The Hedgehog signaling pathway is a signaling pathway that transmits information to embryonic cells required for proper cell differentiation. Different parts of the embryo have different concentrations of hedgehog signaling proteins. The pathway also has roles in the adult diseases associated with the malfunction of this pathway include cancer.
The Hedgehog signaling pathway is one of the key regulators of animal development and is present in all bilaterians. The pathway takes its name from its polypeptide ligand, an intracellular signaling molecule called Hedgehog (Hh) found in fruit flies of the genus Drosophila; fruit fly larvae lacking the Hh gene are said to resemble hedgehogs. Hh is one of Drosophila's segment polarity gene products, involved in establishing the basis of the fly body plan. The molecule remains important during later stages of embryogenesis and metamorphosis.
Mammals have three Hedgehog homologues, Desert (DHH), Indian (IHH), and Sonic (SHH), of which Sonic is the best studied. The pathway is equally important during vertebrate embryonic development and is therefore of interest in evolutionary developmental biology. In knockout mice lacking components of the pathway, the brain, skeleton, musculature, gastrointestinal tract and lungs fail to develop correctly. Recent studies point to the role of Hedgehog signaling in regulating adult stem cells involved in maintenance and regeneration of adult tissues. The pathway has also been implicated in the development of some cancers. Drugs that specifically target Hedgehog signaling to fight this disease are being actively developed by a number of pharmaceutical companies.
Discovery
In the 1970s, a fundamental problem in developmental biology was to understand how a relatively simple egg can give rise to a complex segmented body plan. In the late 1970s Christiane Nüsslein-Volhard and Eric Wieschaus isolated mutations in genes that control development of the segmented anterior-posterior body axis of the fly; their "saturation mutagenesis" technique resulted in the discovery of a group of genes involved in the development of body segmentation, helping to found the field of evolutionary developmental biology. In 1995, they shared the Nobel Prize with Edward B. Lewis for their work studying genetic mutations in Drosophila embryogenesis.1995 Nobel Prize for discovery of the genetic control of early embryonic development
The Drosophila hedgehog (hh) gene was identified as one of several genes important for creating the differences between the anterior and posterior parts of individual body segments. The fly hh gene was independently cloned in 1992 by the labs of Jym Mohler, Philip Beachy, Thomas B. Kornberg and Saigo Kaoru. Some hedgehog mutants result in abnormally-shaped embryos that are unusually short and stubby compared to wild type embryos. The function of the hedgehog segment polarity gene has been studied for influence on the normally polarized distribution of larval cuticular denticles as well as features on adult appendages such as legs and antennae. Rather than the normal pattern of denticles, hedgehog mutant larvae tend to have "solid lawns" of denticles (Figure 1). The appearance of the stubby and "hairy" larvae inspired the name 'hedgehog'.
Fruit fly
Mechanism Insect cells express a full size zinc-finger transcription factor Cubitus interruptus (Ci), which forms a complex with the kinesin-like protein Costal-2 (Cos2) and is localized in the cytoplasm bound to cellular microtubules (Figure 2). The SCF complex targets the 155 kDa full length Ci protein for proteosome-dependent cleavage, which generates a 75 kDa fragment (CiR). CiR builds up in the cell and diffuses into the nucleus, where it acts as a co-repressor for Hedgehog (Hh) target genes. The steps leading to Ci protein proteolysis include phosphorylation of Ci protein by several protein kinases; PKA, GSK3β and CK1 (Figure 2). The Drosophila protein Slimb is part of an SCF complex that targets proteins for ubiquitylation. Slimb binds to phosphorylated Ci protein.
In the absence of Hh (Figure 3), a cell-surface transmembrane protein called Patched (PTCH) acts to prevent high expression and activity of a 7 membrane spanning receptor called Smoothened (SMO). Patched has sequence similarity to known membrane transport proteins. When extracellular Hh is present (Figure 3), it binds to and inhibits Patched, allowing Smoothened to accumulate and inhibit the proteolytic cleavage of the Ci protein. This process most likely involves the direct interaction of Smoothened and Costal-2 and may involve sequestration of the Ci protein-containing complex to a microdomain where the steps leading to Ci protein proteolysis are disrupted. The mechanism by which Hh binding to Patched leads to increased levels of Smoothened is not clear (Step 1 in Figure 3). Following binding of Hh to Patched, Smoothened levels increase greatly over the level maintained in cells when Patched is not bound to Hh. It has been suggested that phosphorylation of Smoothened plays a role in Hh-dependent regulation of Smoothened levels.
In cells with Hh-activated Patched (Figure 3), the intact Ci protein accumulates in the cell cytoplasm and levels of CiR decrease, allowing transcription of some genes such as decapentaplegic (dpp, a member of the BMP growth factor family). For other Hh-regulated genes, expression requires not only the loss of CiR but also the positive action of uncleaved Ci to act as a transcriptional activator. Costal-2 is normally important for holding Ci protein in the cytoplasm, but interaction of Smoothened with Costal-2 allows some intact Ci protein to go to the nucleus. The Drosophila protein Fused (Fu in Figure 3) is a protein kinase that binds to Costal-2. Fused can inhibit Suppressor of Fused (SUFU), which in turn interacts with Ci to regulate gene transcription in some cell types.
Role
Hedgehog has roles in larval body segment development and in formation of adult appendages. During the formation of body segments in the developing Drosophila embryo, stripes of cells that synthesize the transcription factor engrailed can also express the cell-to-cell signaling protein Hedgehog (green in Figure 4). Hedgehog is not free to move very far from the cells that make it and so it only activates a thin stripe of cells adjacent to the engrailed-expressing cells. When acting in this local fashion, hedgehog works as a paracrine factor. Only cells to one side of the engrailed-expressing cells are competent to respond to Hedgehog following interaction of Hh with the receptor protein Patched (blue in Figure 4).
Cells with Hh-activated Patched receptor synthesize the Wingless protein (red in Figure 4). If a Drosophila embryo is altered so as to produce Hh in all cells, all of the competent cells respond and form a broader band of Wingless-expressing cells in each segment. The wingless gene has an upstream transcription regulatory region that binds the Ci transcription factor in a Hh-dependent fashion resulting in an increase in wingless transcription (interaction 2 in Figure 3) in a stripe of cells adjacent to the stripe of Hh-producing cells.
Wingless protein acts as an extracellular signal and patterns the adjacent rows of cells by activating its cell surface receptor Frizzled. Wingless acts on engrailed-expressing cells to stabilize the stripes of engrailed expression. Wingless is a member of the Wnt family of cell-to-cell signaling proteins. The reciprocal signaling by Hedgehog and Wingless stabilizes the boundary between parasegments (Figure 4, top). The effects of Wingless and Hedgehog on other stripes of cells in each segment establishes a positional code that accounts for the distinct anatomical features along the anterior-posterior axis of the segments.
The Wingless protein is called "wingless" because of the phenotype of some wingless fly mutants. Wingless and Hedgehog function together during metamorphosis to coordinate wing formation. Hedgehog is expressed in the posterior part of developing Drosophila limbs. Hedgehog also participates in the coordination of eye, brain, gonad, gut and tracheal development. Downregulation of hedgehog has been implicated in reduced eye development in the amphipod Gammarus minus.
Annelids Hedgehog is also involved in segmentation in the annelid worms; because parallel evolution seems unlikely, this suggests a common origin of segmentation between the two phyla. Whilst Hh does not induce the formation of segments, it seems to act to stabilize the segmented fields once they have appeared.
Vertebrates Mechanism
Sonic hedgehog (SHH) is the best studied ligand of the vertebrate pathway. Most of what is known about hedgehog signaling has been established by studying SHH. It is translated as a ~45kDa precursor and undergoes autocatalytic processing (Process "1" on Figure 5) to produce an ~20kDa N-terminal signaling domain (referred to as SHH-N) and a ~25kDa C-terminal domain with no known signaling role. During the cleavage, a cholesterol molecule is added to the carboxyl end of the N-terminal domain, which is involved in trafficking, secretion and receptor interaction of the ligand. SHH can signal in an autocrine fashion, affecting the cells in which it is produced. Secretion and consequent paracrine hedgehog signaling require the participation of Dispatched (DISP) protein (Process "2" on Figure 5).
When SHH reaches its target cell, it binds to the Patched-1 (PTCH1) receptor (Process "3" on Figure 5, the blue molecule). In the absence of ligand, PTCH1 inhibits Smoothened (SMO), a downstream protein in the pathway (Process "4"). It has been suggested that SMO is regulated by a small molecule, the cellular localization of which is controlled by PTCH. PTCH1 has homology to Niemann-Pick disease, type C1 (NPC1) that is known to transport lipophilic molecules across a membrane. PTCH1 has a sterol sensing domain (SSD), which has been shown to be essential for suppression of SMO activity. A current theory suggests that PTCH regulates SMO by removing oxysterols from SMO. PTCH acts like a sterol pump and removes oxysterols that have been created by 7-dehydrocholesterol reductase. Upon binding of a Hh protein or a mutation in the SSD of PTCH, the pump is turned off allowing oxysterols to accumulate around SMO.
This accumulation of sterols allows SMO to become active or stay on the membrane for a longer period of time. This hypothesis is supported by the existence of a number of small molecule agonists and antagonists of the pathway that act on SMO. The binding of SHH relieves SMO inhibition, leading to activation of the GLI transcription factors (Process "5"): the activators Gli1 and Gli2 and the repressor Gli3. The sequence of molecular events that connect SMO to GLIs is poorly understood. Activated GLI accumulates in the nucleus (Process "6") and controls the transcription of hedgehog target genes (Process "7"). PTCH1 has recently been reported to repress transcription of hedgehog target genes through a mechanism independent of Smoothened.
In addition to PTCH1, mammals have another hedgehog receptor, PTCH2, whose sequence identity with PTCH1 is 54%. All three mammalian hedgehogs bind both receptors with similar affinity, so PTCH1 and PTCH2 cannot discriminate between the ligands. They do, however, differ in their expression patterns. PTCH2 is expressed at much higher levels in the testis and mediates desert hedgehog signaling there. It appears to have a distinct downstream signaling role from PTCH1. In the absence of ligand binding PTCH2 has a decreased ability to inhibit the activity of SMO. Furthermore, overexpression of PTCH2 does not replace mutated PTCH1 in basal cell carcinoma.