Katow H et al. (2020), Initial report of γ-aminobutyric acidergic loco...

ECB-ART-48634

Sci Rep 2020 Jan 21;101:778. doi: 10.1038/s41598-020-57567-w.

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Initial report of γ-aminobutyric acidergic locomotion regulatory system and its 3-mercaptopropionic acid-sensitivity in metamorphic juvenile of sea urchin, Hemicentrotus pulcherrimus.

Katow H , Yoshida H , Kiyomoto M .

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The γ-aminobutyric acid (GABA) signal transmission system (GSTS) contributes to larval swimming through the regulation of ciliary beating. However, whether this system also contributes to the primary podia (PP)-generated motility of juveniles remained unclear. The present study aimed to elucidate the involvement of the GSTS in the motility of metamorphic juveniles (juveniles) (1) by immunohistochemically elucidating the location of molecular constituents of the PP, and (2) by inhibiting the activity of GΑΒΑ decarboxylase (GAD) with 3-mercaptopropionic acid (3-MPA). During metamorphosis, the echinus rudiment protrudes its PP out of the body surface in 8-arm plutei. The PP expresses immunopositive signal (-IS) of GAD, GABA, GABAA receptor and tropomyosin, and is constituted with the GABA-IS negative distal tip and the GABA/GAD-IS gaiter region. The latter radiates distal projections to the disc that contains a GAD-IS cellular network. The juvenile body cavity houses a GABA/βIII-tubulin-IS Penta-radial ring (PrR) that extends branches into each PP and several bridges to the GAD/GABA-IS Penta-radial plate (PrP) on the oral side but does not reach to the gaiter region. 3-MPA reversibly inhibits the juvenile motility and GABA-IS expression in the PrR/PrP complex. This indicates that the complex is the major contributor to the GABAergic motility in juveniles.

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Genes referenced: LOC100887844 LOC579659 tubgcp2
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	Figure 1. Motility of juveniles with or without 3-MPA. (A) Summary of the assay procedures. (B-1) Representative scanner image of juveniles in 24-well plates that were incubated in normal seawater (Control), 1 μM 3-MPA (+3MPA), a mixture of 1 μM 3-MPA and 10 μM GABA (+3MPA/GABA) or in 1 μM 3-MPA before washing out with plain seawater (3MPA-W). (B-2) Representative scanner image of juveniles replaced with fresh plain seawater (Control), fresh 3-MPA (+3-MPA), 3-MPA or GABA (+3-MPA/GABA) or plain seawater (3-MPA-W). Red circles, juveniles that unchanged their location from the initial position during the period indicated on the left side boxes. (C) Statistical analysis of the proportion of juveniles that changed their position (creeped) during the incubation period. Asterisk; Subjected pair of assay results with significant difference (P = 0.0013). Vertical bars; s.d.
	Figure 2. Immunohistochemistry of primary podia (PP) development in the echinus rudiment (EcR) of 8-arm plutei. (A) Left-side trunk. Inset (a), higher magnification of a box (a). Arrow; ciliary band-associated strands. Arrow head; connection between the anterior epaulette (asterisk) and the GAD-immunopositive (GAD-IS) blastocoelar network. Dual asterisks; posterior epaulette. (B) Higher magnification shown by a box (b,c) in (A). Arrow; ciliary band-associated strand. Asterisk; anterior epaulette. (C) Optical cross-section of EcR shown by a box (b,c) in (A). Arrow; GAD-IS PP surface. Asterisk; anterior epaulette. (D,E) Older plutei than (A). Arrow; anus muscle ring. (D) PP-encased EcR. (D1) Higher magnification of a box (d1,d2) in (D) shows GABAAR-IS PP. (D2) The same area as (D1) shows faint tropomyosin (TM)-IS PP at a similar region to GABAAR-IS sites in (D1). (E) Slightly older pluteus than (D). (E1) Higher magnification of a box (e1,e2) in (E) shows faint GABAAR-IS PP. (E2) Same area as (E1) shows intensified TM-IS PP. (F) Further developed GAD-IS PP than (E). (F1) Asynchronous GABA-IS expression among PP. (F2) Merged image between (F) and (F1). Both ISs are missing at the tip of PP (arrowheads). (G1,G2) Three-D image of trunk. aEpt; anterior epaulette. pEpt; posterior epaulette. Asterisk; larval opening. (G1) Outer surface view by ImageJ. Red arrowheads; ciliary band-associated strand. (G2) Inside out image of (G1) by Avizo. (H-H2) Optical cross-section of a PP shown by box (h) in (G1). Red arrowhead; periphery of the opening. (H) Optical cross-sections of GAD-IS image. (H1) TM-IS image. (H2) Merged image between (H) and (H1). la; larval arm. N; nuclei, Ph; pharynx. Stm; stomach. TM; tropomyosin. Scale bars = 100 μm (A,D,E,F), 50 μm (B,C,D1,E1), 75 μm (G1), 20 μm (H).
	Figure 3. Snapshots of video movie of simultaneous activity of the primary podia (PP) and the ciliary beating of epaulets of 36-day post fertilization larva (A–D) during very late metamorphosis and 37-dpf juvenile (E). (A–D) Synchronized up-and-down movements of cilia of anterior (blue arrow) and posterior epaulets (black arrows). (E) Larval arms retreated (red arrow) by leaving spicules (green arrows). Numbers at the upper left corner; the time started from (A–D). Scale bars = 150 μm.
	Figure 4. GABA/GAD-IS patterns in the primary podia (PP) of a 3-day post metamorphosis juvenile. (A1) GABA-IS PP. (A2) GAD-IS on PP (arrowheads) and the larval body surface (arrows). (A3) Merged image between (A1) and (A2). (B) Negative control with rabbit pre-immune serum did not detect GAD-IS at the PP (arrowheads) or any part of the body surface. (C1–C3) Higher magnification of a PP shown by a box (c) in (A3). (C1) GABA-IS PP is accompanied by a packed spiral pattern and the distal projections (arrows). (C2) GAD-IS dots around the distal tip of PP (arrows). (C3) Merged image between (C1) and (C2). (D1–D3) Higher magnification of a longitudinal optical cross-section of a PP shown by a box (d) in (C1). (D1) GABA-IS distal projections radiate into the disc. (D2) GAD-IS spots in the disc (arrows). (D3) Merged image between (D1) and (D2) shows that the tip of GABA-IS distal projections of PP are double stained with GAD-IS spots (arrows). (E) Three-D image of distal tip of a PP by Amira shows close localization of GAD-IS fibers with the GABA-IS distal tip of PP in the disc. (F) Higher magnification of the distal tip of PP shown by a box (f) in (E) without nuclei staining. The distal tips of GABA-IS projections co-localize with GAD-IS fibers (arrowheads). (G1–G3) Relaxed PP. Inset; higher magnification of a longitudinal optical cross-section shown by a box (g2-3). Arrows; cell body. (G1) GABA/GAD-IS spiral rings around the proximal end of PP. (G2,G3) Higher magnification of a box (g2-3) in (G1). (G2) GABA-IS spiral rings. (G3) GAD-IS spiral rings. L; luminal epithelium of PP. Scale bars = 75 μm (A1,B), 25 μm (C1), 15 μm (D1), 50 μm (G1), 20 μm (G2), 10 μm (Insets).
	Figure 5. Layered distribution of the GABA-IS area and the tropomyosin (TM)-IS muscle of PP of 5-day post metamorphosis juvenile. (A) Oral side view of the juvenile. (B1–B3) Higher magnification image of a PP indicated by a box (b) in (A). (B1) GABA-IS outermost layer on the luminal epithelium with a weak IS gap in between (arrow). (B2) An immuno-negative dark layer (arrow) surrounds the TM-IS layer on the outer surface of the luminal epithelium. (B3) Merged image between (B1) and (B2) positions the central luminal epithelium, TM-IS layer, and the outermost GABA-IS layer (arrow). (C) Three-D image of a PP by Amira. Arrows; GABAAR-IS projections. White arrowheads; circumoral TM-IS muscle ring. Red arrowhead; TM-IS peripheral area of the distal disc (disc). (D) Three-D reconstructed optical cross-section of a PP by Amira at the gaiter region indicated by a dotted line (d) in (C) shows the concentric distribution of TM-IS muscle layer and the GABAAR-IS layer on the luminal epithelium (asterisk). (E) Concentric distribution of GABAAR layer and GABA-IS layer on the luminal epithelium (asterisk) via an immuno-negative space (arrow) shown by a stacked confocal laser scanning micrograph of an optical cross-section of a PP. Two IS areas are partially overlapped (yellow arrow). (F) Schematic summary of a PP cross-section shows the concentric distribution of the GAD/GABA-IS layer (light brown), GABAAR-IS layer (red) and TM-IS muscle layer (orange). Dark-blue; luminal epithelium. Gray; lumen of PP. Light blue; the outer most epithelium of PP. Scale bars = 100 μm (A), 30 μm (B1), 25 μm (C,E).
	Figure 6. GABAergic PrR/PrP complex in the body cavity of 3-day post-metamorphosis juvenile. (A–C) Stacked optical cross-sections made perpendicularly to the oral-aboral axis at three levels. Triangle with 35°; the intersection between PrR and PrP at 35°. (A) Thirty-micrometer thick image at the level between 78 μm–108 μm from the oral surface (ventral side) of the body shows GABA-IS PrR/PrP complex. (B) Six-micrometer thick image at 72 μm–78 μm level shows pentagonal alignment of proximal tropomyosin (TM)-IS muscle regions of PP. Arrows; GABA-IS proximal area of PP. Arrowheads; outer periphery of GABA-IS PrP. (C) Six-micrometer thick image at 30 μm–36 μm level shows pentagonal TM-IS triangular shapes (red arrows) that accompanied GABA-IS proximal dots (arrows). (D–G) Three-D image of a PP by Amira showed by a box (d–g) in (A) with various angles (numbers shown at the upper right corner). Curved arrow; rotation direction shown by a curved-arrow (e–g). Arrowhead; a branch of Penta-radial projection of PrR. cmr; circumoral muscle ring. disc; disc of PP. gp; gaiter of PP. (H–J) 3-D images of (A,B) rotated as shown by a curved arrow (i,j). Asterisk; muscle of PP. Circle or rectangle of dotted-line; PrR/PrP complex area. (H) Aboral view (dorsal view). GABA-IS PrR is embedded in PP muscle. (I) Side view after rotation shows PrR on the aboral side and PrP on the oral side (ventral side). (J) Oral view shows PrP resided in the middle of Penta-radially arranged PP muscles. (K) Schematic summary showing dorso-ventrally aligned PrR-PrP complex at the center of body cavity (oval of pale-blue gradation). PP or pp; primary podium. PrP; Penta-radial plate. ppm; primary podium muscle. PrR; Penta-radial ring. Scale bars = 75 μm (A,H).
	Figure 7. Immunohistochemical property of the GABA-IS Pent radial-ring (PrR)/Penta-radial plate (PrP) complex. (A–D) Extracted image of a PrR/PrP complex shown by a box and circles in Fig. 6H–J. (A) Side view shows connections between PrR and PrP by several GABA-IS cables (arrows). (B) Optical horizontal cross-section shown by a box (b) in (A). The PrR was localized on the tropomyosin (TM)-IS PP muscle (left) and its five arms diagonally crossed PrP between PP. The PrR was clearly visualized after digital deletion of the TM-IS area (right). Triangle with 35°; intersection between PrR and PrP at 35°. (C) Optical horizontal cross-section shown by a box (c) in (A). The PrP alone is visualized between the TM-IS PP (left). The PrP is eminently seen after digital removing the TM-IS area (right). (D) A perpendicular optical cross-section of the PrR/PrP complex (left) shown by a dotted box on the right image of PrR on the aboral side and PrP on the oral side. (E1–E3) Colocalization of GABA-IS and βIII-tubulin-IS of 3-dpm juvenile PrR. Fluorescein colors were switched using ImageJ to retain the consistency with the GABA-IS of the others in this figure plate. (E1) GABA-IS/Nuclei (G/N). (E2) βIII-tubulin-IS/Nuclei (βIII/N). (E3) Merged image between (E1) and (E2). Red arrows: central conical rise of PrP. Scale bars = 30 μm (A), 50 μm (E1), 100 μm (B,D).
	Figure 8. 3-MPA-sensitive and-insensitive GABA-IS organs in juveniles. (A) GABA-IS Penta-radial nerve ring (PrR)/Penta-radial plate (PrP) complex in Control juvenile. (B) GABA-IS is inhibited at the PrR by 3-MPA, while it is not at the gaiter region of all primary podia (pp). (C) GABA-IS is weakened or inhibited at the PrR/PrP complex but is not affected at PP by a mixture of 3-MPA and exogenous GABA. (D) GABA-IS is restored at the PrR/PrP complex after washing with normal seawater. (E) Frequency of GABA-IS expression pattern of juveniles in seawater alone (Control), with 3-MPA (3-MPA), with 3MPA and exogenous GABA (3-MPA/GABA), and in plain seawater after washing out 3-MPA (Fresh SW). GABA-IS patterns A (yellow column), B (gray column), C (deep green column) and D (light brown column) encased in a rectangle are represented respectively by above WMIHC images (A–D). (F) 3-D reconstructed images of PrP. Optical cross-section of the oral side of the PrR/PrP complex by ImageJ. (G) Twenty-six μm thick optical cross-section of the aboral side of (F) shows GABA-IS PrR alone. (H) Fourteen-μm-thick optical cross-section of the oral side of (G) shows GABA/GAD-IS PrP. (I) GAD-IS plexus after the digital removal of GABA-IS from (H). Inset; GAD-IS plexus cells (arrow) by a higher magnification of a box in the mainframe. (J1) Sagittal optical cross-section of GAD-IS plexus at the dotted line (j1–j3) in (H). (J2) GABA-IS plexus of the same area as (J1). (J3) Merged image between (J1) and (J2) shows the GAD-IS region at slightly broader area than that of the oral side (Arrow). Double-headed arrow; Dorsoventral axis. Scale bars = 100 μm (A–D), 50 μm (F–I), 25 μm (Inset of I),10 μm (J1).

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