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Fig. 3 | Neural Development

Fig. 3

From: Netrin-1 directs dendritic growth and connectivity of vertebrate central neurons in vivo

Fig. 3

Protein diffusion after treatment. a Schematic of coronal view of stage 45 Xenopus retinotectal circuit depicting injection sites (red arrows) and spread of injected proteins (violet color). b Coronal section at the level of the optic tectum immunostained with antibodies to netrin-1. Note endogenous netrin immunoreactivity in cell body layer and neuropil. c–g Sections at the level of the optic tectum of tadpoles injected with vehicle, recombinant netrin-1, UNC5H2-Ig, or anti-DCC were immunostained to examine the spread of the injected proteins after treatment. c Quantitative analysis of fluorescence intensity in sections of uninjected tadpoles (Endogenous Netrin) or tadpoles injected with recombinant netrin (Injected rNetrin-1). The relative levels of netrin within the cell body layer and the neuropil are illustrated by the average pixel intensity values along the medial-to-lateral axis of the tectum. The zero value in the X-axis corresponds to the cell body layer-neuropil boundary; negative X-coordinates represent distance from the boundary to the ventricle while positive X-coordinates represent distance from the boundary to the lateral-most neuropil. n = 10 brain sections per group, from four tadpole brains per group, with three 20-pixel-wide line scans quantified per section. Error bars represent the standard error of the mean. d–g Sample coronal sections of tadpoles injected with vehicle (d), recombinant netrin-1 (e), UNC5H2-Ig (f), or anti-DCC (g) immunostained with chick antibodies to netrin-1 and Alexa 488 secondary antibodies to chick IgG (top; d, e) or stained with Alexa 488 secondary antibodies to human IgG (top; f) or mouse IgG (top; g). The pseudo-color images in d–g (bottom) show the relative intensity of the Alexa fluor 488 fluorescence. Pixel intensity values ranged from 0 (black) to 255 (white) as illustrated by the color-scale bar (d, bottom). Note the increased immunofluorescence in the cell body layer and neuropil of netrin-1-treated tadpoles (e) when compared to vehicle-injected controls (d) and with endogenous netrin-1 expression (b). In f and g, the relatively higher fluorescence intensity in the hemisphere that received the injection (red arrows) and the diffusion patterns of the proteins are more evident in the pseudo-color images. In g, white arrows point to fluorescently labeled cells in the injected tectal hemisphere. Scale bars in b, d–g: 50 μm

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