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Figure 1. Schematic diagrams of the general anatomy of starfish (Asterias rubens) larvae (modified after, Murabe et al., 2008) (top half) and main neuronal aggregations of the nervous system (bottom half). In all diagrams anterior is uppermost with the overall body structure shown in yellow and the digestive system shown in orange. (A) bipinnaria larva; dorsal view. (B–D) brachiolaria larva. (B) dorsal view. (C) frontal (ventral) view. (D) left lateral view with ventral side on the left and dorsal on the right. (E) bipinnaria larva; dorsal view. (F) bipinnaria larva; ventral view. (G–I) brachiolaria larva. (G) dorsal view. (H) frontal (ventral) view. (I) left lateral view with ventral side on the left and dorsal on the right. The areas of main neuronal aggregations are highlighted in red in (E–I). The lateral ganglia start to develop at the bipinnaria stage and are located at the base of the anterior projection (solid black arrow labels the left lateral ganglion and dotted black arrow labels the right lateral ganglion; Moss et al., 1994; Elia et al., 2009). Neurons are also concentrated in several regions along ciliary bands, especially in the preoral ciliary band near the mouth (small black arrowhead; Moss et al., 1994; Byrne et al., 2007). In addition to these regions, the brachiolaria stage develops neuronal aggregations in the brachia tips (black arrowheads) and stems (white arrowheads), in the adhesive disk (double arrow), and in the basi-epithelial nerve plexus underlying the disk (white arrow; Barker, 1978; Murabe et al., 2008; Elia et al., 2009). a, anus; abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; m, mouth; lbr, lateral brachium; pol, postoral lobe; prl, preoral lobe; s, stomach.
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Figure 2. Localization of L-type SALMFamide (S1) precursor transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A–E) bipinnaria larva; (F,G) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (A) dorsal view showing paired groups of 2–3 stained cells on the left (solid arrow) and right (dashed arrow) sides of the anterior projection. (B) detail of the anterior projection (boxed region in A) showing stained cells on the left and right sides (solid and dashed arrows, respectively). (C) left lateral view showing three stained cells at the base of the anterior projection (solid arrow). (D) detail of the anterior projection (boxed region in C) showing stained cells at the base of the anterior projection (solid arrows). (E) dorsal view of the anterior projection showing absence of staining in a larva incubated with sense probes. (F) left lateral view showing expression in solitary cells of the brachia (black and white arrowheads) and around the adhesive disk (white arrow), and in a group of tightly packed cells in the dorsal region of the larva (black solid arrow). (G) detail of the attachment complex, showing stained cells present in both the tip (black arrowheads) and the stem (white arrowheads) of a lateral brachium. In addition to a group of cells under the adhesive disk (white arrow), there are also scattered solitary stained cells (small black arrowheads) that appear to be located along the ciliary bands of the preoral lobe and a group of stained cells in the dorsal region of the larva (black solid arrows). Inset shows the attachment complex of a brachiolaria larva incubated with sense probe control. abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; lbr, lateral brachium; pol, postoral lobe; prl, preoral lobe; s, stomach; st, stem of brachium; t, tip of brachium. Scale bars: 200 μm (A,C,F, inset of G), 50 μm (B,D,E,G).
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Figure 3. Localization of F-type SALMFamide (S2) precursor transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A–G) bipinnaria larva; (H,I) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (A) early bipinnaria, dorsal view showing stained cells on the left (solid arrow) and right (dashed arrow) sides of the anterior projection. (B) detail of the anterior projection (boxed region in A) showing stained cells on the left and right sides (solid and dashed arrows, respectively). (C) later bipinnaria, left lateral view showing two symmetrical groups of cells along the left (solid arrow) and right (dashed arrow) sides of anterior projection. (D) detail of the anterior projection (boxed region in C) showing stained cells on the left and right sides (solid and dashed arrows, respectively). (E) later bipinnaria, dorsal view showing stained cells along the margin of the anterior projection, with solid and dashed arrows highlighting cells on the left and right side, respectively. (F) detail of the anterior projection (boxed region in E) showing stained cells on the left and right sides (solid and dashed arrows, respectively). (G) left lateral view of anterior projection showing absence of staining in a larva incubated with sense probes. (H) left lateral view, showing expression in brachia (white arrowheads), near the adhesive disk (white arrow), and in a ganglion on the dorsal side (black arrow). Inset shows the attachment complex of a brachiolaria larva incubated with sense probe control. (I) detail of the attachment complex (boxed region in H), showing expression in separate cells along the brachium stems (white arrowheads) and arms (small black arrowhead). Groups of stained cells are located close to the adhesive disk (white arrows) and on the dorsal side (black arrow) of the larva. abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; lbr, lateral brachium; prl, preoral lobe; s, stomach; st, stem of a brachium; t, tip of a brachium. Scale bars: 200 μm (A,C,E,H, inset of H), 100 μm (B,D,F,G,I).
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Figure 4. Localization of asterotocin precursor transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A,B) bipinnaria larvae, showing no detectable expression of asterotocin precursor transcripts (A, left lateral view; B, frontal view). (C–E) brachiolaria larva. The schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (C) left lateral view, showing expression in the tips of the brachia (black arrowheads) and near the adhesive disk (white arrow). (D) detail of the attachment complex (boxed region in C) showing stained cells in the brachium tips (black arrowheads), the adhesive disk (double arrow), and adjacent to the adhesive disk (white arrow). (E) detail of the attachment complex showing absence of staining in a larva incubated with sense probes. abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; m, mouth; lbr, lateral brachium; pol, postoral lobe; prl, preoral lobe; s, stomach; st, stem of a brachium; t, tip of a brachium. Scale bars: 200 μm (A–C), 100 μm (D,E).
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Figure 5. Localization of NGFFYamide precursor transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A–C) bipinnaria larva; (D–F) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (A) frontal view, showing solitary stained cells along the ciliary band of the preoral lobe (small arrowheads). (B) detail of the mouth region, showing expression in solitary cells (small arrowheads) along the ciliary bands of both preoral and postoral lobes. (C) detail of the mouth region showing absence of staining in a larva incubated with sense probes. (D) middle part of the body and the attachment complex, showing absence of staining with sense probes. (E) left lateral view, showing stained cells scattered throughout the larval body (small arrowheads) and under the adhesive disk (white arrows). (F) attachment complex and the middle part of the body, showing stained cells along the rims (small arrowheads) and under the adhesive disk (white arrows). abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; m, mouth; lbr, lateral brachium; pol, postoral lobe; prl, preoral lobe; s, stomach. Scale bars: 100 μm (A–D,F), 200 μm (E).
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Figure 6. Localization of ArTRHP transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A) bipinnaria larva, frontal view showing no detectable expression of ArTRHP transcripts; (B–E) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (B) left lateral view, showing stained cells along the brachium stems (white arrowheads), near the adhesive disk (white arrows) and throughout the preoral lobe (small black arrowheads). (C) detail of a lateral brachium; note the presence of stained cells (white arrowheads) in the stem, but not in the tip of the brachium. (D) detail of the attachment complex; stained cells are present in the brachium stem (white arrowheads) and near the adhesive disk (white arrows). (E) attachment complex and the middle part of the body, showing no staining with sense probes. abr, anterior brachium; ad, adhesive disk; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; m, mouth; lbr, lateral brachium; pol, postoral lobe; prl, preoral lobe; s, stomach; st, stem of a brachium; t, tip of a brachium. Scale bars: 200 μm (A,B,E), 50 μm (C,D).
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Figure 7. Localization of ArGnRHP transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A) bipinnaria larva, left lateral view, showing no detectable expression of ArGnRHP transcripts; (B–D) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (B) left lateral view, showing stained cells close to the adhesive disk (white arrow) (C) detail of the attachment complex (boxed region in B) showing two groups of stained cells near the adhesive disk (white arrows). (D) absence of staining in a brachiolaria larva incubated with sense probes. a, anus; abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; lbr, lateral brachium; prl, preoral lobe; s, stomach; st, stem of a brachium; t, tip of a brachium. Scale bars: 200 μm (A,B,D), 100 μm (C).
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Figure 8. Localization of ArCTP transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A) bipinnaria larva, frontal view, showing no detectable expression of ArCTP transcripts; (B–D) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (B) left lateral view, showing stained cells in the adhesive disk (double arrow) and adjacent to the adhesive disk (white arrows). (C) detail of the attachment complex; note stained cells within the adhesive disk (double arrows) and adjacent to it (white arrow). (D) absence of staining in a brachiolaria larva incubated with sense probes. abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; m, mouth; lbr, lateral brachium; pol, postoral lobe; prl, preoral lobe; s, stomach; st, stem of a brachium; t, tip of a brachium. Scale bars: 200 μm (A,B,D), 100 μm (C).
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Figure 9. Localization of ArCRHP transcripts in larvae of the starfish Asterias rubens using whole-mount in situ hybridization. (A) bipinnaria larva, frontal view, showing no detectable expression of ArCRHP transcripts; (B–D) brachiolaria larva; the schematic drawings illustrate the distribution of stained cells (see Figure 1 for labeling). (B) left lateral view, showing stained cells along the brachia (black and white arrowheads) and near the adhesive disk (white arrow). (C) detail of the attachment complex showing absence of staining in a larva incubated with sense probes. (D) detail of the attachment complex (boxed region in B) showing stained cells in tips (black arrowheads) and stems (white arrowheads) of brachia and close to the adhesive disk (white arrows). abr, anterior brachium; ad, adhesive disk; ap, anterior projection; ar, adult rudiment; ba, bipinnaria arms; e, esophagus; g, gut; m, mouth; lbr, lateral brachium; prl, preoral lobe; s, stomach; st, stem of a brachium; t, tip of a brachium. Scale bars: 200 μm (A,B), 100 μm (C,D).
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