David A. Bumcrot

Proteolytic processing yields two secreted forms of sonic hedgehog.

Sonic hedgehog (Shh) is expressed in tissues with known signalling capacities, such as the notochord, the floor plate of the central nervous system, and the zone of polarizing activity in the limb. Several lines of evidence indicate that Shh is involved in floor plate induction, somite patterning, and regulation of anterior-posterior polarity in the vertebrate limb. In this report, we investigate the biochemical behavior of Shh in a variety of expression systems and embryonic tissues. Expression of mouse Shh in Xenopus oocytes, COS cells, and baculovirus-infected insect cells demonstrates that in addition to signal peptide cleavage and N-linked glycosylation, chicken and mouse Shh proteins undergo additional proteolytic processing to yield two peptides with molecular masses of approximately 19 kDa (amino terminus) and 27 kDa (carboxy terminus), both of which are secreted. In transfected COS cells, we show that the 19-kDa peptide does not accumulate significantly in the medium unless heparin or suramin is added, suggesting that this peptide associates with the cell surface or extracellular matrix. This retention appears to depend on sequences in the carboxy-terminal part of the peptide. Finally, detection of the 19-kDa product in a variety of mouse and chicken embryonic tissues demonstrates that the proteolytic processing observed in cell culture is a normal aspect of Shh processing in embryonic development. These results raise the possibility that amino- and carboxyl-terminal regions of Shh may have distinct functions in regulating cell-cell interactions in the vertebrate embryo.

Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein

Loss of substantia nigra dopaminergic neurons, which develop from the ventral region of the midbrain, is associated with Parkinsons disease. During embryogenesis, induction of these and other ventral neurons is influenced by interactions with the underlying mesoderm of the notochord and the floor plate, which lies at the ventral midline of the developing CNS. Sonic hedgehog encodes a secreted peptide, which is expressed in notochord and floor plate cells and can induce appropriate ventral cell types in the basal forebrain and spinal cord. Here we demonstrate that Sonic hedgehog is sufficient to induce dopaminergic and other neuronal phenotypes in chick mesencephalic explants in vitro. We find that Sonic hedgehog is a general ventralizing signal in the CNS, the specific response being determined by the receiving cells. These results suggest that Sonic hedgehog may have utility in the induction of clinically important cell types.

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.

Somite Differentiation: Sonic signals somites

Sonic hedgehog, a secreted signalling molecule known to play a role in the patterning of the central nervous system and the limb in vertebrates, also controls differentiation of the somites.

Sonic hedgehog: making the gradient

The amino-terminal peptide of Sonic hedgehog is a cell-tethered molecule, which nevertheless seems to provide a developmental signal that acts at a distance and has different effects depending on its concentration. Recent structural data suggest that zinc-dependent proteolysis may somehow be involved in Sonic hedgehogs function.