Fig. 5

Late growth of the amphioxus brain. a–f Proliferation patterns in the brain throughout early and late development assessed by PHH3 (red) immunohistochemistry in whole mount preparations. PHH3 is first detected in the brain of amphioxus at the mid-neurula stage (8 somites: 8S) (a: dorsal view). Thereafter, PHH3 persists in the ventricular zone of the growing brain as shown in 1GS embryos (b: dorsal view), in 3-month-old embryos (5GS) (c: dorsal view and d: lateral view) and in pre-metamorphic embryos (10GS) (e: dorsal view and f: lateral view). At all stages, mitotic cells (PHH3+) are localised in the ventricular surface (outlined in e and f) and only post-mitotic cells (Edu: white labelling in c and d) are pushed outwards away from the ventricle. Arrowheads in c and d indicate Edu+ cells that divided in the ventricle and migrated basally. Occasionally, it is possible to observe the apical-basal translocation of daughter cells, as indicated by an arrow in the inset in e. g Lateral view of the ventricular system of the adult brain showing the presence of at least three ventricles. The image is taken from a whole brain stained and clarified using confocal microscopy. Staining in grey shows the nuclei of the brain cells. Staining for acetylated tubulin in green reveals ventral neuropil and the ciliated lumen of the ventricles. h Frontal sections of amphioxus brains at the levels indicated in the rest of the panels, showing the growth and development of the ventricular system. The dorsal side of the brain begins thickening at pre-metamorphic stages (10GS) and continues to grow to show a very different morphology in adults. Colour code: blue, brain nuclei; green, neuropil and intraventricular cilia (acetylated tubulin). All scale bars are 20μm, except in h that are 10 μm. Abbreviations: 8S 8 somites neurula, 1GS one-gill-slit embryo, 5GS five-gill-slit larva (3 months old), 9GS nine-gill-slit larva (pre-metamorphic), 10GS ten-gill-slit larva (pre-metamorphic), DF dorsal fin, IO infundibular organ, N notochord