Fig. 4

Localization of RTE-2_OV mRNA by in situ hybridization. a Bright-field micrographs of the coronal sections of the supraesophageal (SEM) and subesophageal (SUB) masses hybridized to digoxigenin-labeled LINE antisense (refer to the plane indicated by the dashed lines in b). The five gyri of the vertical lobe (dorsal in SEM) appear positively marked by the RTE-2_OV mRNA. Several cells in the cortical layers of the SUB appear also stained after in situ hybridization. Neuropils of SEM and SUB do not show a signal. b Schematic outline of the parts of the octopus brain for SEM and SUB (sagittal plane) and the optic lobe (OL; horizontal plane). Axes illustrating dorso-ventral and antero-posterior (SEM and SUB) and antero-posterior and left-right (OL) orientations with respect to the octopus body plan. Black letters indicate approximate levels of the sections provided in the other panels of the figure. c Detail of a gyrus of the vertical lobe (SEM) with densely packed amacrine cells showing a positive signal. d A similar signal in the gyri of the vertical lobe and some scattered positive cells in the sub-vertical lobe. e Section at the level of the sub-frontal lobe with densely packed amacrine small cells showing a strong positive signal. In the SUB, we observed f positive cells (20–25 μm in diameter) in the pallovisceral lobe and some larger neurons (40–50 μm) belonging to typical motor-center cellular types. g Cells (20–25 μm) belonging to the ventral side of the anterior pedal lobe and at the level of the dorsal brachial lobe (h) where some larger cells (up to 50 μm) are also marked after ISH. The small cells pertaining to these areas do not show positivity. Details of horizontal sections of the O. vulgaris optic lobe (i, j, k; in areas indicated in b): i Outer layer rich in intensely positive cells (small amacrine cells, < 5 μm), j inner medulla with scattered LINE mRNA-expressing cell bodies (up to 10 μm), and k cell bodies of the peduncle complex at the level of the median and posterior lobules of the olfactory lobe (cells of about 10 μm). Scale bars, 100 μm and 500 μm in a. Schematic drawings in b not to scale