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Figure 1.
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Antedon mediterranea embryogenesis on parental pinnules and in vitro. Embryos attached to adult genital pinnules (a, d and g) develop synchronously with those maintained in vitro (b,c,e,f,h-l). (a–c) 4-cell stage; (d–f) gastrula stage (24 hpf); (g–i) pre-hatching larva (72 hpf); (j–l) hatching (100 hpf) (k) and swimming doliolaria larva (j,l). * indicates ciliary band; arrowhead, apical tuft; adp, adhesive pit; v, vestibulum. Scale bar in (a) = 100 µm (same for d,g); scale bar in (b) = 100 µm (same for e,h,k); scale bar in (c) = 50 µm (applies to f,i,l); scale bar in (j) = 500 µm.
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Figure 2.
. Developmental timeline of Antedon mediterranea embryogenesis. Graphical representation of A. mediterranea development at 17 ± 1°C: zygote stage (fertilization to 2 hpf), 2-cell stage (2–2.5 hpf), 4-cell stage (2.5–3.5 hpf), eight-cell stage (3.5–4 hpf), 16-cell stage (4–5 hpf), 32-cell stage (5–6 hpf), blastula stage (6–9 hpf), gastrula (9–36 hpf), uniformly ciliated stage (36–48 hpf), band formation stage (48–72 hpf), pre-hatching doliolaria stage (72–100 hpf) and swimming doliolaria stage (≥100 hpf).
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Figure 3.
. Morphological characterization of Antedon mediterranea embryogenesis. (a–p) Confocal z-projections of embryos labelled with phalloidin during the cleavage period: (a) zygote (1 hpf); (b) 2-cell stage (2 hpf); (c,d) 4-cell stage (2.5 hpf), in lateral (c) and frontal view (d); (e,f) 8-cell stage (3.5 hpf), in lateral view with animal pole on the top (e) and animal view (f); (g) 16-cell stage (4 hpf), in animal view; (h) 24-cell stage (4.5 hpf), in lateral view with animal pole on the top; (i,j) the two sides of a 32-cell stage (5 hpf); (k, l) the two sides of a 48-cell stage (5.5 hpf); (m,n) the two sides of a 64-cell stage (6 hpf); (o) mid-sagittal optical section of a blastula stage (6.5 hpf), showing the internal cavity; (p) initial gastrula (9 hpf). (q–t) Light microscopy of A. mediterranea embryos undergoing gastrulation: (q) sagittal section of an initial gastrula (9 hpf) with elongating cells at the vegetal pole (outlined with dashed lines); (r) sagittal section of a mid-gastrula (20 hpf), with asterisk indicating the blastopore at the vegetal pole; (s) late gastrula (36 hpf), the blastopore is closed and the archenteron is narrow (outlined with dotted line); (t) ciliated larva (48 hpf) with coelomic cavities. a, archenteron; bc, blastocoel; cc, coelomic cavities; e, ectoderm; em, ento-mesoderm; m, mesoderm. Scale bar in (a) = 100 µm (applies to all images).
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Figure 4.
. External and internal ciliogenesis in Antedon mediterranea embryos. Confocal z-projections of embryos immunolabelled with anti-β-tubulin antibodies (a–d) (green) or anti-acetylated α-tubulin antibodies (e–l) (green) and co-stained with 4′,6-diamidino-2-phenylindole (DAPI) (nuclei, grey). Maximum projections (a–d,g,i,j) and longitudinal sections (e,f,h,k,l) of representative samples. Anti-β-tubulin antibodies marked developing cilia in late gastrula (a), uniformly ciliated larva (b), band formation (c) and swimming doliolaria (d) stages. (a–d) are ventral views. Asterisks and lines in (c) indicate band and interband cells, respectively. Cilia in the ectoderm and in coelomic cavities were also immunoreactive to anti-acetylated α-tubulin in gastrula (e), uniformly ciliated larva (f), pre-hatching larva (g) and swimming doliolaria (i–k); (e,g,i) are dorsal views, (h,k) are longitudinal views and (j) is a ventral view. Magnification of a ciliated band in a swimming doliolaria larva is shown in (l); cilia departing from inner cells are indicated (arrowheads). ac, axocoel; acp, axocoel projection; adp, adhesive pit; at, apical tuft; ehc, enterohydrocoel; hc, hydrocoel; hp, hydropore; lsc, left somatocoel; rsc, right somatocoel; sc, somatocoel; scp, somatocoel projection; v, vestibulum. Scale bars in (a,d,e,g) = 50 µm (apply to a–k); scale bar in (l) = 10 µm.
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Figure 5.
. Ossicle development in Antedon mediterranea. Confocal z-projections of specimens immunolabelled with antibodies against phosphorylated Smad1/5/8 (pSmad1/5/8) (yellow) and acetylated α-tubulin (green) and co-stained with DAPI (nuclei, grey). Maximum projections (a–d) and mid-sagittal sections (e–h) of pre-hatching doliolaria larvae, early (a,e) and late (b,f) stage, swimming doliolaria larvae (c,g) and of specimens at the cystidean stage (d,h). At all stages considered, the pSmad1/5/8 antibodies labelled the developing ossicles of the crinoid skeleton. ad, attachment disk; bp, basal plate; col, columnar ossicles; op, oral plate; sp, skeletal plates. Scale bar in (a) = 100 µm (applies to all images).
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Figure 6.
. Development and cell type composition of the larval nervous system of Antedon mediterranea. Confocal z-projections of immunolabelled embryos and larvae showing axonal development (a–e) and neurotransmitter distribution (f–k) at several developmental stages. In most panels, nuclei are co-stained with DAPI (grey). While neuron projections are not detected at 36 hpf (late gastrula) (a) anti-β-tubulin antibodies (green) marked growing neurites at uniformly ciliated (b), band formation (c) and swimming doliolaria (d,e) stage. (d) is a longitudinal and (e) a dorsal view. In (c,d) the apical neural plexus is indicated with arrowheads. Serotonin (green) is localized in the anterior-dorsal apical organ and in dorsal and lateral neurites in swimming doliolaria larvae (f, g). (f) is a dorsal and (g) a lateral view. Apical organ cells are bottle-shaped, project into the anterior plexus (g(i)), and are concentrated dorsally (g(ii)). Double arrowheads in (g(i)) indicate a single bottle-shaped cell. GABA immunoreactivity (red) was detected anteriorly in swimming doliolaria larvae (h) (ventral view) in bottle-shaped cells that project locally into the apical plexus (h(i)) and were located ventrally around the adhesive pit (h(ii)). Double immunostaining for glutamate (orange) and acetylated α-tubulin (green) shows glutamatergic neurons and fibres anteriorly and along the body of the swimming doliolaria larvae. (i) is a dorsal, (j) a ventral and (k) a lateral view. Arrows indicate fibres and cells of the basiepithelial neural plexus. Antero-dorsally, large cells are arranged around the apical tuft (k(i)–k(iii)), while, ventrally, small cells are organized around the adhesive pit (k(iv)). adp, adhesive pit; at, apical tuft; D, dorsal; np, neural plexus; V, ventral; v, vestibulum. Scale bars in (a,d,f) = 50 µm (apply to all images).
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Figure 7.
. Molecular patterning of the apical region during Antedon mediterranea development. Confocal z-projections showing co-expression of Ame_FoxQ2 (cyan), Ame_Six3/6 (yellow) and Ame_Lhx2/9 (red) during crinoid development using in situ HCR. All specimens were co-stained with DAPI (nuclei, grey). Ame_Six3/6 and Ame_FoxQ2 are co-expressed in the anterior/apical portion of the ectoderm at 20 hpf (early gastrula) (a), and 26 hpf (late gastrula) (b), while starting at the uniformly ciliated stage (c,d) (showing, respectively, a longitudinal and a dorsal view, the latter at the level of the dashed line in (c)) expression of the genes form two concentric domains that persist in the pre-hatching (e) and doliolaria (f–h) stages. At larval stage, Ame_Lhx2/9 is co-expressed with both Ame_FoxQ2 (g) and Ame_Six3/6 (h). Scale bar in (a) = 50 µm (applies to all images).
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Figure 8.
. Formation of the post-metamorphic nervous system in Antedon mediterranea. Confocal z-projections of specimens immunolabelled with anti-β-tubulin (green) antibodies (a,d,g,j), or anti-glutamate (orange) and anti-acetylated α-tubulin (green) antibodies (i,l), or labelled with probes against Ame_Six3/6 (yellow) (b,e,h,k) and Ame_Lhx2/9 (red) (c,f). All specimens are co-stained with DAPI (nuclei, grey). Signal for all genes disappears in the apical region after settlement (a–c). At the cystidean stage (d–f), the post-metamorphic nervous system forms: neurites are labelled with β-tubulin (d) and the tube feet ectoderm starts to express Ame_Six3/6 (e) and Ame_Lhx2/9 (f). Transverse sections for each image are shown at the level indicated by the dashed lines. At the pentacrinoid stage (g-l), β-tubulin marks neural fibres in the stalk nerve, radial nerve primordia, circumoral nerve ring and tube feet, including the papillae (g,j); Ame_Six3/6 is expressed on the oral side of the tube feet ectoderm (h,k); glutamate is localized in tube feet, neurons of the epidermal plexus, aboral nerve centre and stalk nerve (i,l), while acetylated α-tubulin shows cilia in the hydrocoel of the tube feet, the somatocoel and the intestine (i). Panels (j), (k) and (l) show specific portions of the pentacrinoid indicated by dashed lines/boxes in, respectively, (g), (h), and (i). ac, axocoel; adp, adhesive pit; anc, aboral nerve centre; bnp, brachial nerve primordia; conr, circumoral nerve ring; hc, hydrocoel; i, intestine; pa, papillae; sn, stalk nerve; tf, tube feet; v, vestibulum. Scale bars in (a,d,g) = 50 µm (applies to all images).
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Figure 9.
. Comparison of echinoderm larval nervous system development. (a) Expression data and neural populations in the swimming doliolaria larva (lateral view). (b) Comparison of expression of FoxQ2 and Six3/6 across echinoderms. 5HT: serotonin; GABA: γ-aminobutyric acid; Glut: glutamate; SALMFa: SALMFamide neuropeptide.
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