Suppl. Fig. 7. from A feather star is born: embryonic development and nervous system organization in the crinoid Antedon mediterranea

Morphological and molecular characterization of crinoid metamorphosis. Confocal projections showing localization of phosphorylated Smad1/5/8 (red) (A, B, E), acetylated α-tubulin (D, G), and glutamate (G) immunoreactivity and expression of Ame_Six3/6 (yellow) (A, B, E) and Ame_Lhx2/9 (red) (C, F) in doliolaria (A), settled (B-C), cystidean (D-F), and pentacrinoid (G) stages. A) At the doliolaria stage, Ame_Six3/6 marks the apical portion of the ectoderm as well as the axocoel and its anterior projection, which is located outside of the future stalk ossicles in the mesenchyme. B) After settlement, ectodermal expression of Ame_Six3/6 is lost, while Ame_Six3/6 remains expressed in the axocoel and its projection. C) At this stage, Ame_Lhx2/9 is also lost from the ectoderm, but its expression remains in the hydrocoel, which already shows a pentaradial organization. D) During metamorphosis, most of the organs undergo a 90° rotation, so that, at the cystidean stage, the vestibule and the hydrocoel are localized below each other on the oral surface. Five sets of three projections subsequently direct orally from the hydrocoel and push on the vestibular ectoderm, forming the first 15 tube feet. As the hydrocoel is highly ciliated, these projections are visible by acetylated α-tubulin staining. Moreover, the somatocoels are also ciliated and can be visualized surrounding the enteric sac and elongating into the stalk in the center, surrounded by the developing stalk nerve. E) At this stage, Ame_Six3/6 is also expressed in the center of the stalk, indicating that it starts to be expressed in the right somatocoel. This domain is different from the previous axocoelic one, as it is found within the ossicles. F) In the medial (i.e., closer to the center) wall of the hydrocoel, at the base but not the tip of the forming tube feet, strong Ame_Lhx2/9 expression is detectable. Both Ame_Six3/6 and Ame_Lhx2/9 are highly expressed on the oral surface of the vestibule, at the level of the external surface of the tube feet (E-F). G) Additional details of glutamatergic neuron distribution in the pentacrinoid. In the tube feet, glutamatergic cells are found in the ectoderm that surrounds the ciliated hydrocoel and in the papillae from which short cilia emerge. Moreover, sparse glutamatergic cells are found in the stalk nerve. Legend: ac = axocoel; acp = axocoel projection; adp = adhesive pit; hc = hydrocoel; pa = papillae; rsc: right somatocoel; sn = stalk nerve; tfh = tube feet hydrocoel. Scale bar: 50µm.