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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References
Johnson, R. L. & Tabin, C. The long and short of hedgehog signaling. Cell 81, 313–316 (1995).
McMahon, A. P. More surprises in the Hedgehog signaling pathway. Cell 100, 185–188 (2000).
Chuang, P. & Kornberg, T. B. On the range of hedgehog signaling. Curr. Opin. Genet. Dev. 10, 515–522 (2000).
Chen, Y. & Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell 87, 553–563 (1996).
Chuang, P. T. & McMahon, A. P. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397, 617–621 (1999).
Goodrich, L. V., Jung, D., Higgins, K. M. & Scott, M. P. Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube. Dev. Biol. 211, 323–334 (1999).
Nakamura, T. et al. Induction of osteogenic differentiation by hedgehog proteins. Biochem. Biophys. Res. Commun. 237, 465–469 (1997).
Pepinsky, R. B. et al. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998).
Bumcrot, D. A., Takada, R. & McMahon, A. P. Proteolytic processing yields two secreted forms of sonic hedgehog. Mol. Cell. Biol. 15, 2294–2303 (1995).
Porter, J. A. et al. The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature 374, 363–366 (1995).
Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).
Ericson, J., Morton, S., Kawakami, A., Roelink, H. & Jessell, T. M. Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 87, 661–673 (1996).
Cooper, M. K., Porter, J. A., Young, K. E. & Beachy, P. A. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280, 1603–1607 (1998).
Incardona, J. P., Gaffield, W., Kapur, R. P. & Roelink, H. The teratogenic Veratrum alkaloid cyclopamine inhibits Sonic hedgehog signal transduction. Development 125, 3553–3562 (1998).
Tickle, C. Morphogen gradients in vertebrate limb development. Semin. Cell Dev. Biol. 10, 345–351 (1999).
Yang, Y. et al. Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb. Development 124, 4393–4404 (1997).
Lopez-Martinez, A. et al. Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage. Curr. Biol. 5, 791–796 (1995).
Marti, E., Takada, R., Bumcrot, D. A., Sasaki, H. & McMahon, A. P. Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121, 2537–2547 (1995).
Wang, B., Fallon, J. F. & Beachy, P. A. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100, 423–434 (2000).
Riddle, R. D., Johnson, R. L., Laufer, E. & Tabin, C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401–1416 (1993).
Chang, D. T. et al. Products, genetic linkage and limb patterning activity of a murine hedgehog gene. Development 120, 3339–3353 (1994).
Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1–47 (1969).
Burke, R. et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell 99, 803–815 (1999).
The, I., Bellaiche, Y. & Perrimon, N. Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol. Cell 4, 633–639 (1999).
Guy, R. K. Inhibition of sonic hedgehog autoprocessing in cultured mammalian cells by sterol deprivation. Proc. Natl Acad. Sci. USA 97, 7307–7312 (2000).
Porter, J. A., Young, K. E. & Beachy, P. A. Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255–259 (1996).
Robbins, D. J. et al. Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein COSTAL2. Cell 90, 225–234 (1997).
Bell, S. M., Schreiner, C. M. & Scott, W. J. Transspecies grafting as a tool to understand the basis of murine developmental limb abnormalities. Methods Mol. Biol. 136, 219–226 (2000).
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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Supplementary Figure 5
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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
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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
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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
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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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DOI: https://doi.org/10.1038/35079648
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