SUMMARY Hedgehog (Hh) proteins regulate important developmental processes including cell proliferation and differentiation. While Patched acts as the main Hh receptor, in Drosophila, Hh signaling absolutely requires the additional Hh-binding proteins Ihog and Boi. Here we show that, unexpectedly, cerebellar granule neuron progenitors (CGNPs) lacking Boc and Cdon, the vertebrate orthologs of Ihog and Boi, still proliferate in response to Hh. This is because in their absence, Gas1, a Hh-binding protein not present in Drosophila, mediates Hh signaling. Consistently, only CGNPs lacking all three molecules Boc, Cdon, and Gas1 have a complete loss of Hh-dependent proliferation. In a complementary manner, we find that a mutated Hh ligand which binds Patched1 but neither Boc, Cdon, nor Gas1 cannot activate Hh signaling. Together, this demonstrates an absolute requirement for Boc, Cdon, and Gas1 in Hh signaling and reveals a distinct requirement for ligand-binding components that distinguishes the vertebrate and invertebrate Hh receptor systems.

Sonic hedgehog (Shh) and its main receptor Patched (Ptc) are implicated in both neural development and tumorigenesis1, 2. Beside the classic morphogen activity of Shh, Shh is also a survival factor3, 4. Along this line, Ptc has been shown to function as a dependence receptor, inducing apoptosis in the absence of Shh, while its pro-apoptotic activity is blocked in Shh presence5. Here we show that, in the absence of its ligand, Ptc interacts with the adaptor protein DRAL/FHL2. DRAL/FHL2 is required for the pro-apoptotic activity of Ptc both in immortalized cells and during neural tube development in chick embryo. We demonstrate that, in the absence of Shh, Ptc recruits a protein complex that includes DRAL, the CARD containing domain proteins TUCAN or NALP1 and the apical caspase-9. Ptc triggers caspase-9 activation and enhances cell death via a caspase-9-dependent mechanism. Thus, we propose that, upon absence of its ligand Shh, the dependence receptor Ptc serves as the anchor for a caspase-activating complex that includes DRAL, a CARD domain containing protein and caspase-9.

Netrin-1 was recently proposed to control tumorigenesis by inhibiting apoptosis induced by the dependence receptors DCC (Deleted in colorectal cancer) and UNC5H. While a loss of these dependence receptors’ expression has been described as a selective advantage for tumor growth and progression in numerous cancers, recent observations have shown that some tumors may utilize an alternative strategy to block dependence receptor-induced programmed cell death: the autocrine expression of netrin-1. This alternative strategy has been observed in a large fraction of aggressive breast cancers, neuroblastoma, pancreatic adenocarcinoma, and lung cancer. This observation is of potential interest regarding future targeted therapy, since in such cases interfering with the ability of netrin-1 to inhibit DCC or UNC5H-induced cell death is associated with apoptosis of netrin-1-expressing tumor cells in vitro, and with inhibition of tumor growth or metastasis in different mouse tumor models. The understanding of the mechanism by which netrin-1 inhibits cell death is therefore of interest. Here, we show that netrin-1 triggers the multimerization of both DCC and UNC5H2 receptors, and that multimerization of the intracellular domain of DCC and UNC5H2 is the critical step to inhibit the pro-apoptotic effects of both of these receptors. Taking advantage of this property, we utilized a recombinant specific domain of DCC that (i) interacts with netrin-1 and (ii) inhibits netrin-1- induced multimerization, to trigger apoptosis in netrin-dependent tumor cells.

During cerebellar development, Sonic hedgehog (Shh) signaling drives the proliferation of granule cell precursors (GCPs). Aberrant activation of Shh signaling causes overproliferation of GCPs, leading to medulloblastoma. Although the Shh-binding protein Boc associates with the Shh receptor Ptch1 to mediate Shh signaling, whether Boc plays a role in medulloblastoma is unknown. Here, we show that BOC is upregulated in medulloblastomas and induces GCP proliferation. Conversely, Boc inactivation reduces proliferation and progression of early medulloblastomas to advanced tumors. Mechanistically, we find that Boc, through elevated Shh signaling, promotes high levels of DNA damage, an effect mediated by CyclinD1. High DNA damage in the presence of Boc increases the incidence of Ptch1 loss of heterozygosity, an important event in the progression from early to advanced medulloblastoma. Together, our results indicate that DNA damage promoted by Boc leads to the demise of its own coreceptor, Ptch1, and consequently medulloblastoma progression.

The notion of "morphogens" is an important one in developmental biology. By definition, a morphogen is a molecule that emanates from a specific set of cells that is present in a concentration gradient and that specifies the fate of each cell along this gradient. The strongest candidate morphogens are members of the transforming growth factor-beta (TGF-beta), Hedgehog (Hh), and Wnt families. While these morphogens have been extensively described as differentiation inducers, some reports also suggest their possible involvement in cell death and cell survival. It is frequently speculated that the cell death induction that is found associated with experimental removal of morphogens is the manifestation of abnormal differentiation signals. However, several recent reports have raised controversy about this death by default, suggesting that cell death regulation is an active process for shaping tissues and organs. In this review, we will present morphogens, with a specific emphasis on Sonic Hedgehog, a mammalian member of the Hh family, not as a positive regulators of cell differentiation but as key regulators of cell survival.