The Role of Glycosylphosphatidylinositol Biosynthesis and Remodeling in Neural and Craniofacial Development A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Molecular and Developmental Biology Graduate Program of the University of Cincinnati College of Medicine by Marshall Lukacs B.A. Case Western Reserve University June 2019 Committee Chair: Rolf Stottmann, Ph.D. 1 Abstract Glycosylation is the most abundant posttranslational modification though its role in development is highly understudied. One form of glycosylation involves the anchorage of nearly 150 proteins to the cell membrane by glycosylphosphatidylinositol (GPI). Over thirty genes are involved in the biosynthesis and remodeling of the GPI anchor. Mutations in these genes cause an array of genetic disorders called Inherited GPI Deficiencies (IGDs) with broad clinical phenotypes including epilepsy, craniofacial defects, heart defects, and premature death. This thesis utilized several mouse genetic mouse models to test the requirement for GPI biosynthesis in neural and craniofacial development. We found that the Cleft Lip/Palate, Edema, and Exencephaly (Clpex) mutant mouse phenotype is caused by a hypomorphic mutation in the GPI remodeling gene Post-GPI Attachment to Proteins 2 (Pgap2). We found Pgap2 is required for the survival of neural crest cells and the cranial neuroepithelium. We showed that trafficking of a GPI-anchored survival factor for these cells, Folate Receptor 1, requires Pgap2 for proper localization on the cell membrane. Supplementation with folinic acid to overcome the defective FOLR1 trafficking partially rescued phenotypes in the Clpex mutant. As we established the role of Pgap2 in neural and craniofacial development, we sought to determine the requirement for GPI biosynthesis in these tissues by using a more tailored genetic approach. Pgap2 is required for the one of the final steps in GPI biosynthesis but to completely abolish GPI biosynthesis, we utilized a conditional allele of Phosphatidylinositol Glycan Anchor Biosynthesis Class A, Piga, which is a component of the first committed step in GPI biosynthesis. Others have shown that deletion of Piga results in almost total absence of GPI biosynthesis. Thus we crossed the conditional Piga 2 allele to Wnt1-Cre, neural crest cell specific, and Nestin-Cre, central and peripheral nervous system specific, mice to generate tissue specific knockouts in the developing face and brain, respectively. Knockout of Piga in neural crest cells resulted in craniofacial skeletal hypoplasia and cleft palate confirming the cell autonomous requirement for GPI biosynthesis in neural crest cells for their survival. Conditional ablation of Piga in the central and peripheral nervous system produced a number of interesting phenotypes. We found Piga, Nestin-Cre conditional knockout mice gained less weight than their wildtype littermates and died prematurely before weaning. These mice developed neurological decline, tremor, and an ataxic gait. Behavioral tests showed they are ataxic though they showed no defect in motor strength. Immunohistochemical analysis of the cerebellum showed a defect in Purkinje cell dendritic branching in conditional knockouts. RNA-sequencing of the cerebellum identified a signature of Purkinje cell developmental delay and a strong signature of hypoxia. These data demonstrate that the cerebellum and motor coordination is uniquely sensitive to GPI deficiency. This thesis expands our understanding of the requirement for GPI biosynthesis in the development of the brain and face, the two organ systems most affected by GPI deficiency. 3 4 Acknowledgements I would like to first thank my mother without whom this work would really not be possible. My mother has supported me unfailingly in my endeavors and supported me in every way. She always believed in my dreams and supported me unconditionally. Four sentences here is really incapable of expressing my gratitude to my mom, she’s an inspiration. I’d also like to thank my mentor, Rolf Stottmann, who had to tolerate my stubborn, critical personality. I’m sure it was a challenge to handle and he always let me pursue my own intellectual interests. Rolf is a very supportive mentor and was always looking out for new opportunities for me. My committee has been very supportive and pushed me to think critically about my data. I’d like to thank my friend Laura Schapiro who threw a party for me when I passed my qualifier even though none of my medical school friends knew what the qualifier meant. It was incredibly thoughtful and kind of her. I have to thank Sandra Zoubovsky and our weekly therapy/hot sake sessions which helped me survive graduate school. I’d like to thank Tia Roberts who spent a summer with me and did an amazing job in the lab. Lastly, I’d like to thank my best friend Matt Sievers who dragged me out of the lab to escape and explore my queer side. 5 Table of Contents Title Page 1 Abstract 2 Acknowledgements 5 Table of Contents 6 Figures and Tables 8 Chapter 1. Introduction A. Summary 10 B. Congenital Disorders of Glycosylation 31 C. GPI Biosynthesis and Remodeling 36 D. Models of Inherited Glycosylphosphatidylinositol Deficiency 43 E. Craniofacial Development 48 F. Neural Development 54 G. Potential Mechanisms of Neurological Defects in CGD 63 H. Potential Mechanisms of Craniofacial Defects in CGD 68 I. References 72 Chapter 2. Glycosylphosphatidylinositol Biosynthesis and Remodeling are Required for Neural Crest Cell Survival A. Abstract 84 B. Introduction 85 C. Results 87 D. Discussion 114 E. Materials and Methods 119 F. Supplemental Figures 127 G. References 131 Chapter 3. CNS Glycosylphosphatidylinositol Deficiency Results in Delayed White Matter Development, Ataxia, and Premature Death in a Novel Mouse Model A. Introduction 137 B. Materials and Methods 140 C. Results 146 6 D. Discussion 162 E. Supplemental Figures 167 F. References 168 Chapter 4. Discussion A. Summary of Findings 172 B. Future Directions: Role of GPI biosynthesis in CNS development 175 C. Future Directions: The role of GPI biosynthesis in other organs 180 D. Future Directions: Determine the pathophysiology of Postnatal neurological defects in IGD 187 E. Future Directions: Therapy for IGD 188 F. Concluding Remarks 191 G. Acknowledgements 192 H. References 193 7 Figures and Tables 1. Chapter 1. Introduction Table 1. GPI anchored proteins and their knockout phenotypes 11 Table 2.GPI biosynthesis genes, knockout phenotypes, and their putative mechanisms 27 Figure 1. Congenital disorders of glycosylation by type 32 Figure 2. Biosynthesis and remodeling of glycophosphatidylinositol 35 Figure 3. Prevalence of clinical phenotypes in all IGD patients 38 Figure 4. Developmental timeline of facial prominence development 51 Figure 5. Developmental timeline of the secondary palate 54 Figure 6. Developmental timeline of neural tube closure 57 Figure 7. Cerebellum anatomy 60 Figure 8. Development of myelinating oligodendrocytes from 62 Oligodendrocyte precursors. 2. Chapter 2. Glycosylphosphatidylinositol Biosynthesis and Remodeling are Required for Neural Crest Cell Survival Figure 1. The Clpex mutant phenotype is caused by a hypomorphic Mutation in Pgap2 89 Table 1. Exome filtering of the Clpex mutant 90 Figure 2. Pgap2null allele fails to complement Pgap2Clpex allele 91 Figure 3. Pgap2 is dynamically expressed throughout embryogenesis 94 Figure 4. Pgap2 is required for proper anchoring of GPI-APs 97 Figure 5. Trafficking of FOLR1 to the cell membrane requires GPI biosynthesis and remodeling 99 Figure 6. Clpex cNCCs and neuroepithelium undergo apoptosis at E9.5 102 Figure 7. Folinic Acid treatment in utero partially rescues the cNCC apoptosis and cleft lip in Clpex mutants 104 Table 2. RNA Sequencing ToppGene Pathway Enrichment Analysis. 107 8 Table 3. Anterior/Posterior transcription factors differentially expressed in Clpex mutants compared to controls 108 Figure 8. Deletion of Piga in the Wnt1-Cre lineage leads to profound NCC GPI deficiency 110 Figure 9. Conditional knockout of Piga abolishes GPI biosynthesis in NCCs and leads to median cleft lip/palate and craniofacial hypoplasia 113 Table S1. Primers 127 Figure S1. Generation of CRISPR-edited cell lines 128 Figure S2. Clpex mutants are not defective at barrier formation 129 Figure S3. Anterior/Posterior patterning defects in Clpex mutants 129 Figure S4. Embryonic expression of GPI biosynthesis genes 130 3. Chapter 3. CNS Glycosylphosphatidylinositol Deficiency Results in Delayed White Matter Development, Ataxia, and Premature Death in a Novel Mouse Model Figure 1. Piga expression in the CNS 147 Figure 2. Deletion of Piga in the Nestin-Cre lineage results in CNS GPI deficiency 151 Figure 3. CNS GPI deficiency results in cerebellar hypoplasia, decreased weight gain, and decreased survival 153 Figure 4. CNS GPI deficiency results in hindlimb clasping, ataxia, and tremor 156 Figure 5. CNS GPI deficiency delays white matter development 158 Figure 6. CNS GPI deficiency impairs Purkinje cell arborization and alters the cerebellar transcriptome 161 Table S1. PCR Primers 167 Figure S1. Piga Mosaic cKO phenotype videos 168 4. Chapter 4. Discussion Figure 1. The requirement for GPI biosynthesis in diverse tissues during 186 development 9 Chapter 1. Introduction Summary The glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification required for the cell surface expression of nearly 150 distinct proteins (Table