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Fig. 1. Sulf1 is co-expressed with Shh in the floor plate of the neural tube in Xenopus tropicalis and is required for normal Shh signalling. in situ hybridisation shows the normal expression pattern of Sulf1 (A,C,E, G, I, K) and Shh (B, D, F, H, J, L) at stages 15 (A–D) 23 (E–H), and 35 (I–L). Arrows in (A, B, E, F, I, J) indicate level where the vibratome sections were taken at these same stages and are shown in (C, D, G, H, K, L). Sulf1 is expressed in the paraxial mesoderm at Stage 15 (A, C) while Shh is expressed in the floor plate and notochord (B, D). By stage 23, both Sulf1 and Shh are expressed in the floor plate (E–H), and this co-expression is still apparent at stage 35 (I–L). Knockdown of Sulf1 using a splice blocking morpholino oligo shows a reduced level of ptc expression in the neural tube (N, P) as compared to those injected with an equal amount of a control morpholino (M, O). |
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Fig. 5. Sulf1 is required for the normal distribution of Shh protein in vivo. (A–D) Immunostaining embryos unilaterally injected with a control morpholino with 5E1reveals Shh protein in the notochord (NC) and the neural tube (NT), with highest levels in the floor plate (FP). In these control embryos the level of Shh protein in the neural tube drops off sharply away from the FP and is not detected within the dorsal neural tube (C). (E–H) Unilateral knockdown of Sulf1 in vivo leads to a change in the distribution of Shh protein on the injected side (*) where there is reduced Shh protein detected which is detected much further dorsally in a more diffuse pattern than in controls (compare G with C). (I) Quantification of the immunohistochemistry reveals a marked change in the diffusion of Shh away from its source in the absence of Sulf1 expression. The level of Shh in controls embryos (blue) immediately adjacent to Shh producing cells is high; this drops to almost zero within ~20 μm. In the absence of Sulf1(red), the level of ligand adjacent to producing cells is lower than in controls, but this level remains higher much further from the source, only dropping to zero ~50 μm from the source cells. Graph represents average grey level across the width of the neural tube on the injected side. Area measured for quantification shown. Mean values from a number of samples are shown (CMO n=5, AMO n=7). |
