Katow H, Katow T, Yoshida H, Kiyomoto M.

	Figure 1. Hp-Htt expression during early development by immunoblotting. Lysates of embryos at the sBL stage (lane 1), the pL stage (lane 2), the 2aPL stage (lane 3), and the 4aPL stage (lane 4) were probed with anti-Hp-Htt Ab. For the negative control, 4aPL lysate was probed with a rabbit pre-immune serum (lane 5). An Ab-positive band was detected at around the 350 kDa region at the sBL and pL stages (arrow A). In 2aPL a new smaller band at the 165 kDa region was detected (Arrow B). In 4aPL, a further two new smaller bands (small arrows at 192 kDa and 75 kDa regions) appeared, in addition to the initial 165 kDa band. Anti-Hp-Tjp1-antibody detected the protein expression pattern of the pL stage (lane 6) and the 4aPL stage (lane 7). A single Ab-binding band was detected at the 150kDa region (Arrow C). The lysate was probed with a rabbit pre-immune serum (lane 8).
	Figure 2. Fluorescent (A,B) and Confocal microscopic images (C–G) of Hp-Htt expressing cells and primary mesenchyme cells from the sBL stage to the pL stage. (A) Swimming blastula with an Hp-Htt-positive signal (green) in the Epith-2-positive ectoderm (red). (B) A higher magnification image of a region shown by the rectangle (b) in (A) shows an Hp-Htt-positive region in the nuclei of the ectoderm. (C) Confocal microscopy of mBL showing primary mesenchyme cells (PMC, red) that have invaginated into the blastocoel and the Hp-Htt-positive cells in the ectoderm (green). (D) Higher magnification image of the ectoderm indicated with a rectangle (d) in (C). (E*) Higher magnification of the cytoplasmic Hp-Htt-positive dots shown by a squire (e) in (D) shows the signal in the nuclei (blue) and the cytoplasm near the nucleus. Supplemental animation (Figure S2E*) shows a regional difference between the Hp-Htt-positive nuclear spot and the blurred Hp-Htt-positive area around the nuclei. (F) Ventral view of a late pL by fluorescent microscopy depicts the Hp-Htt-positive blastocoelar cells (green) around the archenteron (Arc) and the mouth opening (Mouth). Ant: Anterior, Post: Posterior. (G) A triple-stained image of (F) shows the Hp-Htt-positive signal (green) in the GAD-positive (red) cells (arrows). (H) Statistical analysis of spatiotemporal Hp-Htt-positive signal spreading from the ectodermal cells (blue columns) to the blastocoelar cells (red columns) during development from the mBL stage to the pL stage (Prsm). eG: early gastrula stage, mG; mid-gastrula stage. Red arrow; a tiny proportion of Hp-Htt-positive blastocoelar cells in eG. Scale bars = 50 µm (A,C,F), 15 µm (B), 10 µm (D), 5 µm (E*). (D) and (E*): Refer to Supplemental Figure S2E.
	Figure 3. Confocal microscopy of triple- and double-stained plutei [Hp-Htt; red, serotonin (5HT); green, Serotonin receptor (5HThpr), green, GAD; green, and nuclei; blue)] showing the merged distribution of Hp-Htt-expressing cells and the CBAS during the pluteus stage. Hp-Htt-positive cells distribution was seen in the blastocoel on the basal surface of the ectoderm, and along the larval skeleton. (A) Ventral view of the triple stained 2aPL. Arrow: Hp-Htt-positive cells along the spicule. (B) Dorsal view. The Hp-Htt-positive cells being infiltrated into the ectoderm around the serotonergic apical ganglion. Arrow: Hp-Htt-positive cells were aligned along the arm spicule. (C) Higher magnification image of an apical ganglion region shown by a rectangle (c, d) in (B). Some of the Hp-Htt-positive cells near the apical ganglion shifted their position to the apical surface of the ectoderm. (D*) Animation of the same area as (C) shows spatial relations between Hp-Htt-positive cells and the apical ganglion. (E) In 4aPL, Hp-Htt-positive signals and GAD-positive signals were detected together in the CBAS, and in some of the blastocoelar cells. (F) Higher magnification of a rectangle (f, g) in (E) shows the CBAS (arrow). Long arrow: Hp-Htt-positive spicule tip area. Inset (f’): A higher magnification image of a rectangle (f’) in (E) showing a cytoplasmic Hp-Htt-positive signal along the axonal region and near the nuclei (arrow) in the CBAS. (G) The same area as (F) showing a GAD-positive signal (arrows). (H) A 3 µm thick optical section at around the middle of the stomach indicated by a rectangle (h) in (E) shows higher magnification of the Hp-Htt-positive stomach wall (arrows). (I) Four-arm pluteus triple stained with pre-immune rabbit serum (red) as a negative control of Hp-Htt-staining and anti-5HThpr antibody as a marker of the CBAS. (J) Six-arm pluteus triple stained for Hp-Htt, GAD, and DNA. These two signals of Hp-Htt and GAD are converged to the CBAS (arrows). The same signal conversion is seen at the CBAS of the epaulette (Ep). (K) Five µm thick optical cross-section around the middle of the stomach showing higher magnification of the stomach indicated by a rectangle (k) in (J). Arrows are the Hp-Htt-positive apical surface of the stomach. (L) Higher magnification image of a region shown by a rectangle (l, m, n) in (J) that depicts the perikaryon of the CBAS (arrow). (M) Higher magnification image of a region shown by a rectangle (l, m, n) in (J) of Htt-positive and nuclei image that depicts the perikaryon of the CBAS (arrow). (N) Higher magnification image of a region shown by a rectangle (l. m. n) in (J) double-stained for Hp-Htt and nuclei. Arrow; perikaryon. (N) The same area as (M) double-stained for GAD (green) and nuclei (blue). Scale bars = 50 µm (A,B,E,H,K), 25 µm (C,F), 75 µm (I), 150 µm (J), 20 µm (L), 10 µm (f’). (D*): Refer to Supplemental Figure S3D.
	Figure 4. Shortened larval arm length in Fluorescein-tagged Hp-Htt morpholino (FL-Htt-MO)-treated 4aPL. (A) Fluorescence microscopy of FL-Htt-MO-positive areas (green) in the stomach (Stm), the ectoderm regions (double-headed arrows), and the blastocoelar space (asterisk). Inset: Higher magnification of green glow cytoplasmic region of the ectoderm (arrow) of the larval trunk, shown by a dotted-line rectangle. (B–F) Confocal microscopies double-stained for Htt (red) and nuclei (blue). (B) Control larva. Hp-Htt was detected at the CBAS (arrow). (C) Higher magnification image of a doted-line rectangle (c) in (B) shows CBAS by an arrow. (D) 20 µM FL-Htt-MO-treated larva. Larval arms are distinctively shorter than those in the control larva and CBAS was fragmented (arrows). (E) Higher magnification image of a dotted-line rectangle (e) in (D). The Hp-Htt-positive area shows a fragmented CBAS (arrows). (F) PEG-treated larva extends normal length arms with continuous Hp-Htt-positive signal in CBASs (arrows). (G) Statistical analysis of the arm length shown by columns with error bars depicting the shortened arm length in the FL-Htt-MO-treated larvae. The number of larvae examined is indicated in boxes in the middle of each column. p values were calculated between the two columns connected with solid lines. Inset: The dorsal view of a pseudo-green-colored DAPI-stained larva shows places that were subjected to measurement of the arm length with double-headed arrows. Scale bars = 50 µm (A,G inset), 100 µm (B), 25 µm (C,E), 75 µm (D,F).
	Figure 5. Hp-Htt (red)/BrdU (green)/nuclei (blue) triple stained image of 4aPL. Shortened larval arms due to FL-Htt-MO application occurred, accompanied by decreased BrdU-positive signals, particularly at the larval arm regions. (A) Dorsal view of the larva. Hp-Htt was detected at the CBAS (large arrow) and some of the blastocoelar cells (small arrows). (B) The highly magnified CBAS area shown by a rectangle (b) in (A). BrdU was detected also there (arrows). (C) Hp-Htt/DAPI double stained image of (A). Arrow; CBAS. Hp-Htt-positive blastocoelar cell network (asterisk). (D) At the ectoderm, a smaller number of BrdU-positive signals were detected in the blastocoelar cells (arrows) (E) Ventral view of the FL-Htt-MO treated larva. The number of BrdU-positive signals clearly decreased in the ectoderm, particularly at the tip of arms and in the fragmented CBAS (double-head arrow). (F) The highly magnified area shown by a rectangle (f) in (E). Shorter arm length is clear at the postoral arms (POA). (G) Fragmented CBAS (double-head arrow in F and G). The blastocoelar Hp-Htt-positive cell network (asterisk) is rather unaffected. (H) BrdU-positive signal image extracted from (E) clearly detecting the declined positive signal at the tip regions of the arms (arrows), while those in the blastocoel were not apparently decreased (small arrows). (I) Statistical analysis of average arm length comparison between control larvae and FL-Htt-MO-applied larvae with p-value. The larval number examined are shown in a box on each column. Scale bars = 50 µm (A,C,E,G). 25 µm (B,F).
	Figure 6. Increasing number and types of swirling patterns (arrows) during development. (A) A water current around the body surface with no swirling pattern of the control sBL. (B–E) Control 4aPLs with different numbers of swirling patterns. The number on the upper left corner is that of swirls. (F) Fluorescence microscopy of FL-Htt-MO in the blastocoel of mBL before fixation. (G) An FL-Htt-MO-treated 4aPL generated no water current around the body. (H–J) The swirling patterns of FL-Htt-MO-treated 4aPL. The upper left number shows that of swirls. The arrows point to the swirls. (K) The average number of swirlings of 4aPL of control (Cont), FL-Htt-MO (20 µM MO)-treated, and Endo-Porter PEG (PEG) alone. The numbers in boxes on each blue column indicate those of the samples. Thinner blue vertical bars on the top of each column show the standard deviations. p values are shown between two subjected columns connected with lines. (L) Frequency of the swirling pattern types, from sBL to 4aPL, are shown in the central box, with dark blue for zero swirls to five swirls in the orange column. The control larvae are on the left side and the FL-Htt-MO-treated larvae on the right side of the graph. The numbers in parentheses at the end of the developmental stage names show that of samples that were examined in that stage. The light blue arrow in the control group points to the major swirling pattern group in the control 2aPL. The green arrow shows the major swirling pattern group in the control 4aPL. The dark blue arrow at the top of the dark blue column of the FL-Htt-MO-treated groups shows the major swirling type of the sBL. The brown arrow at the top of the brown column shows the major swirling type of the 2aPL. The light green arrow at the top of the green column in the FL-Htt-MO-treated groups shows the major swirling type of the 4aPL. Scale bars = 100 µm (A), 50 µm (B), 30 µm (F).
	Figure 7. The antigen peptide sequence (red letters) and its location in the Sp-Htt protein (SPU_012067.1_Sp-Hunt: https://www.echinobase.org/entry/; Hp-Htt (HPU_11725_Hp-Hunt: HpBase: http://cell-innovation.nig.ac.jp/Hpul/ [48] (Access on 5 May 2021).The respective sequence is located in the middle section of the protein, and is very similar.

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