The morphogen sonic hedgehog (SHH) regulates central patterning processes along the dorsoventral axis of the emerging forebrain. Secreted from the prechordal plate (PrCP), SHH targets neuroepithelial cells of the overlying forebrain organizer region in the rostral diencephalon ventral midline (RDVM), to establish and further specify ventral identity of the forebrain. Defects in this pathway result in severe developmental forebrain defects including holoprosencephaly (HPE). Besides the canonical SHH receptor patched 1 (PTCH1), several additional cell surface proteins have been identified as being essential for SHH signaling in the neuroepithelium. Jointly, they are referred to as the SHH receptorsome and include LRP2 and GAS1. Mutations in these co-receptors are the cause of familial forms of HPE and related phenotypes, such as in Donnai-Barrow syndrome (DBS), corroborating their importance for SHH-dependent forebrain development in humans. Intriguingly, the structure of SHH coreceptors is highly diverse and they show spatial and temporal differences in expression pattern, arguing for distinct functions of each receptor in SHH-dependent developmental processes. In this thesis, I used human induced pluripotent stem cell (iPSC)-based cell models to recapitulate early steps in neuroepithelial patterning and to elucidate unique roles for LRP2 and GAS1 in these processes. In the first project, I studied a unique missense mutation in LRP2 in two siblings with DBS. To do so, I differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and studied the impact of this mutation on receptor handling of SHH. I demonstrated that the mutant receptor was unable to discharge its ligand SHH, leading to enhanced lysosomal degradation of the mutant receptor bound to its ligand. These studies showed that ligand-induced decay of LRP2 is responsible for the disease phenotype in this family with DBS. Additionally, the results of this study verified the molecular function of LRP2 as SHH co-receptor as it mediates endocytosis and trafficking of the morphogen in forebrain neuroepithelial cells, a process essential for SHH signal reception in this cell type. In the second project, I uncovered a novel function for GAS1 in integrating SHH and NOTCH signaling during early forebrain development. Performing comparative analyses in GAS1- deficient mice and genetically engineered GAS1 knockout (KO) iPSC-derived NPCs, I showed that loss of GAS1 impairs NOTCH-dependent facilitation of SHH signaling and results in a failure to maintain the SHH activity domain in the rostral ventral neuroepithelium. Thus, besides its known function as SHH co-receptor, GAS1 also acts as co-receptor for NOTCH1, enhancing pathway activation which, in turn, promotes maintenance of SHH signaling in the rostral ventral neuroepithelium during forebrain development.
