The dorsal spinal cord marker Pax7 is normally repressed by low concentrations of Hh [22]. highly conserved secreted signaling proteins Gestrinone (for review see [2]). Hedgehog (Hh) proteins are synthesized as approximately 45 kDa precursors that autoprocess in an unprecedented fashion, resulting in the covalent attachment of a cholesterol moiety to the amino-terminal half of the precursor [2]. This processed amino-terminal domain, Hh-Np, is responsible for the activation of a unique and complex signaling cascade that is essential for controlling cell fate throughout development and Gestrinone into adulthood [2]. In mammals there are three Hh-family proteins: Sonic (Shh), Indian (Ihh), and Desert (Dhh). Gene-targeting experiments in mice have demonstrated that the development and patterning of essentially every major organ requires Emr1 input from the Hh pathway [2]. culture systems of neuronal tissues have been used to characterize the biology of the Hh-signaling pathway. Most notably, the neural-plate explant assay has defined the concentration-dependent role that ventrally expressed Shh plays in opposing dorsally expressed bone morphogenetic proteins (BMPs) to pattern the neural tube [2]. The assay demonstrates that Gestrinone the Hh-signaling cascade can distinguish between small concentration differences in the Hh ligand to instruct the differentiation of specific neuronal cell types. Additional insights have been gained by utilizing cultures of postnatal cerebellar neuron precursors [2]. These studies have shown that Hh patterns the cerebellum by promoting proliferation of the granule neuron precursors. Given the role that Hh signaling plays in promoting progenitor-cell proliferation, it is not surprising that misregulation of Hh signaling has been implicated in the biology of certain cancers, in particular basal cell carcinoma (BCC) and medulloblastoma. The Hh-signaling pathway comprises three main components: the Hh ligand; a transmembrane receptor circuit composed of the negative regulator Patched (Ptc) plus an activator, Smoothened (Smo); and finally a cytoplasmic complex that regulates the Cubitus interruptus (Ci) or Gli family of transcriptional effectors. Additional pathway components are thought to modulate the activity or subcellular distribution of these molecules [2]. There is positive and negative feedback at the transcriptional level as the and genes are direct transcriptional targets of activation of the pathway. Smo is a seven-pass transmembrane protein with homology to G-protein-coupled receptors (GPCRs), while Ptc is a twelve-pass transmembrane protein that resembles a channel or transporter. Consistent with its role as an essential pathway inhibitor, removal of Ptc renders the Hh pathway constitutively ‘on’, independent of the Hh ligand. Similarly, specific point mutations in the transmembrane helices of Smo are capable of constitutively stimulating the pathway, efficiently bypassing Ptc inhibition [3]. At present, a controversy surrounds the mechanism by which Ptc inhibits Smo. Although early studies suggested a simple, direct, stoichiometric regulation, more recent data support a more complicated indirect or catalytic model [2]. And although it has been shown that Hh directly interacts with [4] and destabilizes [5] Ptc, the downstream molecular events Gestrinone remain obscure. In particular, little is known about the means by which Ptc exerts its inhibitory effect on Smo, or how Smo communicates with the cytoplasmic Ci/Gli transcription element complex. Through a ‘chemical genetic’ approach of identifying and studying the mechanism of action of small-molecule agonists (and antagonists), we hoped to uncover some of the complexities.
