See-Star: a versatile hydrogel-based protocol for clearing large, opaque and calcified marine invertebrates.

Studies of morphology and developmental patterning in adult stages of many invertebrates are hindered by opaque structures, such as shells, skeletal elements, and pigment granules that block or refract light and necessitate sectioning for observation of internal features. An inherent challenge in studies relying on surgical approaches is that cutting tissue is semi-destructive, and delicate structures, such as axonal processes within neural networks, are computationally challenging to reconstruct once disrupted. To address this problem, we developed See-Star, a hydrogel-based tissue clearing protocol to render the bodies of opaque and calcified invertebrates optically transparent while preserving their anatomy in an unperturbed state, facilitating molecular labeling and observation of intact organ systems. The resulting protocol can clear large (> 1 cm3) specimens to enable deep-tissue imaging, and is compatible with molecular techniques, such as immunohistochemistry and in situ hybridization to visualize protein and mRNA localization. To test the utility of this method, we performed a whole-mount imaging study of intact nervous systems in juvenile echinoderms and molluscs and demonstrate that See-Star allows for comparative studies to be extended far into development, facilitating insights into the anatomy of juveniles and adults that are usually not amenable to whole-mount imaging.

Molecular evidence of anteroposterior patterning in adult echinoderms.

The origin of the pentaradial body plan of echinoderms from a bilateral ancestor is one of the most enduring zoological puzzles1,2. Because echinoderms are defined by morphological novelty, even the most basic axial comparisons with their bilaterian relatives are problematic. To revisit this classical question, we used conserved anteroposterior axial molecular markers to determine whether the highly derived adult body plan of echinoderms masks underlying patterning similarities with other deuterostomes. We investigated the expression of a suite of conserved transcription factors with well-established roles in the establishment of anteroposterior polarity in deuterostomes3-5 and other bilaterians6-8 using RNA tomography and in situ hybridization in the sea star Patiria miniata. The relative spatial expression of these markers in P. miniata ambulacral ectoderm shows similarity with other deuterostomes, with the midline of each ray representing the most anterior territory and the most lateral parts exhibiting a more posterior identity. Strikingly, there is no ectodermal territory in the sea star that expresses the characteristic bilaterian trunk genetic patterning programme. This finding suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals and provides a developmental rationale for the re-evaluation of the events that led to the evolution of the derived adult body plan of echinoderms.