Fig. 7

Involvement of the N-sulfo-rich HS clusters in Wnt8 internalization. a Time-lapse imaging of internalization of mV-Wnt8 puncta. Subapical region (basal to the tight junction) of the superficial cell of the Xenopus gastrula (st. 11.5) was observed. In the vicinity of mV-Wnt8 expressing cells (asterisk), puncta at the cell boundary (white and yellow arrowheads) and inside cells (orange arrowheads) were observed. The time point of each picture was as indicated. A budding and subsequently internalized punctum (tracked by a cyan arrowhead) was generated from a punctum (yellow arrowhead) on the cell membrane. See also Supplementary Movie 1. b Colocalization of internalized puncta of Wnt8 with N-sulfo-rich HS clusters. Wnt8-Myc expressing cells were as indicated (*), and the others were receiving cells. The puncta of Wnt8-Myc inside cells near Wnt8-Myc expressing cells are considered to be internalized, because the subapical region (basal to the tight junction) but not the apical cell surface was observed (arrowheads or open arrowheads). Upper panels, co-immunostaining of Wnt8-Myc and N-acetyl HS. Staining of N-acetyl HS was absent from internalized puncta of Wnt8-Myc (open arrowheads). Lower panels, co-immunostaining of Wnt8-Myc and N-sulfo HS. Colocalization of N-sulfo-rich HS clusters and Wnt8-Myc inside cells (arrowheads) was observed, suggesting involvement of N-sulfo-rich HS cluster in Wnt8 internalization. Images are a representative of at least two independent experiments. Amounts of injected mRNA (ng/embryo): mV-wnt8, 1.0; wnt8-myc, 0.25. Scale bars, 10 μm (a); 20 μm (b)