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Front Zool
2016 Jan 01;13:27. doi: 10.1186/s12983-016-0159-8.
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Immunohistochemical and ultrastructural properties of the larval ciliary band-associated strand in the sea urchin Hemicentrotus pulcherrimus.
Katow H
,
Katow T
,
Yoshida H
,
Kiyomoto M
,
Uemura I
.
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BACKGROUND: The swimming activity of sea urchin larvae is dependent on the ciliary band (CB) on the larval surface and is regulated by several neurotransmitters, including serotonin (5HT), dopamine, and γ-aminobutyric acid (GABA). However, the CB signal transmission mechanism remains unknown. The present study investigated the structural relationship between the CB and external signal receptors by immunohistochemical and transmission electron microscopic analyses of sea urchin, Hemicentrotus pulcherrimus, larvae.
RESULTS: Glutamate decarboxylase (GAD; GABA synthetase) was detected in a strand of multiple cells along the circumoral CB in 6-arm plutei. The GAD-expressing strand was closely associated with the CB on the oral ectoderm side. The ciliary band-associated strand (CBAS) also expressed the 5HT receptor (5HThpr) and encephalopsin (ECPN) throughout the cytoplasm and comprised 1- to 2-μm diameter axon-like long stretched regions and sporadic 6- to 7-μm diameter bulbous nucleated regions (perikarya) that protruded into the oral ectoderm side. Besides the laterally polarized morphology of the CBAS cells, Epith-2, which is the epithelial lateral cell surface-specific protein of the sea urchin embryo and larva, was expressed exclusively by perikarya but not by the axon-like regions. The CBAS exposed its narrow apical surface on the larval epithelium between the CB and squamous cells and formed adherens junctions (AJs) on the apical side between them. Despite the presence of the CBAS axon-like regions, tubulins, such as α-, β-, and acetylated α-tubulins, were not detected. However, the neuroendocrine cell marker protein synaptophysin was detected in the axon-like regions and in bouton-like protrusions that contained numerous small ultrastructural vesicles.
CONCLUSIONS: The unique morphology of the CBAS in the sea urchin larva epithelium had not been reported. The CBAS expresses a remarkable number of receptors to environmental stimuli and proteins that are probably involved in signal transmission to the CB. The properties of the CBAS explain previous reports that larval swimming is triggered by environmental stimuli and suggest crosstalk among receptors and potential plural sensory functions of the CBAS.
Fig. 1. The multicellular strand of the larval arms co-expresses glutamate decarboxylase (GAD) and the 5HT receptor (5HThpr). a The oral ectoderm and the aboral ectoderm are bordered by a multicellular strand that expresses GAD (green) and 5HThpr (red) in the 6-arm pluteus. ala, anterolateral arms; arrow, tip of arm; l-pda, left posterodorsal arm; l-, r-poa, left- and right-postoral arms; Mo, mouth. b Fifteen μm thick optical section of the part of the arm indicated by the dotted box in (a) shows GAD and nuclei. Apparent smeared green on the larval surface reflects blastocoelar GAD cells. c Same area as (b) shows 5HThpr and nuclei. d Merged image between (b) and (c) shows GAD and 5HThpr expression in the same place as (b, c). Arrows, perikarya regions
Fig. 2. The ciliary band-associated strand (CBAS) resides on the oral ectoderm side of the ciliary band. (a) Schematic aboral view of 6-arm pluteus of (B). Gray area, oral ectoderm; l- and r-ala, left- and right-anterolateral arms; l- and r-pda, left- and right-posterodorsal arms; l- and r-poa, left and right-postoral arms. (b) Confocal laser scanning microscopic image of triple-stained whole-mount immunohistochemistry of the r-pda indicated by box (b) in (A) shows the 5HT receptor (5HThpr)-expressing CBAS (red) on the oral ectoderm side (Oral) of the dopamine receptor (DRD1)-expressing ciliary band (CB; green dots). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue; DAPI). (c) High-magnification image of the CB of the r-pda indicated in (B) shows characteristic multiple lines of DRD1-positive basal bodies of the cilia. Perikarya (arrows) protrude into the oral ectoderm side (Oral). Aboral, aboral ectoderm area
Fig. 3. Immunohistochemical cell surface property of the ciliary band-associated strand (CBAS). (a) Schematic of arm tip region indicated by box (Fig. 3) in Fig. 2a. Red line, CBAS with perikarya (red dots). Vertical bar (b–l), approximate region of image (b–l). (b) Glutamate decarboxylase (GAD) expression in the CBAS. (c) Epith-2 expression in the perikaryon (green arrow). (d) Merged image between (b) and (c). (e) Hybrid Super Resolution software (HSR)-processed image of (d) shows locations of the optical cross-sections (dotted lines). (f–j) Optical cross-sections indicated by vertical dotted lines (f–j) in (e). (k) HSR-processed 3D reconstructed image of the CBAS. (l) The other side of image (k) after rotation mostly covered by the Epith-2 signal. (m) Schematic of cross-section of arm indicated by bar (m) in (a). Bcl, blastocoel. Nuclei in (d–l) were stained with 4′,6-diamidino-2-phenylindole (blue). Aboral, aboral ectoderm regiuon; CB, ciliary band area; green arrow, perikaryon; Oral, oral ectoderm region; red arrow, axon-like region; white arrow, apical protrusion
Fig. 4. Transmission electron micrograph of the ciliary band (CB) of 6-arm pluteus. (a) Columnar CB cells (blue) adjacent to squamous cells on the aboral side (Aboral) and the rectangular cell (red) on the oral side (Oral). Inset, toluidine blue-stained thick cross-section of the arm shown by (a–e). (b) High-magnification of the region indicated by box (b) in (a). The rectangular cell possesses adherens junctions (AJ) between a CB cell (arrow 1) and a squamous cell (arrow 2). (c) High-magnification of the region indicated by box (c) in (a). Squamous cell adjacent to the CB cell (arrow). (d) Cross-section of the rectangular cell (red) adjacent to the CB cell (blue). (e) Cross-section of the rectangular cell (red) adjacent to the CB cell (blue) 40 μm away from the image in (d). (f) Cross-section of the perikaryon of the rectangular cell (red) adjacent to CB cells (blue). (g) High-magnification of the apical cytoplasm of the CB cells indicated by box (g) in (a). Arrows, basal bodies; double-arrow, AJ. (h) High-magnification of an AJ (arrow) between the rectangular cell and adjacent squamous cell shown by box (h) in (d). (i) High-magnification of an AJ (arrow) between the rectangular cell and the CB cell indicated by box (i) in (e). Aboral, the aboral ectoderm region; green arrows in (b, d-f), electron-translucent vesicles; N, nucleus in (a), (d–f); Oral, the oral ectoderm region, red arrows in (b, d, e), electron-dense vesicles
Fig. 5. Three-dimensional (3D) tomographic reconstruction of the ciliary band (CB)-associated strand (CBAS) of the postoral arm. (a) Toluidine blue-stained thick cross-section of a larval arm of a 6-arm pluteus. Transmission electron microscopy (TEM, B–E) was applied to the region indicated by box. The larval arm was flattened during sample preparation. (b) TEM image of the first proximal section (0 μm) at the area indicated by box in (a). (c) TEM image of the last distal section 32 μm from the first section (B). (d) Stacked image of nine semi-serial cross-sections (0–32) by the Amira image processor. The CBAS (red) constitutes the initial bulbous proximal sections (0–8) to the long and thin stretch of strand towards the proximal sections (12–32). Squamous epithelium (SqE; yellow) encapsulates the major surface of the larval arm except the apical surface of the CB and the narrow strip of CBAS (red). (e) Smoothened 3D topographic reconstruction of (d). (b) and (c), proximal end indicated by (B) and distal end indicated by (c). Aboral, aboral ectoderm region; arrows, the apical protrusions; Blc, blastocoel; Oral, oral ectoderm region
Fig. 6. Transmission electron micrograph of the apical protrusion of the ciliary band-associated strand (CBAS, red). (a) The apical protrusion from the CBAS perikaryon was approximately 0.4 μm in diameter (short arrow). Its tip was obliquely decapitated approximately 1.8 μm from the base in this particular thin section. N, nucleus. (b) High-magnification of box (b) in (A). (c) High-magnification of box (c) in (b). Aboral, aboral ectoderm region; CB, the ciliary band (CB) cells; long arrows, basal bodies in the CB cells [blue in (a)]; Oral, oral ectoderm region; white arrow, small vesicles
Fig. 7. Immunochemical detection of encephalopsin (ECPN) in the ciliary band-associated strand (CBAS). (a) Immunoblotting of the 4-arm plutei lysate. Lane 1, rabbit anti-Hp-ECPN pAb; Lane 2, rabbit pre-immune serum. Arrow, 76 kDa region. (b–) Confocal laser scanning micrographs of whole-mount immunohistochemistry of 4-arm pluteus. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). (b) Triple-stained aboral view of 4-arm pluteus. (c–e) High-magnification image of a postoral arm indicated by box (c–e) in (B). (c) ECPN (green) at the CBAS. (d) 5HThpr (red) at the CBAS. (e) Merged image between (c) and (d). (f) Arm of a 6-arm pluteus triple-stained with rabbit pre-immune serum (green, negative), mouse anti-5HThpr (red), and DAPI. Arrows in (c–f), perikarya of the CBAS; Bcl, blastocoel
Fig. 8. Immunochemical detection of synaptophysin (Syn) in the ciliary band-associated strand (CBAS). (a) Immunoblotting of the 6-arm plutei lysate was probed with rabbit anti-sea urchin-Syn pAb (lane 1) and detected a single band in the 30-kDa region (arrow). Lane 2: Rabbit pre-immune serum. (b–e) Confocal laser scanning micrographs of whole-mount immunohistochemistry of a 6-arm pluteus. (b) Left side view of the pluteus. The CBAS was positive for Syn (green) and the 5HT receptor (5HThpr; red). (c–e) High-magnification of the CBAS indicated by box (c–e) in (B). (c) Syn expression. (d) 5HThpr expression. (e) Merged image between (c) and (d) with nuclei stained with 4′,6-diamidino-2-phenylindole (blue). Green arrows; apical protrusions from the CBAS; white arrows, perikarya; yellow arrow, granular expression of Syn in the axon-like region of the CBAS
Fig. 9. Immunohistochemistry of cytoplasmic tubulins in the ciliary band (CB)-associated strand (CBAS). a Immunoblotting of the 6-arm plutei lysate with mouse anti-β-tubulin monoclonal antibody (mAb) (lane 1), mouse anti-acetylated α-tubulin mAb (lane 2), mouse anti-α-tubulin mAb (lane 3), and mouse pre-immune serum (lane 4). Arrow indicates the 63-kDa region. b–h Confocal laser scanning micrographs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). b One-μm thick single optical longitudinal cross-section of the triple-stained CBAS. Double-arrow, cilia; red arrow, the CBAS positive to anti-GAD pAb; white arrow, cytoplasmic β-tubulin (green). c–e and g; apical surface views of the larval arms. c Twelve-μm thick stacked image of the CBAS (red arrow). d, e Color channel arranged images of (c). d Twelve-μm thick stack image of the CBAS stained with anti-GAD pAb (red arrow). e Twelve-μm thick stack image stained with anti-β-tubulin mAb (green). f Triple-stained 21-μm thick longitudinal stack image of optical cross sections of larval arm. The CBAS was stained with anti-GAD pAb (red). Yellow arrow, cytoplasmic α-tubulin (green). g Triple-stained 28-μm thick stack image of optical cross sections of the CBAS that was stained with anti-GAD pAb (red, red arrow). CB cilia were stained with mouse anti-acetylated α-tubulin (green). White arrow, CB cilia. h Fourteen-μm thick stack image of optical cross sections of mouse pre-immune serum-treated (green, not shown) larval arm. Blc, blastocoel; Ep, epithelium of the larval arm
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