原位杂交结合半薄切片对石鳖贝壳发育相关细胞的观察研究OA北大核心CSTPCD

Polyplacophoran mollusks(chiton)have eight serially arranged shell plates,which have important de-velopmental and evolutionary implications and are considerably different from those in other molluscan lineages,such as gastropods and bivalves.Previous studies have revealed that shell-formation cells of Acanthochitona rubrolineata are irregularly arranged,making it difficult to discern single cells under ordinary microscopy.In this study,we initially investigated shell-formation tissues through in situ hybridization.Engrailed-positive cells were observed in the ridges of the central and marginal regions of the shell field.Expressions in the ridges exhibited a striped pattern,and the expression in the marginal region restricted the periphery of the shell field.However,the morphology and arrangement patterns of these cells could not be clearly discerned.Moreover,larval cells could be better recognized in semithin sections,which was still limited.Therefore,we combined semithin sectioning and in situ hybridization to further improve cellular resolution.Semithin sectioning of the larvae after in situ hybridization revealed intact tissue morphology with discernable cell outlines,and the engrailed-positive cells could be clearly distinguished.These cells had high nucleus-to-cytoplasm ratios and exhibited an overall V-shaped arrangement pattern.Additionally,the engrailed-positive cells in the central and marginal regions of the shell field exhibited different arrangement patterns and cell shapes.These results demonstrated that combining in situ hybridization and semithin sectioning could improve the resolution at the cellular level.Thus,the proposed strategy will help investi-gate the cellular mechanisms underlying chiton shell formation and the roles of shell-formation genes.Moreover,the proposed strategy can help further elucidate the origin and evolution of molluscan shells.