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Fig. 4 Polysialic acid (PSA) degradation in the central nervous system of EndoN-treated embryos impairs migration from the cerebellar rhombic lip. All panels show lateral views, anterior is to the left. Panels A-D show PSA expression in cerebellar primordia after phosphate buffered saline (PBS) and EndoN treatment at 33 hours postfertilization (hpf). A,B: Control embryos injected with PBS show PSA immunoreactivity after 2 hr (A) and 24 hr (B). Note the weaker expression levels at 57 hpf are due to endogenous down-regulation of PSA. C,D: EndoN injection at 33 hpf completely abolished PSA expression already after 2 hr (C), lasting up to 24 hr (D). Panels E-J are maximum intensity projections (50-μm stacks recorded every 12 min) showing migrating neuronal progenitors in the differentiating cerebellum after PBS (E-G) and EndoN treatment (H-J), recorded by in vivo time-lapse confocal microscopy. E: A group of neuronal progenitor cells at the upper rhombic lip (URL) in the control embryo is marked by a white arrowhead 2 hr post-injection. F: These cells migrated approximately halfway between URL and midbrain-hindbrain boundary (MHB) after 6 hr, while others have reached the MHB (white arrowhead). G: After 11 hr, cells in the ventral cerebellar region become stationary and form a cluster (white arrowhead, see Supplementary Movie S1). H: After injection of EndoN into the hindbrain ventricle, neuronal progenitors marked by an arrowhead are positioned at the URL or are polarized and span across the cerebellum between the URL and MHB (white arrowheads). I,J: Eight hours later (I), these neuronal progenitor cells are stalled at the URL and remain in this position (J) for up to 17 hr of time-lapse recording (see Supplementary Movie S2). K,L: The migratory routes of five individual uncGFP-expressing neuronal progenitor cells in PBS (K) and EndoN (L) -treated embryos were traced using the manual tracking tool plug-in of ImageJ1.34, the migratory direction is marked by an arrowhead. K: Anteroventral migration of cells in a PBS-injected embryo. L: Reduced migration of cells in EndoN-injected embryo. M: Quantification of neuronal progenitor migration from the cerebellar rhombic lip in PBS- and EndoN-treated embryos. While 84% of neuronal progenitors (n = 5 movies, 37 cells analyzed) in PBS-treated embryos were migratory, covering an average distance of almost 30μm during a 4-hr period, only 23% of neuronal progenitors (n = 5 movies, 47 cells analyzed) in EndoN-treated embryos were migratory and covered a similar average migration distance. Cells were classified as nonmigratory when they moved less than one cell diameter (approximately 10μm) during the observation period.