The Roles of Tubulins in the Developing Mouse Brain A thesis submitted to the Division of Graduate Studies and Research of the University of Cincinnati In partial fulfillment of the requirements for the degree of Master of Science In the Department of Molecular Genetics, Biochemistry, & Microbiology of the College of Medicine 2018 Elizabeth A. Bittermann B.S, Anderson University, 2016 Committee Chair: Rolf W. Stottmann, Ph.D. Abstract The tubulin genes form a superfamily composed of eight alpha-tubulins, nine beta-tubulins, and two gamma-tubulins. Together these form microtubules which are important for cell motility, membrane structure, cilia structure, and neuron extension support. The alpha- and beta-tubulins alternate to form cylindrical structures, which are nucleated by gamma-tubulins, to bind laterally and form the tube structure of the microtubule. Elongation of the microtubule is critical for neurite extension during neuronal migration. Neuronal migration is, in turn critical for proper brain development. Neurons have both radial and tangential migration routes which are crucial for proper brain architecture. To date, mutations have been found in ten of the human tubulin genes. Eight have been linked to brain phenotypes including polymicrogyria, lissencephaly, enlarged ventricles, and microcephaly. TUBA1A is one of these, with two homologs which have almost identical amino acid sequences. TUBB2A and TUBB2B are two of the eight genes with brain phenotypes, which have almost identical amino acid sequences. There have only been four mouse models of tubulin mutations to model the different brain phenotypes, but none were designed to precisely recapitulate variants found in humans. All four mice were made through ENU screens. The severity of these phenotypes is surprising as the high homology between genes suggests they may be able to compensate for each other. Here we used CRISPR-CAS9 genome editing and created five novel alleles with the deletion of Tubb2a, Tubb2b, and Tuba1a. We also acquired a null allele of Tuba8. Tubb2ad3964, Tubb2ad4223, Tubb2bd4183, and Tuba8em1J mice were all viable and fertile in the homozygous state, with no difference in size. Tuba1a homozygous loss led to embryonic lethality. Deletion of Tuba1a produced mice that had enlarged ventricles with loss of the intermediate zone of the cortex, and about a 25% incidence of cleft palate. The Tuba1aquas was previously identified through an ENU screen with an R215* nonsense mutation. Tuba1aquas ii mutants have a phenotype similar to the Tuba1a null, with enlarged ventricles and a loss of intermediate zone. A complementation test between Tuba1ad4353/wt and Tuba1aquas/wt produced no live animals with both mutations, confirming the Tuba1a R215* mutation to be an allele of Tuba1a. Preliminary molecular characterization of these Tuba1a phenotypes indicated an increase in proliferation and differentiation of neurons in Tuba1aquas mutants. Similar analysis of the Tuba1ad4353 mutants also suggested a possible decrease in proliferation and an increase in differentiation of neurons. Overall, Tuba1a is critical for brain development, but Tubb2a and Tubb2b have the ability to compensate for each other. iii Acknowledgments I would like to thank Dr. Rolf Stottmann for all the help and guidance he has provided while I have been in his lab. His willingness to answer every question, no matter how basic, has been a wonderful help in furthering my love of genetics and biology. I am also sincerely grateful to Ryan Liegel, a postdoc in the lab who helped me get started. In addition, thank you to Chelsea Menke who helped with the mouse work and Dr. William Miller and Dr. David Wieczorek for their willingness to be part of my committee. Lastly, I would like to thank my family who is a constant source of support. iv v Table of Contents Abstract Acknowledgments Table of Contents List of Figures and Tables List of Abbreviations Chapter I. Introduction Microtubules: Structure and Function Tubulins Genes in Human and Mouse The Neurons Role in Brain Development Tubulinopathies Tubulins in the Mouse Tubb2b-eGFP Mouse Tuba1a Mice Tubb2b Mouse Tubg Mouse Chapter II. Not All Tubulins are Created Equal: Tuba1a is Required for Brain Development Abstract Introduction What is a Tubulin? TUBA1A, TUBB2A, TUBB2B Phenotypes Gene Similarities No Null Alleles Published Hypothesis Materials and Methods Results Discussion Chapter III. Future Directions vi References vii Figures and Tables Figure 1. Structure of the microtubule. Figure 2. Tuba1* and Tubb2* genes are adjacent to each other on their respective chromosomes. Figure 3. Amino acid sequences between tubulin proteins are almost identical. Figure 4. Cell fate is determined by the division of neural progenitors. Figure 5. Migration of neurons radially from the ventricular zone (VZ) or tangentially from the ganglionic eminences form the cortex. Table 1. CRISPR sgRNA guide, PCR primer, and sequencing primer sequences. Figure 6. PCR and Sanger sequencing confirm successful deletion of Tubb2a, Tubb2b, Tuba1a and exon 2 of Tuba8. Figure 7. Second alleles of Tubb2a and Tuba1a deletions was confirmed through PCR and Sanger sequencing. Table 2. Survival at weaning of mice with deletions of Tubb2a, Tubb2b, Tuba8, and Tuba1a. Figure 8. Homozygous deletion of Tubb2a or Tubb2b has no significant difference on the weight of adult mice. Figure 9. Homozygous deletion of Tubb2a, Tubb2b, and exon 2 of Tuba8 has no morphological or histological effect on the adult mouse brain. Table 3. Embryonic survival of Tuba1a deletion mutants (E14.5-E18.5). Figure 10. Loss of Tuba1a causes edema, hemorrhaging, enlarged ventricles and layering defects. Table 4. Survival stats of Tuba1ad4353/wt x Tuba1aquas/wt at weaning and embryonically (E17.5). Figure 11. Tuba1aquas/quas mutants show significant cortical malformations as early as E14.5. Figure 12. Quas allele confirmed as a causative allele of Tuba1a through a complementation test. Figure 13. Tuba1aquas mutants show an increase in proliferation and increased differentiation. Figure 14. Tuba1ad4353 mutants show a possible decrease in proliferation and an increase in differentiation. viii List of Abbreviations CCHMC: Cincinnati Children’s Hospital Medical Center CGE: caudal ganglionic eminence CP: cortical plate E: embryonic day GCPs: gamma-tubulin complex proteins IHC: immunohistochemistry IZ: intermediate zone LGE: lateral ganglionic eminences MAPs: microtubule-associated proteins MGE: medial ganglionic eminences MT: microtubules MTOCs: microtubule organizing centers PFA: paraformaldehyde RMS: rostral migratory stream SNP: single nucleotide polymorphism SVZ: sub-ventricular zone Tuba1*: Tuba1a, Tuba1b, Tuba1c Tubb2*: Tubb2a, Tubb2b VZ: ventricular zone WT: wild-type ix Chapter I. Introduction 10 Microtubules: Structure and Function The cytoskeleton of the cells is, in part, composed of microtubules. Microtubules are an important part of the cell, not just for their shape, but also for their function. Their uses include transport of different materials or signals and the composition of mitotic spindles which pull chromosomes apart during cell division. In addition, microtubules provide structure to the cell membrane, structure to cilia, and are paramount to cell motility (1). Most often they are a group of thirteen linear filaments that are arranged to form hollow cylinders (2). Filaments are composed of alternating alpha- and beta-tubulin monomers, connecting laterally to form the cylinder (Figure 1). Between each alpha- and beta-tubulin monomer is one molecule of GTP. If the GTP is bound to the alpha-tubulin, it stays as GTP. If the GTP is bound to the beta-tubulin monomer, the GTP can be hydrolyzed to GDP and then exchanged for a new GTP. The hydrolysis of GTP to GDP allows for the next alpha-tubulin to bind and elongate the microtubule. Thirteen cylinders then group together to provide the structure of cilia, mitotic spindles, and the support for neuron extensions in the form of axons and dendrites (1). Figure 1. Structure of the microtubule. The microtubule structure is composed of gamma- tubulins at the base, with alpha-tubulins and beta- tubulins composing the long filament structure. Image adapted (3). 11 Microtubules are a dynamic structure that grows and shrinks depending on the needs of the cell. Their base is at the centriole, where a network of gamma-tubulin rings and gamma-tubulin complex proteins (GCPs) form a microtubule-organizing center (MTOC). The GCPs either make a gamma-tubulin small complex or a gamma-tubulin ring complex. These complexes, especially the gamma-tubulin ring complex, form a structure that is similar to the final shape of the microtubule filaments. Gamma-tubulins are attached to these complexes and together they nucleate the microtubule, which allows the filaments to bind together (4, 5). Gamma-tubulins mark the minus-end of the microtubule, while the end that is most often growing is the plus-end. The growth and destruction of microtubules helps to move neurons to the proper layers of the brain as it grows and develops. Movement of neurons is caused by the extension of a process, which is created by the growth of the microtubule, followed by the nucleus being pulled along. Since the microtubules are anchored to the centrioles, a structure can be formed around the nucleus which allows the microtubules, along with the help of actin filaments, to be able to pull or push the nucleus to follow the neurite extension (6). Disruptions in the microtubule structure can have drastic effects