FUNCTIONAL CHARACTERIZATION OF THE SHH RECEPTORSOME USING HUMAN INDUCED PLURIPOTENT STEM CELL MODELS

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

LRP2 controls sonic hedgehog-dependent differentiation of cardiac progenitor cells during outflow tract formation

Conotruncal malformations are a major cause of congenital heart defects in newborn infants. Recently, genetic screens in humans and in mouse models have identified mutations in LRP2, a multi-ligand receptor, as a novel cause of a common arterial trunk, a severe form of outflow tract (OFT) defect. Yet, the underlying mechanism why the morphogen receptor LRP2 is essential for OFT development remained unexplained. Studying LRP2-deficient mouse models, we now show that LRP2 is expressed in the cardiac progenitor niche of the anterior second heart field (SHF) that contributes to elongation of the OFT during separation into aorta and pulmonary trunk. Loss of LRP2 in mutant mice results in depletion of a pool of sonic hedgehog-dependent progenitor cells in the anterior SHF due to premature differentiation into cardiomyocytes as they migrate into the OFT myocardium. Depletion of this cardiac progenitor cell pool results in aberrant shortening of the OFT, the likely cause of CAT formation in affected mice. Our findings identified the molecular mechanism whereby LRP2 controls maintenance of progenitor cell fate in the anterior SHF essential for OFT separation, and why receptor dysfunction is a novel cause of conotruncal malformation

Induced pluripotent stem cell-based disease modeling identifies ligand-induced decay of megalin as a cause of Donnai-Barrow syndrome

Donnai-Barrow syndrome (DBS) is an autosomal-recessive disorder characterized by multiple pathologies including malformation of forebrain and eyes, as well as resorption defects of the kidney proximal tubule. The underlying cause of DBS are mutations in LRP2, encoding the multifunctional endocytic receptor megalin. Here, we identified a unique missense mutation R3192Q of LRP2 in an affected family that may provide novel insights into the molecular causes of receptor dysfunction in the kidney proximal tubule and other tissues affected in DBS. Using patient-derived induced pluripotent stem cell lines we generated neuroepithelial and kidney cell types as models of the disease. Using these cell models, we documented the inability of megalinR3192Q to properly discharge ligand and ligand-induced receptor decay in lysosomes. Thus, mutant receptors are aberrantly targeted to lysosomes for catabolism, essentially depleting megalin in the presence of ligand in this affected family

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations

Cardiac Subtype-Specific Modeling of Kv1.5 Ion Channel Deficiency Using Human Pluripotent Stem Cells

The ultrarapid delayed rectifier K+ current (IKur), mediated by Kv1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of Kv1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in Kv1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial Kv1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K+ current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of Kv1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSCs

Generation and cardiac subtype-specific differentiation of PITX2-deficient human iPS cell lines for exploring familial atrial fibrillation

Loss-of-function mutations in the PITX2 transcription factor gene have been shown to cause familial atrial fibrillation (AF). To potentially model aspects of AF and unravel PITX2-regulated downstream genes for drug target discovery, we here report the generation of integration-free PITX2-deficient hiPS cell lines. We also show that both PITX2 knockout hiPS cells and isogenic wild-type controls can selectively be differentiated into human atrial cardiomyocytes, to potentially uncover differentially expressed gene sets between these groups

Cardiac Subtype-Specific Modeling of K(v)1.5 Ion Channel Deficiency Using Human Pluripotent StemCells

The ultrarapid delayed rectifier K+ current (I-Kur), mediated by K(v)1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of K(v)1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in K(v)1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial Kv1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K+ current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of K(v)1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSC