biochemistry of morphogens
Biochemistry of Morphogens ¨C Hedgehog (Hh) Members of the Hedgehog family of signalling molecules mediate many important short- and long- range patterning processes during invertebrate and vertebrate development. In the fly, a single hedgehog gene regulates segmental and imaginal disc patterning. In contrast, in vertebrates a hedgehog gene family is involved in the control of left-right asymmetry, polarity in the CNS, somites and limb, organogenesis, chondrogenesis and spermatogenesis. The hedgehog gene encodes a novel membrane-linked inductive ligand that plays an important role in the local patterning of many tissues in both vertebrates and invertebrates. Hedgehog was first identified by a genetic screen in Drosophila, in the late 1970s and early 80s. since 1993 several hedgehog homologues have been isolated from various vertebrate species. One hedgehog gene has been identified in Drosophila and other invertebrates, multiple hedgehog genes are present in vertebrates, with differences between vertebrate species. Modification of a secreted hedgehog precursor yields a cell-tethered inductive signal. The primary hedgehog translation product includes a signal peptide with a highly conserved N-terminal region and a more divergent C-terminal domain. The signal peptide is cleaved to yield a 45-kDa precursor protein. This precursor protein is thought to form all the biologically active hedgehog peptides. Cleavage of this secreted precursor protein produces a 20 ¨C kDa N-terminal fragment, which is associated with the plasma membrane and contains the inductive activity, and a 25kDa C-terminal fragment. The N terminal Hh fragment doesn¡¯t contain any hydrophobic sequences, and it stays tightly associated with the surface of cells in which it was synthesised while the C-terminal peptide is freely diffusible, both invitro and invivo. ¡¤ The thiol side chain of the cystein at position 258 attacks the carbonyl carbon of the glycine at position 257, forming an internal thioester linkage between Gly-257 and Cys-258. The C terminal domain then facilitates the addition of cholesterol to Gly-257, splitting the molecule into two fragments. ¡¤ The N-terminal signalling fragment is then tethered to the membrane via its attached hydrophobic cholesterol moiety. ¡¤ ( in addtion to above notes) > This internal thioester intermediate is subsequently cleaved in a nucleophilic substitution. Its likely that the nucleophile is a small lipophilic molecule, which becomes covalently bound to the C-terminal end of N-peptide, tethering it to the cell surface. ¡¤ As a result of this tethering, a high local concentration of N-terminal hedgehog peptide is generated on the surface of the hedgehog-producing cells. It is this N-terminal peptide that is both necessary and sufficient for short and long range signalling activities in Drosophila and vertebrates. (copy fig 23-12) Both in vivo and in vitro experiments have been conducted to see whether the cholesterol moiety is needed for the biological activity of the Hh. 1. an N terminal fragment lacking cholesterol, produced in E.coli using recombinant DNA techniques, is sufficient to induce specific neuronal cell fates in the vertebrate spinal cord. 2. studies in drosophila using hh transgenes encoding the full length molecule or an untethered N-terminal domain also indicate that the cholesterol linkage is not required for inductive activity. 3. in wild-type flies, Hh is expressed in a single cell in each parasegment in the developing embryo and induces the expression of another patterning gene called wingless (wg) only in its immediate neighbouring cell. When normal membrane-tethered Hh was overexpressed within this cell using a cell-specific promoter, expression of Wg protein was still restricted to the immediate neighbouring anterior cell, though it was expressed at increased levels. 4. in contrast to that experiment, the expression of the untethered N-terminal Hh fragment in the same cell led to a high level of wg expression in cells more than a cell diameter away. The studies therefore show that the cholesterol linkage is not needed for the inductive activity of Hh. However, by having it, it acts to spatially restrict the Hh signal in the developing embryo. This spatial restriction of the inductive signal plays a crucial role in patterning both vertebrate and invertebrate tissues. Binding of Hedghog to the Patch Receptor Relieves inhibition of Smo. Smo is a gene that acts in the Hh signalling pathway and encodes a trans-membrane protein with seven membrane spanning domains similar to G protein-coupled receptors. Because ot was observed that the smo and hh mutant phenotypes were similar in the drosophila fly embryo were very similar and thus it was hypothesised that Hh binds to smo. ¡¤ Biochemical studies havnt shown the direct evidence for the interaction between these two. ¡¤ Tissue culture studies however have shown that Hh can bind to cells overexpressing patched (Ptc).