Hh and Wnt signalling (L5) Flashcards
What does Hh and Wnt signalling play a role in?
Involved in patterning many tissues and the development of many organs in both vertebrates and invertebrates. Also involved in the regulation of stem cell fate/decisions and in the maintenance and initiation of cancer
What is Hh and when was it discovered?
Hedgehog gene was first defined as a mutation that showed defects in segmentation in Drosophila. Loss of the gene leads to so-called segment polarity phenotype, in each segment, the naked cuticle is lost, creating an embryo full with denticles. So Hh is a segmentation polarity gene discovered in the 1989s by Nusslien-Volhard and Wieschaus
In what way do Hh and Wg act together?
Hh acts in a reciprocal loop with the signalling molecule Wg. During segmentation patterning in flies, Hh and Wg maintain each other’s expression in an autoregulatory loop. Since these genes are very dependent on each other, the loss of Wg also leads to a loss of Hh and therefore gives a very similar phenotype. Wg was originally discovered as Lnt1 in mice, but was basically the same thing, so was combined with Wg to make its new name Wnt. both signals are present in multicellular organisms (except C. elegans doesn’t have a Hh signal)
Vertebrates often have more than one orthologue of genes in these siganlling pathways. there are about 18 Wnts in vertebrtes and 7 in drosophila.
How is a Hh signal created?
Hh genes are translated with an N-terminus signal sequence that targets them to the secretory pathway. The signal sequence is removed and then the protein undergoes an autoproteolytic cleavage catalysed by the C-terminal part of the protein (it has no other role that promoting this cleavage). The N terminus part is then coupled with a cholesterol molecule. One further modification is made at the N-terminus, where a palmitoyl group is added. These modifications will render the Hh quite insoluble and target it to the membrane. The hydrophobicity of the signal would make it impossible for the molecule to leave the cell, and would therefore only allow for signalling to neighbours. the action of the dispatched protein (a 12 TMSD protein) and scube (in vertebrates) glycoproteins are important for long-range signalling. Alternatively, cytonemes might be involved (molecules needed for this). In the ECM HSPGs have also been shown to be required for the long-range effect of Hh signalling
How is a Wnt signal created?
Wnts are also produced with a signalling sequence that is cleaved off when it enters the secretory pathway. Wnt is first modified by palmitoylation and palmitoleic acid modification of ser209. the hydrophobicity of the modifications makes Wnt insoluble. Again, lipoproteins particles of cytonemes are involved in presenting the ligand to other cells and the wntless 7 pass transmembrane proteins may be involved in getting Wnt to the membrane and allowing it to reach other cells.
What are cytonemes?
Long cellular protrusions that the wnt producing cell uses to touch the other cells and signal to them to change their behaviour.
When was the signalling transduction mechanism of Hh first proposed and what molecules are thought to be involved?
Signal transduction model first proposed by Hooper and Scott in 2005. There are 2 crucial transmembrane proteins in the Hh pathway. In Drosophila, they’re mo and Ptc. In vertebrates, they’re Ptch1 and 2. The Patched gene is a 12-pass transmembrane protein that can bind Hh. It is similar to the dispatched gene. Surprisingly, it acts in a negative way in that it continuously inhibits a positively acting component called Smoothened (a 7-pass transmembrane protein) when the ligand is absent. So Smo is basically inhibited by Ptc.
Explain in what ways Ptc and Smo interact with each other.
Experiments in drosophila show that Ptc and Smo don’t work on a one-to-one basis. A single Ptc molecule can inhibit a large number of Smo molecules. Ptc somehow keeps Smo from getting to the cell surface. It is thought to regulate trafficking of Smo to a compartment where Smo gets degraded. When Ptc binds to Hh, they both get internalised and degraded and Smo can now get trafficked to the cell surface. Overall, there are three changes that occur to Smo- relocation, accumulation and phosphorylation. However, these events are so tightly coupled it is not possible to say what causes what. Sp overall - Ptc regulates the subcellular distribution and stability of Smo.
Explain the Hh signalling in mammals.
In mammals, it appears the primary cilium plays a role in the signalling process. IN the absence of Hh signal, Ptch1 is localised to the cilium of the cell and Smo is excluded from the territory. As a result of Hh binding to Ptc, it is removed from the cilium, allowing Smo to accumulate there and initiate signalling. the importance of the cilium for signalling has been discovered in mice where mutations that disrupt cilia formation were found to impair Hh signalling. the cellular antennae may be focal points for Hh sensing.
What mechanism allows Hh genes to be repressed when there is no Hh binding?
In the absence of Hh, two complexes exist that keep the TF Ci responsible for the effects of Hh signalling outside the nucleus. One contains Costal2, a kinesin-like a molecule that acts as a scaffold protein and fused a serine-threonine kinase. The other contains the Ci and repressor of the fused gene (Sufu) a gene without clear domains. Under the influence of the first complex when bound to Smo, three other kinases can act of Ci. They form a complex consisting of casein kinase 1, protein kinase A and GSK3beta. The transcription factor Ci which as a full-length gene is a transcriptional activator is processed under the influence of kinases to a shorter form via Slimb. This involved ubiquitination. The short form acts as a transcriptional repressor and therefore Hh target genes are actively repressed.
How are Hh target genes activated when Hh binding occurs?
When there is ligand present, the interaction of Ci with the three kinases is somehow blocked, and a full-length Ci is released that will actively promote transcription of target genes. Data from vertebrates indicates that this activation could involve phosphorylation, but the exact nature of this is unknown. Furthermore, its thought that phosphorylation of Sufu by fused promoted formation of the active form of Ci.
In what way does the Hh signal act on its own pathway?
It causes negative feedback where the induction of Ptc is used to limit its activation. It can also feedback positively by inducing Gli1. Gli can’t be proteolyzed into a receptor, so it continually activates the pathway. The induction of Ptc is also seen in Drosophila, but Ci (gli homolog) is not induced.
How is Hh used in wing development?
In the Drosophila wing imaginal disk, Hh is expressed in the posterior compartment and diffuses into the anterior where it induces expression of decapentaplegic (dpp). Dpp is related to BMP/TGFb like genes. This molecule helps to pattern the wing. These wing disks are easy to manipulate genetically and their study has contributed a lot to look at how Hh signalling works.
How is Hh involved in neural development and limb development?
The notochord and floor plate are sources of Hh protein and together, these 2 structures pattern the neural tube. Different cell types are induced depending on the amount and duration of Hh signal. Hh signalling is active in the posterior limb bud, where is forms the zone of polarising activity that can confer posterior identity to the forming limb. It is also involved in its outgrowth.
How do Hh mutations cause diseases?
Hh loss of function is connected to a number of congenital diseases. e.g. Holoprosencephaly - a loss of ventral brains structures, as a result the eyes fuse. Cyclopamine is a chemical that can inhibit smoothened and block Hh signalling. Misregulation of Hh in limbs can cause syndactyly (partially joined digits). Ectopic activation of Hh can cause cancer - e.g. basal cell carcinoma, medulloblastoma and rhabdomyosarcomas. Activation of the pathway can occur by inactivation of Patched1 or Sufu or by rare mutations that activate the Smo gene independently of the presence of Ptc1.
BCCs are the most common form of cancer and are ussually accompanied by a loss of Ptc. Patients of Gorlin’s syndrome or Nevoid basal cell carcinoma inherit a defective Ptc. In these people, loss of the other healthy Ptc is quite common and can cause many times of skin cancers.
