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A freely diffusible form of Sonic hedgehog mediates long-range signalling

Abstract

The secreted protein Sonic hedgehog (Shh) exerts many of its patterning effects through a combination of short- and long-range signalling1,2,3. Three distinct mechanisms, which are not necessarily mutually exclusive, have been proposed to account for the long-range effects of Shh: simple diffusion of Shh, a relay mechanism in which Shh activates secondary signals, and direct delivery of Shh through cytoplasmic extensions, termed cytonemes. Although there is much data (using soluble recombinant Shh (ShhN)) to support the simple diffusion model of long-range Shh signalling1,2, there has been little evidence to date for a native form of Shh that is freely diffusible and not membrane-associated. Here we provide evidence for a freely diffusible form of Shh (s-ShhNp) that is cholesterol modified, multimeric and biologically potent. We further demonstrate that the availability of s-ShhNp is regulated by two functional antagonists of the Shh pathway, Patched (Ptc) and Hedgehog-interacting protein (Hip)4,5,6. Finally, we show a gradient of s-ShhNp across the anterior–posterior axis of the chick limb, demonstrating the physiological relevance of s-ShhNp.

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Figure 1: Identification of a soluble form of Shh.
Figure 2: Soluble Shh activity is inhibited by Shh functional antagonists.
Figure 3: -ShhNp is a highly potent, multimeric form of ShhNp.
Figure 4: -ShhNp exists in an anterior–posterior gradient.

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Acknowledgements

We thank the members of the Robbins' laboratory and A. J. Capobianco, Y. Sanchez, T. Doetschman, L. A. Woollett, S. M. Bell, X. Lin and K. E. Yutzey for discussions. D.J.R. is a recipient of a Burroughs Wellcome Career Development Award.This work was supported by Grants from the National Institutes of Health.

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Correspondence to David J. Robbins.

Supplementary information

Supplementary Figure 5

(JPG 34 KB)

Soluble Shh activity is inhibited by two mechanistically distinct inhibitors of Shh signaling. a, Cyclopamine (2.4 µM) inhibits s-ShhNp induced AP activity in C3H10T1/2 cells and does not inhibit BMP4 or Gli1 activity (data not shown). b, Shh conditioned media induces a Gli-luciferase reporter in Shh-Light2 cells. Confluent Shh-Light2 cells were incubated with Shh conditioned media or control media, produced under serum free conditions, for 48 hrs in the presence or absence of the inhibitory mAb 5E1 (1 µg/ml).

SupplementaryFigure 6

(JPG 18 KB)

Shh is lipid modified. a, s-ShhNp was immunoprecipitated using the 5E1 mAb, then treated with 50 mM KOH/95% methanol for 10 min at 37 ËšC to remove any attached cholesterol. Treatment resulted in a shift in the mobility of s-ShhNp, upon SDS-PAGE followed by immunoblotting (compare lane 3 and lane 4), in which s-ShhNp now migrates like the unmodified ShhN (lane 1). Lane 2 shows ShhNp immunoprecipitated from the lysate of Shh transfected cells. b, 293T cells were incubated with [3H]-cholesterol in serum-free Optimem (Gibco) for 24 hrs before and 48 hrs after transfection. Shh was immunoprecipitated from the cell lysate, or conditioned media collected from 293T cells transfected with Shh or a vector control. Tritium labeled Shh was detected the cell lysate and conditioned media from Shh transfected cells. s-ShhNp appears to be a cholesterol modified form of Shh, which is freely diffusible and highly potent. To verify that s-ShhNp was also palmitoylated we mutated the palmitoyl acceptor site of Shh (ShhC25-A) to examine its effect on Shh activity and solubility. If s-ShhNp was palmitoylated, we anticipated that ShhC25-A would produce soluble Shh with reduced activity, which is what we observed (data not shown). Thus, we suggest that this soluble form of Shh contains both of the lipid modifications previously thought to keep Shh membrane associated, and suggest a model to account for the solubility of this s-ShhNp (see Fig 4).

Supplemenatry Figure 7

(JPG 152 KB)

The posterior region of chick limb buds produces s-ShhNp. The posterior 1/3 fragment of chick limb buds were cultured in a 24-well plate to allow s-ShhNp to diffuse out of the limb tissue. Media was collected after 16-24 hrs, and added to C3H10T1/2 cells in the presence or absence of 5E1 mAb (1 µg/ml) (left panel) or cyclopamine (2.4 µM) (right panel). AP activity was determined by histochemical staining.

Supplementary Figure 8

(JPG 122 KB)

Evidence for a s-ShhNp anterior-posterior gradient across the limb bud. The anterior 1/3 and posterior 1/3 fragments of chick limb buds were cultured separately in a 24-well plate. Conditioned media was collected after 16-24 hrs, then added to C3H10T1/2 cells. AP activity was determined five days later by a, histochemical staining or b, a liquid AP assay. The data shown in a and b were generated from different experiments. Shh-N conditioned media or control media, from Bosc cells, were used as the positive and negative controls.

In this figure anterior conditioned media has greater AP activity than that incubated with 5E1, which is similar to our current Fig 4b. However, in this experiment media not incubated with anterior tissue gave similar AP activity to that of anterior tissue conditioned media inhibited with the 5E1 mAb. If the activity seen from anterior tissue was due to endogenous Shh from C3H10T1/2 cells it should be the same as conditioned media collected in the absence of anterior tissue, and it is not. Additionally, anterior tissue had 4-5 times more activity than conditioned media made in the absence of limb tissue using a more quantitative liquid AP assay.

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Zeng, X., Goetz, J., Suber, L. et al. A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411, 716–720 (2001). https://doi.org/10.1038/35079648

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