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Figure 1. CBAS patterning process during the 4aPL stage. (A) Confocal laser scanning microscopy (CLSM) of the right antero-lateral arm showing a mass of blastocoelar GAD-positive cells (GADC, asterisk) egressed in part onto the larval body surface at the arm tip (arrows). (*B.1) 3D-reconstructed image of (A). Part of the egressed GADCs extend as a fragmental stripe of preoral CBAS (preoc) on the apical surface of the antero-lateral arm tip. The other GADCs are egressed onto the other direction and extended to form a short preoral CBAS (preoc). (B.2) Rotated image of (B.1) toward the direction shown by a curved arrow, b.2, in (B.1), showing the fragmental stripe of preoral CBAS. Part of the right lateral CBAS is seen (r-latc). (B.3) Rotated image of (B.1) as shown by the curbed arrow b.3 in (B.1). GADCs extend as a stripe on the larval arm surface. (C.1) CLSM image of the left post-oral arm. A posteriorly extended fragmental CBAS (l-latc) retains perikarya on the apical surface (p1) and basally in the ectoderm (p2). (*C.2) 3D-reconstructed image of (C.1). (C.2, inset) Rotated image of (C.2) as shown by a curbed arrow. (D) CLSM of the oral side of the larva shows the left- and right-post-oral arm region (l-poa, r-poa). Two CBASs are seen between the aboral ectoderm (aoe) and the circumoral ectoderm (coe). (E) Higher magnification of the post-oral CBAS (poc) with perikaryon, shown by a rectangle (e) in (D). (F) Higher magnification of the left lateral CBAS (l-latc), shown by a rectangle (f) in (D). (*G) Aboral view of 3D-reconstructed image of the same area, as shown by a rectangle (g) in (D). The poc and the l-latc are separated from the blastocoelar GADCs (asterisk), while some blastocoelar GADCs are egressed at the tip of the arm (arrow). (H) The poc in (G), viewed from the direction shown by a yellow arrow (h) in (G). (I) The l-latc shown in (G), viewed from the direction shown by a yellow arrow (i) in (G). (J) Fully formed circumoral CBAS and those on the oral side antero-dorsal-epauletes (ad-e) and posterior-epaulet (po-e). * Refer to Supplemental Figure S1(B.1,C.2,G). Scale bars = 25 μm (A, C.1), 10 μm (D–F), 100 μm (J).
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Figure 2. Circumoral CBAS interruption at the oral side of the upper lip field of 4aPL. (A) Confocal laser scanning microscopy (CLSM) of preoral CBAS (preoc) shows suspension at the upper lip field (white arrows). (*B) 3D-reconstructed image of (C) shows the oral side view around the serotonergic apical ganglion (AG, green). The terminal ends of the left- and right-preocs are shown with yellow arrows. White arrow; one of the bilateral arms from the apical ganglion. (C) CLSM of (B). The serotonergic apical ganglion (AG) extends an axon toward the right antero-lateral arm (r-ala) beneath the ectoderm. (D) Higher magnification of the rectangle (d) in (C) shows several cell processes from the perikaryon reaching to the preoral CBAS (white arrows). Red arrow: preoral CBAS. Scale bars = 30 μm (A), 25 μm (C), 5 μm (D). * Refer to Supplemental Figure S2B.
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Figure 3. Schematic summary of the CBAS pattern formation. (A) Schematic process of the CBAS pattern formation from the 3 days post-fertilization, 4-arm pluteus stage (3dpf-4aPL) to 24dpf-8aPL, immediately before metamorphosis. The long arrow on 24dpf 8aPL: suspended CBAS near the apical ganglion. (B) Increasing cell number of the CBAS from 3dpf-4aPL to 10dpf-6aPL, with error bars. Asterisk: the number of post-oral CBAS (poc)-constituting cells is statistically significantly larger than that of r- and l-latc. p = 0.0001 < 0.05. The number on each column is the total number of larvae subjected to count the cell number in the CBAS.
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Figure 4. Heterogeneous expression intensities of cytoplasmic proteins among the post-oral-arm CBAS forming cells in 4aPL. (A) The intensity of GAD expression among the four cells is different; #3 cell expressed the most intensively, while #4 cell was the weakest, with the other two cells in between. (B) 5HThpr was expressed the most intensively in the #1 and #3 cells, while the other two cells were weaker. (C) Merged image between (A) and (B). Scale bar = 30 μm.
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Figure 5. Pattern formation of GAD-positive CBAS (red) along the Netrin-positive stripe in 4aPL. (A) Confocal laser scanning microcopy (CLSM) of the post-oral CBAS (poc). (B) The Netrin stripe along the poc. (C) Merged image between (A) and (B). Inset: pre-immune serum of anti-Netrin Ab applied poc (arrow) area. (D) CLSM of a post-oral arm region. Netrin (green) layer was on the apical surface and on the spicule surface (Spcl) in the blastocoel (Bcl). GAD-positive CBAS (red) is seen beneath the Netrin-positive layer on the apical surface (arrow). (E) 3D-reconstructed image of (D). Thin vertical rectangles locate the following cross-sectional images of the area shown by (F1) to (F5). (F1–F5) These cross sections consistently located the CBAS (white arrowheads) on the circumoral ectoderm side of the Netrin strip (green arrowheads), as is seen with the flat oval cut-surface. (G) CLSM of a longitudinal center line section of a post-oral arm used for 3D-reconstruction images (H) and (I). (H) The CBAS (red) is seen beneath the Netrin-positive layer (NS, green). (I) 3D image of (G) that was tilted along the curved arrow in (H) showing the arm surface from the direction pointed by a yellow arrow in (G), indicating the Netrin stripe (NS) on the CBAS. (J) CLSM of the whole-body of the aboral side of a larva, and the left-lateral CBAS (l-latc) extends from the left antero-lateral arm (l-ala) to the left post-oral arm (l-poa). (*K) 3D image of (J) shown by a rectangle (k) in (D) locates l-latc (red) between the Netrin stripe (NS) and circumoral ectoderm area (Oral). Scale Bars = 25 μm (A), 10 μm (D, E, F1), 25 μm (G). * Refer to Supplemental Figure S5K.
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Figure 6. Confocal laser scanning microscopy of the Netrin stripe formed on the oral side of the DRD1-expressing ciliary band in 4aPL, and lack of the Netrin stripe in very early 2aPL. (A) Aboral side view. A Netrin-positive signal is detected on the apical side of the DRD1-expressing circumoral ciliary band. Inset: higher magnification of the right antero-lateral arm shown by a rectangle in (A). (B) Oral side of the post-oral CBAS. Inset: Netrin/DRD1 stained image of the rectangle in (B) shows a partially overlapped Netrin stripe and DRD1-positive ciliary band. Inset: Netrin stripe on the circumoral ectoderm side of the DRD1-positive ciliary band. (*C) Close to the end of the prism stage. The DRD1-positive ciliary band is not associated with the Netrin stripe but remained in the blastocoel (red arrows) in this developmental stage. Inset: higher magnification of the ciliary band region (arrow) shown by the rectangle in (C). Scale bars = 50 μm (A), 25 μm (insets of A and C), 30 μm (B, C), 15 μm (inset of B).
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Figure 7. Confocal laser scanning microscopy of 4dpf-4aPL shows co-expression of Unc-5 and GAD at the CBASs. (A) GFP:Unc-5-positive post-oral CBAS (poc) and lateral CBAS (latc). Asterisk: GFP:Unc-5-positive cells in the blastocoel. (B) GAD-positive, same area as (A). Inset: GAD-positive cells in the blastocoel. (C) Merged image between (A) and (B). Double arrows: poc; single arrow: lateral CBAS. Scale bar = 30 μm.
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Figure 8. Aboral view of triple-stained 10dpf-6aPL with Unc-5 (red), Netrin (green), and Nuclei (blue). (A) Confocal laser scanning microscopy (CLSM) shows the Unc-5-positive CBAS. Inset: higher magnification of the rectangle shows Unc-5-positive cells in the blastocoel of the arm tip. (B) CLSM of the same larva shows the Netrin stripe (green). (C) Merged image between (A) and (B) shows the CBAS on the Netrin stripe. (*D) 3D-reconstructed image of the area, indicated by a rectangle in (C), was rotated to the direction shown by a curved arrow to show the Unc-5-positive CBAS on the oral side of the Netrin stripe (red arrowheads in C and D). Scale bar = 75 μm. * Refer to Supplemental Figure S8D.
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Figure 9. Application of Netrin peptide interferes with ciliary beating activity and CBAS patterning in 3dpf 4aPL. (A) A major proportion of the peptide-applied larvae formed no swirl (Column A in (E)). (B) A second major proportion of the control larvae formed one swirl (Column B in (E)). (C) A major proportion of control (Column C in (E)) and a distant second major proportion of peptide-applied larvae formed two swirls. (D) A small proportion of control larvae formed four swirls (Column-D in (E)). (E) The frequency of swirls in terms of group distribution was examined in 35 control larvae (blue columns) and 22 peptide-applied larvae (brown columns). Letters on the top of each column represent swirl patterns A–D. (F–I) Fluorescence microscopy of the whole-mount immunohistochemistry of 4aPL. (F) The major CBAS pattern of the control 4aPL acquired the clear poc (arrow). (G) A small proportion of the peptide-applied 4aPL formed CBAS composed only of a few GADCs insertions into the circumoral ectoderm (arrows, rectangle). Inset: enlarged rectangle area. (H) No CBAS was formed in the majority of peptide-applied larva. (I) The frequency of CBAS forms in the control larvae (blue columns, total 20 larvae) and in the peptide-applied larvae (brown columns, total 26 larvae). Letters on the top of columns represent the CBAS forms shown by the WMIHC images (F–H). Scale bar = 60 μm.
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