Formation of the octopus arm crown and development of the arm buds. (A to F) Confocal projections of arm crowns labeled with TO-PRO-3 to visualize the nuclei (cyan). Embryos are oriented in a lateral view, with anterior to the left. (A′ to F′) Sagittal histological sections through individual arms stained with toluidine blue (A′ to D′) and Masson’s trichrome stain (E′ to F′). (A″ to F″) Merged image stacks of median aboral (A″ to D″), sagittal (E)″ and parasagittal (F″) sections of individual arms stained with an antibody against PCNA to visualize proliferating cell nuclei (red) and TO-PRO-3 to highlight cell nuclei (cyan). Inset in (F″) shows a close-up of the entire confocal stack of the arm’s tip. Individual arm buds are oriented with proximal to the left. White arrows in (B) to (C) point at tissue contributing to the eye lid; white arrowheads in (E) to (F) denote the velar webs. Black open arrowhead in (B′) points out the location of a cluster of darker stained, denser cells and in (D′) denotes the basal cells of the future Kölliker’s organ. Dotted lines are indicating the outline of the forming axial nerve cord in (C′) to (D′); asterisk marks the general area of the axial nerve cord. Arrows are pointing at longitudinal muscle fibers; closed arrow heads are marking transverse muscle fibers in (E′) to (F′) and (E″) to (F″). Abbreviations: cm, cell mass; d, dermis; ep, epithelium; e, eye; gza, growth zone of the arm; Ko, Kölliker’s organ; mu, muscle layer; nc, neuronal cells; s, sucker; st, statocyst. Scale bars: A (refers to A to F): 100 μm; A′ (refers to A′, B′ and A″, B″), C′ (refers to D′ to F′ and D″ to F″): 50 μm.