Kevin P. Williams

Identification of a Palmitic Acid-modified Form of Human Sonic hedgehog

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24–197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the α-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Comparative biological responses to human Sonic, Indian, and Desert hedgehog.

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

A dual role for Sonic hedgehog in regulating adhesion and differentiation of neuroepithelial cells.

In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, we investigated whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface beta1-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promoted differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, our experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through different mechanisms, leading to a coordinated regulation of the various cell interactions essential for neural tube morphogenesis.