The Hedgehog (Hh) pathway structure remains mainly conserved from Drosophila to humans, although some differences exist. This section will examine the individual components of the Hh pathway and their regulation in arthropods and in vertebrates.
The secreted protein Hedgehog (Hh in Drosophila, or one of three vertebrate orthologs, primarily Sonic hedgehog, Shh) is synthesized as a precursor protein which undergoes a cholesterol-catalyzed self-cleavage and palmitoylation. The processed N-terminal fragment is then secreted in a process that depends on the 12-spanner Dispatched and is further assisted by Scube. On the receiving end, signaling is triggered when the secreted Hh ligand binds to its membrane receptor, the 12-spanner protein Patched (Ptc). Ptc normally inhibits the 7-pass transmembrane protein Smoothened (Smo). When bound by Hh, Patched is inhibited resulting in Smo activation. Active Smo then triggers the activators of the cytoplasmic steps of the signaling pathway, and ultimately induces transcription of the Hh target genes which include Gli1 and Ptc.
Regulation of Smo is critical for Hh signaling; however, the way Smo is activated and inhibited is poorly understood mechanistically. Ptc does not directly bind Smo, and acts catalytically to inhibit it. The non-stoichiometric inhibition of oncogenic Smo suggests that Ptc regulates Smo indirectly, by affecting the concentration of an intermediate modulator, and several lines of evidence point to a small molecule mediator whose identity remains unknown. Furthermore, in a cellular context, Smo is constitutively active in the absence of Ptc; however, it is not known whether Smo is intrinsically in the active conformation, or is kept active by a ubiquitous activator molecule – and Ptc may act indirectly on Smo by interfering with this activator. Ptc may regulate the abundance of a Smo modulator; nevertheless, in the absence of direct biochemical evidence, the mechanism of Smo inhibition remains an open question.
While Hedgehog, Patched and Smoothened as well as the Ci/Gli transcription factors are conserved from Drosophila to vertebrates, the pathway components downstream of Smo have diverged significantly. Moreover, despite sequence conservation of Smo throughout species, the presence of additional regulatory sequences in the cytoplasmic tail of Drosophila Smo suggests a divergent, specialized mechanism of downstream signaling. In Drosophila and in the absence of signaling, the zinc-finger transcription factor Cubitus interruptus (Ci) forms two separate complexes, one containing the kinesin-like protein Costal2 (Cos2) and the serine/threonine kinase Fused (Fu), and a small fraction exists in a separate complex containing the Suppressor of Fused, Su(Fu); the complexed Ci is then proteolytically cleaved to a repressor form. Cos2 is anchored to microtubules in the cytosol and thus sequesters Ci in the cytosol. Cos2 inhibits the vast majority of available Ci while Sufu only plays a minor role. Pathway activation leads to accumulation of Smo, which binds Cos2 directly; this in turn destabilizes the Ci-Cos2 complex, releases Ci and inhibits the proteolytic cleavage of Ci. The full-length Ci-activator form translocates to the nucleus where it transcribes the hedgehog target genes. Smo binds Cos2 through its C-terminal domain, while the vertebrate Smo does not interact with Cos2; interestingly, the Drosophila C-terminal domain renders vertebrate Smo sensitive to Cos2 in a chimeric protein.
In vertebrates, the Glioma-associated family of transcription factors (Gli1, 2 and 3) are the homologs of Ci. Gli forms a complex with Suppressor of Fused (SuFu) in the cytosol, and is proteolyzed to a short repressor form. Pathway activation leads to destabilization of the Gli-SuFu complex, which inhibits the proteolytic cleavage of Gli and frees the active Gli-A form to translocate to the nucleus and transcribe the Hedgehog target genes. Of the three Gli proteins present in vertebrates, Gli3 is processed robustly to the repressor form in the absence of signaling, while pathway activation stabilizes the fulllength Gli3 activator form which translocates to the cilium along with SuFu. Gli1 only exists in the activator form and is not processed to a repressor, while Gli2 is primarily in the active form but is kept repressed by binding to SuFu in the cytosol. The primary mediator of transcriptional activation is Gli2, while the primary mediator of transcriptional repression is Gli3; as a direct target of Hh signaling, Gli1 is not essential for Hedgehog signaling but forms a positive feedback loop to amplify the transcriptional output of the pathway. Early on in embryonic development, mouse knockouts of the positive regulators Shh, Smo or Gli2 show severe defects in specifying left-right asymmetry, in establishing midline structures such as the notochord or floor plate; later in embryonic development, gross morphological defects include holoprosencephaly, cyclopia, absence of motor neurons in the ventral neural tube, and defects in limb development.
Strikingly, mouse knockouts of the Fu kinase homolog (Stk36) are viable and have normal Hh signaling. The closest vertebrate homologs of Cos2 are Kif27 and Kif7. The mouse Fu homolog interacts directly with Kif27, and together are essential for assembly of the central pair of microtubule dublets in motile cilia, but have no role in Hedgehog signaling. Separately, Kif7 is a ciliary protein which acts downstream of Smo and does not bind Smo directly. In the absence of signaling, Kif7 localizes at the base of the primary cilium; activation of the hedgehog pathway triggers a redistribution of Kif7 along the entire length of the cilium, and it accumulates at the distal tip of the cilium. Furthermore, mutations in the motor domain of Kif7 disable this movement, impair the formation of the Gli3 repressor form, and result in ectopic Hh pathway activation; this is similar to the effect of a complete loss of Kif7, which also recapitulates the Gli3-null phenotype.