discontinuous layers of smooth muscle cells (Fig. 2c). Inner wall: Epithelium and lamellar body secretion. There was a marked change in the ultrastructure of the epithelium at the caudal tip, compared with that found lining the general wall of the posterior chamber. At the tip, the epithelium remained as a single layer of cells with thin apical extensions between adjacent cells serving to maintain a contiguous epithelial layer at the lumen (Fig. 2c,d). However, the cell bodies occupied invaginations that extended into the underlyJournal of Morphology

ing connective tissue (Fig. 2c,d). Typically, the basal portion of the cell narrowed to form a single, filamentous basal process (Figs. 2d,e) which was surrounded by a basal lamina. More than one cell could occupy a single invagination resulting in apical junctional complexes present within the luminal span of that invagination; adjacent epithelial cells were attached by tight junctions and desmosomes (Fig. 2f). The luminal surface of these cells displayed microvilli; no cilia were observed. The nuclei of epithelial cells were lobulated and situated within the apical portion of the cytoplasm (Fig. 3a). Epithelial cells also contained large vesicular bodies, free ribosomes, and rough endoplasmic reticulum (Fig. 3a–c). However, the main features distinguishing the epithelial cells in the caudal tip of the posterior chamber from those of epithelial cells elsewhere in the swim bladder were the presence of lamellar bodies, multivesicular bodies and large vacuoles full of vesicles (Fig. 3b,c). Most vesicles within the vacuoles appeared to have a homogeneous internal structure, but some displayed a lamellar internal organization (Fig. 3c, arrows). Lamellar bodies similar to those shown in Figure 3b,d were observed in epithelial cells of all five sampled swim bladders. Most lamellar bodies had thin, straight or slightly curved lamellae that were arranged in parallel. In several cases, a lamellar body was observed in the process of merging with the apical membrane and extruding its contents into the lumen (Fig. 3d). Figure 4a shows the characteristic membranous whorls of secreted surfactant located in the lumen. Occasionally, these whorls formed a rudimentary organization wherein some lamellae were aligned in parallel (Fig. 4b), however, the regular geometric array of tubular myelin was not found. Fibroblasts and connective tissue. Figure 5 shows ultrastructural details of fibroblasts, at the caudal tip, and demonstrates the variable relationship between these cells and the basal extensions of epithelial cells. The presence of a continuous basal lamina, enveloping the basal extensions of the luminal epithelial cells, completely separated these cells from the connective tissue layer (Fig. 5a). There was no direct contact between epithelial cells and fibroblasts seen in this survey. Some fibroblast cell bodies were located between the epithelial basal processes; others were situated peripheral to these same processes within this layer (Fig. 5b). The fibroblasts had lobular nuclei, were rich in rough endoplasmic reticulum, and extended long thin processes toward the epithelial cells. These processes abutted, but did not broach, the basal lamina (Fig. 5c,d). Fibroblasts also extended thin processes which contacted adjacent fibroblasts (Fig. 5e,f) in the form of direct cell membrane appositions (Fig. 5g). In one thin section, we observed five fibroblasts, connected



Fig. 2. Light-micrographs (a,c) and electron-micrographs (b,d–f) comparing structural differences in the posterior chamber between the caudal wall specialization and that of the adjacent general lateral wall. a) A bright-field micrograph of 1-lm thick plastic section (to orient micrographs in b–f) displays the caudal-most portion of the posterior chamber, taken in the rostral (R) to caudal (C) axis, oblique to the frontal plane. Note the difference in thickness between the regular lateral wall (box b) and the specialized caudal portion (box c) of the swim bladder wall. L-lumen, Bar 5 200 mm. b) Enlargement of region in box b in panel a, showing typical structure of the general posterior chamber lateral wall, excluding the lateral muscle band. The wall consists of a uniformly flat epithelium (E) and a connective tissue layer (CT), surrounding a fibroblast (F), with a small nerve (Nv) at the periphery. The thickness of the swim bladder wall is approximately 6 mm at this position. L 5 lumen Bar 5 2 mm. c–f) Enlargements of specialized caudal wall region in box c of panel a. c) The nature and shape of the epithelium (E) in the caudal specialization differs from that of the thin lateral wall (as seen in panel b) such that the epithelial cell bodies (arrows) extend into the thickened connective tissue layer and are linked by thin apical processes (arrowhead). The swim bladder wall is approximately 40 mm thick at this position. L-lumen, CT-connective tissue layer, SM-smooth muscle layer Bar 5 10 mm. d) The epithelial cell bodies (E) of the caudal specialization extend basal processes (B) into invaginations of the connective tissue layer (CT) and are joined by thin processes (arrowhead) at their apices. The portion of the micrograph enclosed in the box is enlarged in panel e. L-lumen, F-fibroblast Bar 5 2 mm. e) An epithelial cell body (E) extends a filamentous basal process (B) into the connective tissue layer (CT). This process is completely enveloped by a continuous basal lamina (arrowheads). L-lumen, m-microvilli, Bar 5 1 mm. f) Thin apical processes of adjacent epithelial cells overlap and form a tight junction (J) and a desmosome (D). L-lumen, arrowheads-basal lamina Bar 5 200 nm.

Journal of Morphology



Fig. 3. Electron micrographs of vesicular and lamellar bodies in specialized caudal tip epithelial cells. a) A typical epithelial cell has a lobulated nucleus (N) and contains a large vesicular body (v). The areas within boxes b and c are enlarged in panels b and c respectively. L-lumen Bar 5 5 mm. b) Detail of epithelial cell cytoplasm (in box b of panel a) showing lamellar bodies (LB) and a multivesicular body (mvb). Bar 5 500 nm. c) Detail of a vesicular body (v) (from box c in panel a). The contents of most vesicles appeared homogeneous, but some had a lamellar appearance (arrows). Bar 5 1 mm. d) Lamellar body (LB) contents were expressed into the lumen (L) by an epithelial cell (E). arrowheads-basal lamina, B-basal process Bar 5 500 nm.

Journal of Morphology



Fig. 4. Electron micrographs of surfactant configuration. a) The surfactant profiles, within the swim bladder lumen, were usually in the form of whorls (w). Bar 5 2 mm b) Some surfactant profiles exhibited a rudimentary organization characteristic of tubular myelin (arrows). L-lumen, Eepithelial cell Bar 5 500 nm.

serially, spanning a total length of 25 lm (data not shown). Outer wall: Smooth muscle, “peripheral” cells, and vascular elements. Small blood vessels (2–3 erythrocytes in diameter) were observed only in association with the serosal surface of the wall at the caudal tip, as shown in Figure 6a. A discontinuous and irregular layer of smooth muscle cells was embedded in the connective tissue matrix of most areas of the wall at the caudal tip of the posterior chamber; these cells were usually positioned toward the serosal side of the wall as shown in Figure 6b. However, in some regions of the caudal tip wall, a few smooth muscle myocytes were present midwall, appearing to approach the basal extensions of epithelial cells as closely as did some fibroblasts. A population of distinctive cell bodies was located next to the serosal surface of the wall at the caudal tip (Fig. 6b–e). We have named these cells “peripheral” cells (P-cells) due to their location. Their cell body profiles were observed in only a few sections, existing singly or in groups of two or three per section. The overall cell shape ranged from ovoid to lunate with a central, nonlobulated nucleus (Fig. 6b). The cell body had a long dimension ranging between 5 and 10 lm. P-cells contained many free ribosomes and some rough endoplasmic reticulum, as well as highly developed Golgi bodies (Fig. 6c). Distinct round vesicles (200–400 nm in diameter) were present singly or in clusters of three or four per cell (Fig. 6c–e). The content of most of these vesicles appeared homogeneous (Fig. 6d), but some vesicles showed evidence of an internal structure of membranous lamellae or small spherical bodies (Fig. 6e). There did not seem to be a specific intracellular location for these vesicles as they were observed throughout the cytoplasm.

Innervation Ultrastructure. At the caudal tip of the posterior chamber, small nerve trunks (Fig. 7a; see also Fig. 6b) and individual axons (Fig. 7b) were observed only in association with the serosal side of the wall, where they were embedded in connective tissue or in close apposition to P-cells (Fig. 7c). Some of these axon terminals formed synapses with P-cells (Fig. 7d); no synapses were found in association with any other cell type. No neuronal elements were found in contact with the epithelial cells. Immunohistochemistry. Antibodies against zn12 showed that the paired lateral nerves converged to form a plexus at the caudal tip of the posterior chamber, in the region where the epithelial cells that secreted lamellar bodies were observed (Fig. 8a). These lateral nerves appeared to provide the only extrinsic innervation to the plexus. Single axons and small nerves arising from this plexus were distributed in wall tissues surrounding the plexus. In addition, some axon terminals in this region appeared to form a lattice-like network around putative cell bodies (Fig. 8a, inset). In the plexus region, antibodies against NPY labeled axons in both the lateral nerves and within the plexus (Fig. 8b). Furthermore, many putative cell bodies within the plexus were surrounded by NPY-positive fiber terminals (Fig. 8b, inset). Antibodies against vasoactive intestinal polypeptide (VIP) showed a relatively high density of punctate labeling in selective regions of the plexus (Fig. 8c). This punctate labeling was in close approximation to putative cell bodies, some of which also appeared VIP-positive (Fig. 8c, inserts). These putative cell bodies were circular to ovoid in shape and approximately 8–10 mm in diameter. Although the intensity of fluorescence of the VIP-positive punctate elements was consistent in all specimens, the Journal of Morphology

Fig. 5. Ultrastructural organization of caudal wall of posterior chamber. a) An epithelial cell (E) contains a lamellar body (LB), microvilli (m) are present on the apical surface, and a basal process (B) extends into the underlying connective tissue (CT). A continuous basal lamina (arrowheads) separates the epithelial cell from underlying tissue. L-lumen Bar 5 500 nm b) Low-power electron micrograph demonstrates the relationships between the three cell layers of the specialization. The epithelial cells (E) extend basal processes (B) into a connective tissue layer (CT) of fibroblasts (F) set within an extracellular matrix. These fibroblasts extend long, thin processes towards epithelial cells and adjacent fibroblasts. The smooth muscle layer (SM) is present at the external margin of the connective tissue layer. L-lumen bar 5 2 mm c,d). Relationship of a fibroblast process to a basal extension of an epithelial cell c) A fibroblast process (F) abuts the basal extension (B) of an epithelial cell (E) as indicated by the arrow. L-lumen, N-nucleus, mmicrovilli Bar 5 2 mm. d) The fibroblast process (F) indicated in panel c is enlarged here. It abuts, but does not broach, the basal lamina (arrows) of the epithelial cell basal process (B) Bar 5 0.5 mm. e–g) Series of related micrographs detailing the relationship between the processes of two adjacent fibroblasts (F1, F2) shown in panel e. An asterisk is placed in register in all three panels for orientation purposes. The two fibroblasts indicated in panel e (F1 and F2) are in contact as shown in panel f (between arrows) by membrane apposition, panel g (arrow). Bar 5 2 mm in e; 500 nm in f and 100 nm in g.



Fig. 6. Ultrastructure of the outer layer of caudal posterior chamber tip. a) Light-micrograph of a section through the caudal swim bladder wall shows two small-caliber blood vessels (arrows) associated with the serosal surface. Bar 5 10 mm. b) Peripheral cell bodies (P) are found clustered on the serosal side of the smooth muscle layer (SM), at the periphery of the specialization. Two small nerves are situated between the peripheral cell bodies and are indicated by (Nv) and the vertical arrow, respectively. Details of these nerves appear in Fig. 7. Bar 5 2 mm. c) The peripheral cells contain Golgi bodies (G), multivesicular bodies (mvb) and are distinguished by the presence of large vesicles (v). N-nucleus, bar 5 500 nm. d) Vesicles (v) were found throughout the cytoplasm of the peripheral cell; the three shown here are situated adjacent to the cell membrane. Bar 5 200 nm. e) Vesicle content usually appeared homogenous, however, some show internal inclusions of vesicles (upper arrow) or lamellae (lower arrow). Bar 5 200 nm.

fluorescent intensity of putative cell bodies was highly variable among specimens. Antibodies against nNOS clearly labeled putative cell bodies in the plexus (Fig. 9a,c); these were surrounded by zn12-positive axons (Fig. 9b,c). The putative nNOS-positive cell bodies were approximately 4–5 mm in diameter. Moreover, some of these nNOS-positive putative cell bodies appeared to have short neurites. In addition to nNOS-positive neural elements within the plexus, a small number of nNOS-positive elements were observed in the wall adjacent to the caudal tip.

In an attempt to identify potential parasympathetic and sympathetic neural elements associated with the plexus at the caudal tip of the posterior chamber, we used antibodies against ChAT (the enzyme involved in acetylcholine synthesis) and TH (an enzyme in norepinephrine synthesis), as markers for these respective autonomic limbs. There were no ChAT-positive neural elements detected near the specialization and the few THpositive axons present appeared to be associated with regional blood vessels rather than with the plexus (data not shown). Journal of Morphology



Fig. 7. Ultrastructure of innervation on serosal aspect of caudal posterior chamber. a) Enlargement of the nerve profile (labeled Nv) in Fig. 6b. Small nerves (Nv) contain several unmyelinated profiles in varying orientation and size. Bar 5 1mm. b) Enlargement of the region within the box, in panel 7a, shows that this nerve contains axonal profiles (A) and terminals with clear- and densecored vesicles. Bar 5 200 nm. c) Enlargement of region within box in Fig. 6b, showing that neural elements (arrow) are situated between adjacent peripheral cells (P). Bar 5 1mm d) Enlargement of box in panel 7c showing a synapse (arrow) between a nerve terminal and a peripheral cell body (P). A-axon profile Bar 5 500 nm.

Figure 10 shows a summary, schematic diagram of the components of the swim bladder wall. DISCUSSION We have shown that the internal aspect of the caudal tip of the zebrafish swim bladder was lined by an epithelium specialized to secrete surfactant into the lumen as evinced by the exocytosis of lamellar bodies (Fig. 3d). Our results indicated that the innervation to this region consisted of a neural plexus that was formed by the convergence of the terminals of the two lateral nerves onto cell bodies situated at the caudal tip on the external surface of the swim bladder wall (Figs. 8 and 9). The lateral nerves contained NPY-positive and VIP-positive fibers; some cell bodies were nNOS-positive and some were VIP-positive (Figs. 8 and 9). Structural Relationships Epithelium and surfactant. The apical portion of the specialized epithelium in the zebrafish was similar to that of lamellar body-containing cells found in other swim bladders (Copeland, Journal of Morphology

1969; Morris and Albright, 1975; Morris and Albright, 1979) in that there were microvilli, specialized junctions, and extensive lateral interdigitations present (Fig. 2). The cytoplasm contained lamellar bodies, multivesicular bodies, and other organelles possibly involved in surfactant recycling, such as vacuoles (Fig. 3), so that the necessary cellular elements for surfactant processing, and secretion were present. The expressed luminal surfactant in the zebrafish swim bladder was found in the form of whorls that sometimes aligned in regular profiles suggestive of a rudimentary form of tubular myelin organization (Fig. 4). However, it did not conform to the classic geometrical regularity of tubular myelin as seen in other swim bladders such as the snapper (Daniels et al., 2004) so that this refinement of surfactant structure may not be present within the zebrafish swim bladder. Alternatively, our survey simply did not encounter tubular myelin as a conventional fixation protocol, such as that used here, retains only a small portion of the in vivo surfactant layer (Bachofen et al., 2002).


Fig. 8. Fluorescent immunohistochemical labeling demonstrated a nerve plexus at the caudal tip of the posterior chamber, where the lateral nerves converge. Micrographs show the external swim bladder wall with the innervated caudal tip (arrowheads) directed towards the viewer. a) zn-12 immunohistochemistry indicates both lateral line nerves (arrows) converge upon and contribute to the caudal plexus of innervation (arrowhead). Bar 5 20 mm. Insetlabeled axon terminals appear to surround putative cell bodies (arrow) within the plexus. Bar in 8a 5 25 mm for inset. b) NPYpositive axons within both lateral nerve trunks (arrows) form a component of the plexus (arrowhead). Bar 5 20 mm. Inset-NPYpositive fibers are distributed in a lattice pattern and appear to surround putative cell bodies (arrow) Bar in 8b 5 33 mm for inset. c) VIP-positive axons in both lateral nerves (arrows) converge on plexus (arrowhead). Bar 5 20 mm. Insets-labeling for VIP appears in both fibers (left inset, arrow) and cell bodies (right inset, arrow, bar in 8c 5 50 mm for both insets). Note that the insets are subsets of the z-stack shown in panel c and that all three micrographs are in a common orientation.

The basal lamina set a continuous barrier between the luminal epithelial cells and the connective tissue (Fig. 5a). The role of the invaginations, containing the epithelial cell bodies and processes, was not readily apparent but this configuration was encountered at the earliest stage of development examined (day 8 postfertilization; Robertson, unpublished results) and is retained in the adult animal. The invaginations may provide a


microenvironment for the epithelial cells which formed specialized junctions at the luminal surface so that their entire lateral and basal surfaces are partitioned and encompassed by the basal lamina. Some invaginations served as host to more than one cell body or basal process and this close membrane apposition, along with the extensive apical interdigitation, may aid intercellular communication. In addition, the increased surface area provided by the basal extensions may enhance communication between the adjacent epithelial and connective tissue layers as reported in Winata et al., (2009; see also Adamson et al., 1991). Basal processes have been noted in at least one other swim bladder epithelial cell (Australian lungfish, Power et al., 1999) where the authors suggested that the processes anchor the cell body. This likely does not apply to the zebrafish epithelial cell bodies as they did not protrude into the lumen and seem sufficiently supported by their position within the invaginations. Fibroblasts and connective tissue. The epithelium was supported by a connective tissue layer that included fibroblast cell bodies that were large in comparison to the flat fibroblasts of the regular posterior chamber wall (Fig. 5b, see also Fig. 2b). The fibroblasts at the caudal tip extended sinuous processes to abut the basal lamina of the epithelium (Fig. 5). This morphology is similar to that seen in the mammalian lung where fibroblast processes directly contact the surfactant-producing AT II cell basal membrane through apertures in the basal lamina (Vaccaro and Brody, 1981; Sirianni et al., 2003; see also Grant et al., 1983). Lung fibroblasts and the extracellular matrix are instrumental in the reciprocal epithelial:fibroblast interactions that induce cell proliferation, differentiation, maturation, and surfactant synthesis necessary for development and function (Grant et al., 1983, Rannels and Rannels, 1989, Smith and Post, 1989, Adamson et al., 1991, Caniggia et al., 1991, Griffin et al., 1993). In contrast, the swim bladder fibroblasts extended processes to, but not through, the basal lamina (Fig. 5d). These structures do provide an obvious physical pathway for interactions between the two tissues; however, reciprocal communication between these different cell types must be diffusible through the basal lamina in the adult zebrafish. These same cells also demonstrated extensive lateral cell membrane apposition between adjacent fibroblasts (Fig. 5e–g). There was, occasionally, an adherent junction between these processes but the predominant type of contact was composed of lengthy appositions of cell membrane (see Sirianni et al., 2006). Fibroblasts can form a functional cellular network (Furuya et al., 2005) that reacts to mechanical force, transmitted between contiguous cells by adherent junctions, activating stretchsensitive channels (Ko et al., 2001). If this Journal of Morphology



Fig. 9. Patterns of immunostaining by neuronal NOS and zn-12 within the caudal neural plexus. a) Immunolabeling for nNOS stains a cluster of putative cell bodies within the plexus. b) The fiber pattern shown is that of axonal labeling with zn-12 immunohistochemistry. c) Combined nNOS and zn-12 labeling demonstrates that putative nNOS-positive cells are surrounded by zn-12-positive axons. Scale bar in panel a represents 10 lm for all panels.

Fig. 10. Schematic diagram of a section through the specialization at the caudal tip of the posterior chamber wall of the zebrafish swim bladder. Surfactant profiles (s) are found within the swim bladder lumen (L). They are exocytosed from the lamellar bodies (lb) present in the epithelial cells (E). These cells are surrounded by a prominent basal lamina (BL) that encompasses the basal processes (BP) of the epithelial cells. The thin cell processes of fibroblasts (F) extend to contact the basal lamina and each other. A layer of smooth muscle cells (SM) is situated at the outer aspect of the fibroblast connective tissue layer. Peripheral cell bodies (P), containing distinctive vesicles (V) receive synapses from axons (a) originating in nerves (Nv) present near the outer margin of the swim bladder wall. Blood vessels (BV), which contain red blood cells (RBC) are present only on the external facet of the specialization. Diagram is not to scale. The physical separation between the epithelium and the secretory elements (nerve endings, synapses and, possibly, peripheral cells) suggests a paracrine mode of communication.

Journal of Morphology


mechanism is tenable in the zebrafish swim bladder, then positioning such cells beneath and directly adjacent to the basal lamina of the surfactant-producing epithelium may facilitate an increase in the surfactant release, possibly triggered by a mechanical force stretching the swim bladder wall upon inflation. Smooth muscle cells. The smooth muscle cells were quite variable in position as they were sometimes present as a distinctive layer set peripherally in relation to the fibroblasts, they sometimes intermingled with the fibroblasts and yet were completely absent in other areas (Fig. 6b). These fibers are likely the radiating fibers (Rfibers) described in Finney et al. (2006). Proposed relationship of innervation to specialized epithelium. An important part of this work is a description of the structure, distribution, and transmitter content of the elements (Figs. 7 and 8) that compose the neural plexus. Here, we suggest that the nervous system is related to the specialized epithelium by proximity, coincidence, and exclusion. Our supposition, regarding a relationship between the neural plexus, peripheral cells, and lamellar body-containing cells, is that a terminal extension of the nervous system is organized to affect the surfactant properties of a physically isolated, yet neurally integrated, patch of specialized epithelium. An assessment of the validity of this proposed relationship will await the pertinent physiological and pharmacological data. Signal transmission. Extrinsic innervation. The terminal extensions of the two lateral nerves, some containing VIP-positive fibers and NPYpositive fibers, formed interdigitations at the caudal tip. In our observations, these nerves comprised the only extrinsic innervation to this area and so would convey signals from a distance. As the position of cell bodies extending these fibers is not known, we cannot identify the origin of these signals. Intrinsic innervation. There are cell bodies, which contain nNOS (Fig. 9a) and VIP (Fig. 8c, right inset), situated within the distribution extent of the extrinsic fiber interdigitations at the caudal tip and some of these cell bodies appear to be contacted by fibers of the lateral nerves (Figs. 8c and 9c). This evidence suggests a mechanism of neural connection. Nerve signals may evoke a release of NPY or VIP from a distance or act through connections to cause a local release of NO and VIP from the cell bodies present at the tip. This organization permits the release of the neurotransmitters VIP, NPY, and NO, either separately or in combination. Further, the physical separation between the neural elements (extrinsic axons, nNOS, and VIP cell bodies) and the presumptive epithelial target tissue suggests a paracrine mode of secretion, a documented mode of signal transmission in the respiratory system for NO (Rengasamy et al.,


1994) and in the digestive system for VIP and NPY (Chandrasekharan et al., 2013). As the recipient of synapses and possessor of vesicles, it would seem that the peripheral cell is ideally suited and situated to transduce external neural activity into a local chemical signal. However, we do not have evidence that the peripheral cells contain VIP or nNOS. Neurochemical properties and distribution in relation to the specialized epithelium. VIP. Application of exogenous VIP relaxed the smooth muscle from the swim bladders of the cod and the eel (Lundin, 1991) and caused a slight decrease in the tension of the cod swim bladder artery (Lundin and Holmgren, 1984). In the present instance, VIP may act to relax the nearby smooth muscle cells of the radial muscle at the posterior portion of the zebrafish swim bladder; however, it may also affect surfactant production. VIP stimulates the production of surfactant by ATII cells from rat lung explants, through a suspected autocrine or paracrine methods (Li et al., 2004). VIP-type VPAC1 receptors are present in the zebrafish swim bladder; however, their distribution is unknown (Fradinger et al., 2005). Interestingly, human VIP can directly affect surfactant properties by stabilizing lipid layers while the lipids, in turn, convert the VIP molecules to their most biologically active form (Onyuksel et al., 2000). Indeed, just such a mechanism may be used during the zebrafish larval stage where VIP, present in a noncellular distribution, was positioned at the junction of the esophagus and pneumatic duct during a critical period of swim bladder inflation (Robertson et al., 2007). nNOS. There is evidence that NO may cause a vasodilation of swim bladder vasculature in the eel (Schwerte et al., 1999). nNOS and NO distribution is quite varied amongst swim bladders as nNOS is contained within nerve fibers (goldfish, Bruning et al., 1996) and epithelial sheets (catfish, Zaccone et al., 2006) and the gas NO is present within the swim bladder of the cod (Midtvedt et al., 2007). Haddad et al. (1996) found that NO inhibits surfactant synthesis by rat ATII cells; however, Lee et al. (2005) cautioned that the effects of NO on ATII cells seemed model dependent. Here, we simply note the position of the nNOS containing cells in relation to the surfactant secreting cells. NPY. The NPY molecule too has surfactant properties (Minakata et al., 1989; Dyck et al., 2006); however, any possible role with respect to surfactant secreting cells is not immediately obvious in the present context. It is noted that NPY-positive fibers innervate the anteroventral portion of the goldfish swim bladder (Pickavance et al., 1992). TH and ChAT. The notable lack of TH-positive nerves near the structure suggests that the major Journal of Morphology



neural impetus to mammalian ATII cells may not be a primary factor in the zebrafish as this enzyme is essential for the formation of adrenergic neurochemicals. Adrenergic agents may also be delivered through the bloodstream but this represents a general whole body response rather than a specific refined signal targeted solely to the specialized epithelial cells of the swim bladder as the neural plexus/paracrine system suggested here. Similarly, we confirm that there is essentially no cholinergic fiber presence as assayed by the ChAT enzyme. It is our observation that neuronal fibers containing the two traditional autonomic neurotransmitter phenotypes are not physically engaged with the specialization. In principle, then, the zebrafish may differ from the lizard (Wood et al., 1995), bullfrog, fat-tailed dunnart, and lungfish (Wood et al., 2000) for their various surfactant systems respond to one or both of these classic autonomic neurotransmitters. Comparison of the zebrafish swim bladder with those of other fishes. Ostensibly, the swim bladder of the zebrafish is most gainfully compared to that of the goldfish as the gross morphology of this organ in these cyprinids is very similar. They are both double-chambered, constricted at the ductus communicans, with a patent pneumatic duct joining the esophagus to the posterior chamber (Morris and Albright, 1979). However, a recent description of the nerves of the goldfish swim bladder, as assayed by immunochemistry (TH, ChAT, VIP, nNOS, and serotonin), did not report a plexus at the tip of the posterior chamber and concluded that the innervation patterns of the goldfish and zebrafish “do not overlap” (Zaccone et al., 2012; see also Nilsson, 2009). An obvious difference between these two species, that may affect innervation patterns, is that the anterior chamber of the goldfish swim bladder contains a discrete gas gland, a complex of specialized cells that extract and transfer gas from the circulating blood into the swim bladder to aid buoyancy by inflation (Zaccone et al., 2012). Further, to facilitate the transfer of gas, the gland is extensively vascularized by a network of blood vessels which ensures that each gas gland cell is in close proximity to a capillary (Copeland, 1969; Morris and Albright, 1975). The innervation of teleost gas glands and their associated blood supply is rich and complex (Nilsson, 2009) and this fundamental difference may help account for the variation in nerve supply between the two species. In contrast, the zebrafish does not possess an integrated gas gland and, significantly, there was no direct contact observed between the vasculature and the specialized lamellar body cells at the posterior tip as the few blood vessels present ran solely upon the external portion of the swim bladder wall (Fig. 6). Consequently, we do not consider the lamellar body-epithelial cells to be associated Journal of Morphology

with gas deposition. This is an important distinction as fishes such the eel, perch (Prem et al., 2000), and toadfish (Morris and Albright, 1975) do contain lamellar bodies within the cells of the gas gland. Thus, in these fish, gas transfer into the swim bladder may be concomitant with surfactant production, as the level of gas inflation or deflation will determine the volume of the swim bladder and hence the amount of surfactant required for function. In this case, it would be difficult to differentiate the impulse for increased gas production from that for increased surfactant production, if indeed, such a difference exists. It seems that the simple zebrafish swim bladder has allowed a serendipitous glimpse into a possible mechanism of surfactant control without the complications of an association with a gas gland. It is just this anatomical simplicity that makes this physical relationship amenable to the physiological and pharmacological experiments that may establish a direct connection between the innervation and the luminal expression of surfactant. A perspective. We have made comparisons between the mammalian type ATII cells and the zebrafish specialized epithelia as much is known about ATII cells and the biological factors that elicit responses from them (Andreeva et al., 2007). Although there is evidence of some molecular homology between them (Zheng et al., 2011; Cass et al., 2013), the mammalian lung differs from the zebrafish swim bladder in many ways. The lung serves as a respiratory organ that transfers gas by regular volume changes whereas the zebrafish swim bladder retains gas in homeostasis with fish depth to remain buoyant. This fundamental difference, alone, will influence any comparisons made. ACKNOWLEDGMENTS The authors thank Chantelle McGee for her excellent technical assistance. This article is dedicated to the late Dr. Margaret (Peggy) Hansell, a dedicated teacher of histology. LITERATURE CITED Adamson IYR, Young L, King GM. 1991. Reciprocal epithelial: Fibroblast interactions in the control of fetal and adult rat lung cells in culture. Exp Lung Res 17:821–835. Andreeva AV, Kutuzov MA, Voyno-Yasenetskaya TA. 2007. Regulation of surfactant secretion in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 293:L259–L271. Bachofen H, Gerber U, Schurch S. 2002. Effects of fixative on function of pulmonary surfactant. J Appl Physiol 93:911–916. Brooks RE. 1970. Ultrastructure of the physostomatous swimbladder of rainbow trout (Salmo gairdneri). Z Zellforsch Mikrosk Anat 106:473–483. Brown LAS, Longmore WJ. 1981. Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture. J Biol Chem 256:66–72.

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The structure of the caudal wall of the zebrafish (Danio rerio) swim bladder: evidence of localized lamellar body secretion and a proximate neural plexus.

In this study, we present a morphological description of the fine structure of the tissues composing the caudal tip of the adult zebrafish swim bladde...
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