Table 1.
Lectins used.
Fig 1.
AO gel label by lectins WGA or SBA; profile plots.
(A1, B1) Frozen 10 μ cross sections of Polyodon AOs were labeled with biotinylated lectins and streptavidin-DyLight488 fluorescent reporter (Vector). Single raw widefield images, 10x 0.3 NA lens, RGB camera. (A1) WGA-biotin (WGA-b), 5 μg/mL. (B1) SBA-biotin, 5 μg/mL, preabsorbed with chitin hydrolysate solution. -b: Biotinylated lectin. BL: Basal Lamina. D: Dermis between AOs. EN: Electrosensory neuroepithelium. F: Flattened smooth ampulla wall epithelium, deep in AOs, distal of the EN. H: Hypodermis deep to AOs. Red arrowheads: Limits of an EN. SE: Striated ectoderm; a double arrow marks its thickness. Yellow lines: Paths of profile plots, identified by their panel codes. (A2) Profile plot of pixel gray values (8-bit scale) along the left AO in A1, and continuing outside its pore (red *). Apical EN: Maximal emission near the apical face of an EN. (A1, A2) Orange ■: Gap between gel and EN; see Discussion. (A3) Profile plot from left to right across a gel-filled partial AO in A1; maximal fluorescence was from the apical face of non-EN epithelia of the ampulla walls. (B2, B3) Profile plots from B1. SBA+ gel was disturbed in the neck and pore of these AOs.
Fig 2.
Controls: Validation of WGA, SBA selectivity.
Lectin-biotin (-b) conjugates, 5 μg/mL final concentration, and streptavidin-DyLight488 reporter were applied. See Results text for preabsorption protocols. All panels show single widefield images of cross sections of AOs; RGB camera, 10x 0.3 NA lens. Similar image processing was applied to all panels, including deblurring and increased contrast. B: Barb-like protrusions from transitional epithelial cells of AO interior wall. EN: Electrosensory neuroepithelium. F: Flattened epithelial cells of the deep interior ampulla wall in AOs. P: Papillas lining the neck and pore. (A) WGA-biotin labeled gel in the lumen of AOs, the EN, the interior epithelium of the ampulla wall, neck, and pore, and in the skin’s striated ectoderm. (B) Background fluorescence was low when WGA was omitted, but reporter was applied. EN: Autofluorescence from EN receptor cells (see S1A Fig). (C) Nil label when WGA-biotin was preabsorbed (preabs.) with chitin hydrolysate (Chitin Hy.). (D) AO label like A when WGA-biotin was preabsorbed with N-acetyl-D-galactosamine (GalNAc). (E) SBA-biotin labeled AOs and luminal gel. (F) Label by SBA-biotin was abolished, as expected, when it was preabsorbed with N-acetyl-D-galactosamine, its preferred ligand. Also, Fig 1B1 shows labeling of AOs, similar to E, by SBA-biotin preabsorbed with chitin hydrolysate.
Fig 3.
Apical surface views of WGA-labeled EN.
Parallel (en face) views of the apical surface of partial EN, in briefly fixed AOs. (A1) WGA-CF488A conjugate (Biotium) labeled the apical faces of support cells (S). R: Receptor cell. zo: Zona occludens. Red arrow: Support cell with a small-area apical face. Confocal single image, deconvolved, 63x 1.4 NA lens. The ‘black’ end of this image’s gray scale was unmodified. (A2, A3) Profile plot across part of A1. Fluorescence emissions reached zero at a receptor cell’s apical face (R) or a zona occludens (zo), indicating non-label by WGA. (B–D) Widefield stack images. (B) WGA-CF488A label; 60x lens, monochrome camera. Yellow arrow: Region of a support cell’s cilium. A similar zone is marked in A1, yellow arrow. (C) Phalloidin (orange) labeled the zona occludens (zo) joining EN support (S) and receptor (R) cells apically. One kinocilium (k) per receptor cell was labeled by anti-acetylated-α-tubulin (green), which also labeled short cilia (c) on support cells. Monochrome camera, 100x lens, 0.5 μ stack steps. (D) Triple label with WGA-CF488A (blue), phalloidin (red), and anti-acetylated-α-tubulin (green). Monochrome camera, 60x lens, 0.4 μ stack steps.
Fig 4.
WGA / SBA label in electrosensory neuroepithelia.
Transverse cross sections (10 μ) of EN were labeled with lectins WGA (A, B) or SBA (C–E). Tissue fixation or lectin conjugation were varied. Images show flattened projections from widefield partial stacks, with an EN’s apical face ‘up’. The nucleus (n) or cytoplasm (c) of support (s) or receptor (r) cells are indicated. Nuclei of support cells are more apical than nuclei of receptor cells [15]. BL: Basal lamina. L: Lumen of AO. p: Pedicel of support cell [14]. v: Void containing afferent branches [16]. (A, C, D) ‘Hard’ fixation for 24 h preserved the apical microvilli (μv) on EN support cells, labeled using lectin-fluor conjugates; monochrome camera. (B, E) Brief (45 min) fixation, and label with lectin-biotin (-b) conjugates using streptavidin-DyLight488 reporter; RGB camera. (A1–A5) Triple label with WGA-CF488A (green, Biotium, 2 μg/mL), and phalloidin-CF594 (Phal, red, Biotium), and DAPI (blue). (A1–3) WGA label. (A2) Enlarged part of A1, resampled 4x. A support cell (S) was also marked on other panels of A. Arrows: Apical face of receptor cell (R). (A3) WGA label with DAPI+ nuclei. Inset: Local patch of WGA+ microvilli in a single image from another section, resampled 4x. (A4) Phalloidin (Phal) labeled apical microvilli (μv) on support cells. (A5) All 3 labels were superimposed. Yellow color of apical microvilli on support cells indicated co-label by WGA and phalloidin. (B) WGA-biotin (WGA-b) label of EN, 1 μg/mL; stack of 6 images. (C, D) SBA-fluorescein label (Vector, 65 μg/mL). (C) This ampullary organ, its gel, and skin were labeled by SBA, which labeled the EN more brightly than in ampulla wall (W), and labeled the apical (Ap) surface of all AO epithelia. SE: Striated ectoderm. (D) SBA label of EN. Inset: Expanded view of SBA+ apical microvilli on support cells. (E) SBA-biotin label of EN, 5 μg/mL; stack of 3 images. (A, D) 60x lens, 0.5 μ stack steps. (B, E) 100x lens, 1 μ stack steps. (C) 10x 0.45 NA lens.
Fig 5.
Lectins PNA, RCA120, or ConA labeled EN support cells.
(A–E) Lectin-fluor conjugates were applied to 10 μ transverse sections of EN in hard-fixed skin from the rostrum; see keys. Monochrome camera, 60x lens, 0.5 μ stack steps. (A) Lectin PNA-CF488A (Biotium, 48 μg/mL) labeled apical microvilli (μv) on support cells. BL: Expected site of basal lamina. L: Lumen of AO. R: Receptor cell nucleus. S: Support cell nucleus. (B, C) Lectin ConA-CF488A (Biotium, 48 μg/mL) labeled small vesicles throughout support cells, in hard-fixed (B) or briefly fixed (C) EN. a: Apical face of support cell. Arrow: Apical face of receptor cell. Nuclei (n) or cytoplasm (c) of support (s) or receptor (r) cells are labeled. p: Basal pedicel of support cell. (C) This EN was thicker than usual. (D–F) Lectin RCA120-fluorescein (Vector, 10 μg/mL, without added CaCl2 or ZnCl2), labeled Golgi tubules (G) in the apical half of support cells, and faintly labeled apical microvilli (μv). f: Fibrous RCA+ sub-EN layer. (F) En face view of EN, showing DAPI+ nuclei at the center of nests of RCA+ tubules (white).
Fig 6.
Extracellular sockets around receptor cells.
(A, B) En face stack images of EN receptor cell arrays. Some receptor cells were dislodged by the cryostat blade during sectioning, leaving green-fluorescent ‘sockets’. * or ▲: Corresponding sites in A and B; see Results text. RGB camera, 60x lens. (A) Green: Anti-neurofilament-H (NEFH) labeled afferent (aff.) terminals. S: Void corresponding to a support cell. (B) Superimposed anti-parvalbumin-α (PVALBα, orange) label of remaining receptor cells.
Fig 7.
Cross sections of ampulla wall.
b: Biotinylated lectin. B: Barbed wall cells. c: Capillaries near AO. D: Dermis. EN: Electrosensory neuroepithelium. F: Flattened cells of deep ampulla wall. L: Lumen of AO. m: Melanin-containing outer fibrous layer on AOs. P: Papilla cells lining neck and pore. Phal: Phalloidin label. SE: Striated ectoderm. W: Non-EN ampulla wall epithelium. (A) Brightfield cross-section of AO; unstained, 10x phase lens. Partial luminal gel remained at the EN. (B) Cross section of an AO and adjacent skin stained with phalloidin (orange) and WGA-biotin (green). *: AO gel with small particles was visible outside the pore. (C, D) Parallel views of the interior wall of AOs labeled with anti-S100-ß (C) or WGA-CF488A (D), showing different cells types of the ampulla wall epithelium, including flattened cells (F) deep in AOs, transitional cells with barb-like protrusions (B), and papilla cells (P) in the neck and pore. (E–H) Closeup images of cross sections of ampulla wall showing vesicles in wall epithelial cells labeled by a lectin; see keys. Double arrows: Thickness of wall epithelium. (E) WGA-b, 1 μg/mL. (F) ConA-b, 5 μg/mL. (G) RCA-b, 2.5 μg/mL. (H) SBA-b, 5 μg/mL. Arrow: Apical vesicle labeled by SBA-biotin. *: Barb-like SBA+ protrusion from wall cell. (E, H) 100x lens. (F, G) 40x lens. (I) SBA-biotin label of horizontally sectioned ampulla walls; stack projection of a 50 μ section parallel to skin. Blue arrow: En face view of SBA-b+ apical surface labeling on epithelial cells, in a tilted part of the ampulla wall, enlarged in inset 1. Green arrow: SBA-b+ apical label of cross-sectioned epithelial cells of ampulla wall. Red arrow: Sectioned SBA-b+ barb-like protrusions from wall cells, enlarged in inset 2 (resampled 4x).
Fig 8.
Ampulla wall cells: Junction immunolabel and lectin surface label.
(A–I) Parallel views of the apical faces of ampulla wall epithelial cells. See keys and Methods for the probes applied; 40x lens. aff: Afferent branches to EN, labeled by anti-acetylated-α-tubulin (aTub). Arrows: Small circular whorls. b: Border between EN and ampulla wall. Col-1: Anti-collagen-1. EN: Electrosensory neuroepithelium. k: Kinocilium labeled by aTub, on each receptor cell’s apical face. Phal: Phalloidin. r: Receptor cell apical face. TJP2: Anti-tight junction protein 2. WGA: WGA-CF488A (Biotium) conjugate. ZO1: Anti-tight junction protein 1. (B, D) DAPI stain of wall cell nuclei. (D) Lectin DBA-biotin bound to the apical surfaces of wall cells but not zona occludens between cells. (E) Section of an ampulla’s wall; its luminal edge showed barb-like protrusions (*) from wall epithelial cells. (H) Anti-tricellulin (MARVELD2) preferentially labeled vertices (arrowhead), where borders of wall cells intersected. (I) Anti-β-catenin labeled series of small punctate sites along cell borders. (G–I) Strands of dark melanin (m) overlay the wall epithelium.
Fig 9.
Comparison of ampulla wall vs. EN label by WGA.
(A, B1) Cross sections showing the transition (*) from EN to ampulla wall. (A) Triple-label as in Fig 4A5; see keys. The ampulla wall appeared green whereas the EN appeared yellow; see Results text. (B1) Green emissions from WGA-CF488A (Biotium) label; brightened deblurred image, monochrome camera, 40x lens. BL: Basal lamina. L: Lumen of AO. (B2, B3) Transverse profile plots along the blue lines in B1, crossing the wall epithelium (B2) or a support cell of the EN (B3). These plots were scaled identically, and were from the original raw image of B1 converted to 8-bit grayscale. Area under the curve was summed between the pink drop lines; see Results text. a: Apical face of epithelium. (B4) Lengthwise profile plot along the middle of the wall epithelium and EN (yellow line, B1).
Fig 10.
Lectin-labeled papilla cells lined the AO neck.
(A) Cross section of skin showing WGA-CF488A label in the neck and pore of AOs, including a deep AO (left) and an adjacent small AO; 20x 0.5 NA lens. P: Papilla cells covered a ~250 μ-deep band in the neck and pore. *: Transition from papilla cells to flattened ampulla wall cells (W). SE: Striated ectoderm. Small AO: En face view of the interior apical surface of its electrosensory neuroepithelium (EN) and ampulla wall, which included papilla cells (P). Arrow 1: 236 μ depth of the papilla cell band in the deep AO (left side). Arrow 2: 223 μ maximal wall depth of the small AO. Arrow 3: 286 μ total depth of the small AO. Pink construction lines show dimensions of the small AO’s tapered ampulla wall, including its height (142 μ), maximum internal width (218 μ), and the neck’s ID (51 μ), for a model of gel acceleration (Tab 1 in S2 Table). (B–D) Biotinylated (-b) lectins and streptavidin-DyLight488 reporter were used. (B) Stack image parallel to the neck interior of an AO, showing WGA-b+ papillas (P) in the neck (N), and that large WGA-b+ papillas, almost parallel to the wall, continued into the distal ampulla; 10x lens. L: Lumen of AO. (C) Lectin DBA-b (preabsorbed with chitin hydrolysate solution) labeled the interior epithelium of this AO, including papilla cells (P) in the neck (N). DBA-b+ gel was visible in the AO’s lumen; 10x lens. (D) Cross section of short papilla cells (P) labeled by SBA-b in the neck / pore of an AO; single image, 100x lens.
Fig 11.
Lectin labeling of axon sheaths.
(A1) Lectin PNA-biotin (PNA-b) labeled a thin sheath (arrow) around large myelinated ALLn afferent axons, surrounding internodes (IN) and continuing across nodes (N); 40x lens. (A2) Superimposed triple label of same axons. Myelin (red) was labeled by anti-myelin basic protein (MBP), and electroreceptor afferent axons (blue) by anti-neurofilament-H (NEFH). (B) Lectin RCA120-biotin (green) labeled perineurium tissue of a small nerve. Orange: Individual axons labeled by anti-neurofilament-H (NEFH).
Fig 12.
Models of gel secretion in ampullary electroreceptors.
(A) Ampulla of Lorenzini: Blue arrows indicates gel secretion from canal wall cells, along the length of a canal. Cap: The ampulla’s centrum cap, near the start of a canal, also secretes gel [2, 13]. (B1–4) Model of a small AO of Polyodon, based on Fig 10A. (B1) Arrows indicate gel secretion mainly from support cells of the wide EN covering the interior basal pole of each AO. Gel accelerates going toward the pore, due to AO taper. Relative gel velocity (blue curve) was calculated as inversely proportional to the circular cross-sectional area of the ampulla lumen (Tab 1 in S2 Table). (B2) Traced outline of the small AO in Fig 10A. (B3) Simplified scale model of the ampulla wall (pink lines) as a symmetrical funnel-shaped taper, based on the pink dimensional lines in Fig 10A. (B4) To-scale areas of the neck and the maximal ampulla wall width, assumed circular.