Journal: Nature Communications
Article Title: Roles of two types of heparan sulfate clusters in Wnt distribution and signaling in Xenopus
Figure Lengend Snippet: Endogenous Wnt8 shows not only a gradient but also HS-dependent punctate distributions. a The gradient of the endogenous Wnt8 protein from the lateral to mid-dorsal marginal zones at the mid-gastrula stage (st. 11.5). The observed region is indicated by the cyan box (reported localization of wnt8 mRNA in magenta). Embryos were flat-mounted under a coverslip and the image was acquired using automatic tiling with the maximum intensity projection of z -stacks (junctions of tiling appeared darker, arrowheads). b Quantification of the gradient of Wnt8 staining ( a ). The junctions of tiling are indicated by dotted lines, showing the drop in intensity. c A high-resolution image of endogenous Wnt8 staining in the VMZ. wnt8 is expressed in the underlying mesoderm, but not in the superficial layer in Xenopus embryos. An optical section at the subapical region of cells (basal to the tight junction) of the superficial layer is shown (see also Supplementary Fig. 2b, d ). d Exogenous mV-Wnt8 expression and heparitinase treatment. Experimental procedures are illustrated on the left. Embryos were observed at stage 10.5. The source cells are indicated with “*”. e Immunostaining of HS chains with NAH46 (for N- acetyl HS) or HepSS-1 (for N- sulfo HS). Notably HepSS-1 staining shows puncta inside cells (orange arrowheads). f – k The colocalization of endogenous Wnt8 and a subpopulation of HS. Gastrula embryos (st. 10.5) were co-immunostained for Wnt8 and NAH46 epitope ( N- acetyl HS) or HepSS-1 epitope ( N- sulfo HS) at the VMZ ( f , i ). Signal intensities along white arrows were plotted ( g , j ), starting and ending points as indicated by yellow and cyan arrowheads, respectively ( f , i ). Scatter plots show the indicated signal intensities for every pixel along the arrow, presented with correlation coefficients ( r ) ( h , k ). Note N- sulfo-rich HS clusters inside cells, with Wnt8 (orange arrowheads) and without Wnt8 (white arrowheads). Staining of N- acetyl HS was absent from the Wnt8 puncta inside cells (open arrowheads in f ). Images are a representative of at least two independent experiments. Amounts of mRNA (ng/embryo): mV-wnt8 , 1.0; mRFP , 0.50. Scale bars, 100 μm ( a ); 10 μm ( c – f , i ). a.u., arbitrary units
Article Snippet: Heparitinase (25 μU, #100703, Seikagaku Biobusiness, a mixture of heparitinase I and II at 4:1 ratio) was microinjected into the blastocoel of embryos at stage 6.5 (48 cells) .
Techniques: Staining, Expressing, Immunostaining