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.

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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.

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

References

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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.

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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

Chapter I.

Introduction

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).

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 on proper brain development.

Tubulin Genes in Human and Mouse

Tubulin genes make up a superfamily composed of alpha-, beta-, and gamma-tubulins.

There are eight different alpha-tubulins, nine beta-tubulins, and two gamma-tubulins in humans.

In mice, there are seven alpha-tubulins, eight beta-tubulins, and two gamma-tubulins. TUBA1A,

TUBA1B, TUBA1C, TUBB2A, and TUBB2B are of particular interest because of their homology and the human phenotypes associated with three of them. The three alpha-tubulins are immediately adjacent on chromosome 12 in humans and chromosome 15 in mice. The two beta- tubulins are adjacent on chromosome 6 in humans and chromosome 13 in mice with almost a 50kb

12 intergenic region between the two genes. The intergenic regions between the three alpha-tubulins and the two beta-tubulins have no other annotated genes in these areas (Figure 2). However, in these intergenic regions there is one long non-coding RNA that resides between Tuba1a and

Tuba1c, and between Tubb2a and Tubb2b there are two long non-coding RNAs.

Figure 2. Tuba1* and Tubb2* genes are adjacent to each other on their respective chromosomes. Tuba1b, Tuba1a, and Tuba1c are found next to each other on chromosome 15 with a 13kb intergenic region between Tuba1b and Tuba1a, and a 76kb intergenic region between Tuba1a and Tuba1c (A). Tubb2a and Tubb2b are next to each other on chromosome 13 with a 50kb intergenic region between them (B). There are no other annotated genes in these intergenic regions.

What is interesting is the protein sequence similarities between Tuba1a, Tuba1b, and

Tuba1c, and Tubb2a and Tubb2b. In Tuba1a/b/c (Tuba1*) the amino acid sequence similarities are close to 100%. Out of the 451 amino acids in Tuba1a, 449 (99.6%) are identical in Tuba1b and

442 (98%) are identical in Tuba1c (Figure 3A). In Tuba1b, the two amino acids different from

Tuba1a are not in similar positions in the protein. Tuba1c has two fewer amino acids than Tuba1a and has seven different amino acids; four of the seven are clustered at the 5’ end of the protein.

Tubb2a/b (Tubb2*) each have 445 amino acids, and 443 (99.6%) are identical. The two different amino acids are not adjacent in the protein (Figure 3B). Tuba1* and Tubb2* proteins are almost

13 identical between the mouse and human. Sequences for TUBA1A and TUBA1B are 100% identical between the mouse and human, while TUBA1C is 95.8% identical. TUBB2A and TUBB2B proteins sequences are 100% identical between the two species. The homology between the two species makes the mouse a great model for tubulinopathies. However, the homology between the sequences is so high that there is no way to raise antibodies specific to the individual proteins.

A. mTUBA1A MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGK mTUBA1B MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGK mTUBA1C MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGK ************************************************************ mTUBA1A HVPRAVFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLD mTUBA1B HVPRAVFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLD mTUBA1C HVPRAVFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLD ************************************************************ mTUBA1A RIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQVSTA mTUBA1B RIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQVSTA mTUBA1C RIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQVSTA ************************************************************ mTUBA1A VVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLIGQIVSSITA mTUBA1B VVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLISQIVSSITA mTUBA1C VVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLISQIVSSITA ***************************************************.******** mTUBA1A SLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLSVAEITNACFEPAN mTUBA1B SLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLSVAEITNACFEPAN mTUBA1C SLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLTVAEITNACFEPAN **********************************************:************* mTUBA1A QMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFKVGINYQPP mTUBA1B QMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKRSIQFVDWCPTGFKVGINYQPP mTUBA1C QMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFKVGINYQPP ***************************************:******************** mTUBA1A TVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEGMEEGEFSE mTUBA1B TVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEGMEEGEFSE mTUBA1C TVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEGMEEGEFSE ************************************************************ mTUBA1A AREDMAALEKDYEEVGVDSVEGEGEEEGEEY mTUBA1B AREDMAALEKDYEEVGVDSVEGEGEEEGEEY mTUBA1C AREDMAALEKDYEEVGADS--AEGDDEGEEY ****************.** .**::*****

B. mTUBB2A MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGSYHGDSDLQLERINVYYNEAAGNKYV mTUBB2B MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGSYHGDSDLQLERINVYYNEATGNKYV ******************************************************:*****

14 mTUBB2A PRAILVDLEPGTMDSVRSGPFGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELVDSVLDVV mTUBB2B PRAILVDLEPGTMDSVRSGPFGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELVDSVLDVV ************************************************************ mTUBB2A RKESESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYPDRIMNTFSVMPSPKVSDTVV mTUBB2B RKESESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYPDRIMNTFSVMPSPKVSDTVV ************************************************************ mTUBB2A EPYNATLSVHQLVENTDETYSIDNEALYDICFRTLKLTTPTYGDLNHLVSATMSGVTTCL mTUBB2B EPYNATLSVHQLVENTDETYCIDNEALYDICFRTLKLTTPTYGDLNHLVSATMSGVTTCL ********************.*************************************** mTUBB2A RFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTSRGSQQYRALTVPELTQQMFDSKNMM mTUBB2B RFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTSRGSQQYRALTVPELTQQMFDSKNMM ************************************************************ mTUBB2A AACDPRHGRYLTVAAIFRGRMSMKEVDEQMLNVQNKNSSYFVEWIPNNVKTAVCDIPPRG mTUBB2B AACDPRHGRYLTVAAIFRGRMSMKEVDEQMLNVQNKNSSYFVEWIPNNVKTAVCDIPPRG ************************************************************ mTUBB2A LKMSATFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS mTUBB2B LKMSATFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVS ************************************************************ mTUBB2A EYQQYQDATADEQGEFEEEEGEDEA mTUBB2B EYQQYQDATADEQGEFEEEEGEDEA *************************

Figure 3. Amino acid sequences between tubulin proteins are almost identical. MUSCLE alignment of (A) Tuba1 proteins and (B) Tubb2 proteins in the mouse. Amino acid differences are highlighted in yellow.

The Neurons in Brain Development

During the development of the brain, neurons proliferate at the ventricular zone (VZ), migrate out towards the sub-ventricular zone (SVZ), then towards the pial surface to form the cortical plate (CP). Neural stem cells populate the VZ. During mitosis, these stem cells can repopulate the stem cell population or create a differentiating neuron depending on their division.

During mitosis, if the plane of cleavage is oriented perpendicular to the plane of the VZ, then the new cells will continue to be progenitors. If the plane of cleavage is oriented parallel or even oblique to the plane of the VZ, then one new cell will go on to become a differentiated neuron that moves away from the VZ and into the cortical plate, while the other stays a neural progenitor

(Figure 4, 7). These are not the only neurons that are produced to populate the developing cortex

15 of the brain. The lateral ganglionic eminences (LGE), medial ganglionic eminences (MGE), and caudal ganglionic eminences (CGE) are more inferior to the cortex and also produce neurons that will migrate tangentially up and around the developing brain and to populate the cortex (Figure 5,

8). Once these neurons reach the cortex, will migrate radially into the cortical plate. The processes of the neurons in the cortical plate compose the intermediate zone (IZ, 9). Problems with the migration of neurons lead to major consequences to the structure of the developing brain, which can cause intellectual disabilities, seizures, and if severe enough, death.

Figure 4. Cell fate is determined by the division of neural progenitors. Vertical cleavage will produce new stem cells while oblique cleavage will produce a differentiated neuron. Ventricular zone (VZ), sub-ventricular zone (SVZ), cortical plate (CP), neural epithelial progenitor (NEP), radial glial (RG). Imaged adapted (7).

Figure 5. Migration of neurons radially from the ventricular zone (VZ) or tangentially from the ganglionic eminences form the cortex. Neurons from the ventricular zone migrate radially (A), while neurons from the ganglionic eminences migrate tangentially (B) to form the cortical plate. Lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE). Image adapted (9). Tubulinopathies

Tubulin genes have been implicated in so many different brain malformations that the phenotypes associated with tubulins are classified as tubulinopathies. TUBA1A mutations have been mainly known to cause lissencephaly (smooth brain), but have also been associated with enlarged ventricles, pachygyria (fewer folds), microcephaly (small brain), polymicrogyria (extra folds), and a thin cortex (10-32). The other alpha-tubulin that has been implicated in disease is

TUBA4A, associated with familial ALS (33-35). In the beta-tubulin family, there are many more genes that are thought to cause brain phenotypes. TUBB2A has only a few published mutations that have phenotypes involving simplified gyral patterning, enlarged ventricles, pachygyria, and microcephaly (36-39). The homolog of TUBB2A, TUBB2B, is most often associated with polymicrogyria but can also include microcephaly, pachygyria, microlissencephaly (small smooth brain) and ocular congenital cranial dysinnervation disorders (40-48). TUBB3 mutations are

17 commonly associated with cortical dysplasia (improper neuron migration) and fibrosis of extraocular muscles, but patients can also present with polymicrogyria and gyral disorganization

(49-53). Dystonia-4 (disorganized microtubule network) and leukodystrophy are commonly associated with TUBB4A mutations along with enlarged ventricles, cerebellar atrophy, and incomplete myelination in white matter (54-59). TUBB5 has been linked with microcephaly (60).

The final tubulin gene to be associated with a brain phenotype is TUBG1, which has been associated with cortical dysplasia (61). Many of these brain phenotypes also cause epilepsy, intellectual disability, and some are severe enough that they cause the fetus to die in utero. There are two other tubulins that have been associated with phenotypes not related to the brain. TUBB8 has been linked to oocyte maturation defects (62, 63) and TUBB1 has been linked to macrothrombocytopenia (platelet disorder) (64-67). The number of mutations found in these genes, that have been published, makes for very interesting studies to try and understand their functions.

Tubulins in the Mouse

There has been some expression data published for the tubulin genes, but much of this data is not reliable. Most of the data was published before the full set of homologous genes was discovered and was based on Western Blots or RNA in situ hybridization. These techniques rely on the protein or mRNA for a template. The high homology between the tubulin proteins makes it impossible to use Western blots to distinguish between individual proteins. The proteins do not have enough differences that would allow for specific antibodies or RNA probes to distinguish between the different homologs. The expression data published can be used as a guide for a general tubulin group, such as Tuba1*. The available data are not able to differentiate between Tuba1a,

Tuba1b, and Tuba1c. This is also the case for the Tubb2* genes.

Tubb2b-eGFP Mouse

A 2015 paper published some of the most reliable expression data to date (68). qRT-PCR first suggested that Tubb2b had high expression in the E16.5 mouse brain and was significantly reduced in the adult mouse brain. In the adult mouse, expression was noted in the cochlea, eye, lung, and thymus. A Tubb2b-eGFP BAC transgenic mouse was created to show Tubb2b expression. The GFP showed that Tubb2b was expressed embryonically mostly in the central nervous system with some expression in the developing limbs and whiskers. Upon closer inspection of the developing cortex, it was determined that Tubb2b expression was widespread, but enriched in post-mitotic regions that included migrating and differentiating neurons. By co- staining for GFP and neuronal markers, Tubb2b was determined to be expressed in radial glia, intermediated progenitors, migrating neurons, and proliferating cells. The GFP signal was not as strong at P0 or P7, but still was co-expressed within the same group of cells. In the brain of an eight week old mouse, Tubb2b could be seen around the lateral ventricles and within the internal capsule. Together, this all shows that Tubb2b is expressed in developing neurons and is downregulated in the adult brain (68).

Tuba1a Mice

TUBA1A is one of the tubulin genes with the most reported tubulin mutations but yet has very few mouse models. To date, there have only been three Tuba1a mouse models published and none of them were generated to model patient alleles. Each mouse had comparable qualities to a patient phenotype and they each provided interesting information. The first mutant published was the Tuba1aJna/+ (Jenna) mouse, found through an ENU screen, with a p. S140G mutation.

Heterozygous animals had a significant phenotype and heterozygous crosses failed to results in any pregnancies. Initial behavioral testing showed that Tuba1aJna/+ were more hyperactive, less

19 fearful, and failed to build proper nests. Upon closer examination, the heterozygous mice were both smaller, had smaller brains, and brain sections showed abnormal organization of the hippocampus. In vitro studies determined that the S140G mutation impaired the ability of the heterodimer to bind GTP. The abnormal organization of the cortex was caused by an inability to form heterodimers or the creation of unstable microtubules (69).

Further research into the adult mouse hippocampus and dentate gyrus broadened the understanding of this mouse mutant. It was concluded that Tuba1a was expressed in mature granule cells and certain post-mitotic neurons. New neurons were able to differentiate, but were likely ectopic and would lead to the disorganization that was seen in the sub-granular zone in the dentate gyrus (70). When looking at the superior colliculus there was a fusion of the intermediate grey and intermediate white layers. NeuN antibody staining showed impaired radial migration and a decrease in post-mitotic neurons. Caspase-3 antibody staining showed increased numbers of apoptotic cells. The decrease in post-mitotic neurons was attributed to the increased apoptosis

(71).

The most recent paper on the Jenna mutant was published in 2017. This paper focused on the rostral migratory stream (RMS) and olfactory bulbs. While the olfactory bulbs appeared to have normal organization, their size was reduced. The RMS had an accumulation of cells that was likely due to migration defects, which would result in the reduced olfactory bulb size. Interneurons migrate from the RMS out to the olfactory bulbs where they differentiate. In Jenna mutants, cells in the RMS were having trouble migrating, yet were still able to differentiate and mature. To better understand the migration defect, live imaging was used to study the migration pathways of the neurons. Live imaging found in wild-type mice that neurons moved tangentially along the RMS, whereas the mutant neurons were neither parallel nor unidirectional. When neurons migrate, the

20 centrosome will move out and the nucleus is pulled or pushed in the direction of the centrosome.

In the Jenna mutant, neurons had the centrosome move out, however, in a large portion of neurons the nucleus failed to follow and the centrosome moved back to the nucleus. Looking at the structure of Tuba1a and Tubb2b in the microtubule, it was determined that the S140G mutation caused the structure to be straighter and more rigid. The conclusion was that the mutant neurons were slower to move, and did not move in a parallel direction which was likely caused by straighter microtubules (72).

While the most research has been done on the Jenna mouse, there are two other Tuba1a mouse mutations that were found through ENU screenings. The Tuba1Rgsc1736 mouse has a p.

D47G mutation (73) and the Tuba1aND/ND mouse has a p. N102D mutation (74). The Tuba1Rgsc1736 mouse exhibited a behavioral phenotype similar to the Tuba1aJna/+ mouse. Tuba1Rgsc1736/+ mice were more active and paid less attention to new objects placed in their cages. Their brains also had disorganized cortical layers caused by impaired neuronal migration. Very little research was done to discover how the mutation caused the neuronal migration to be impaired. Instead, research focused on how MPH, a common ADHD drug, could partially rescue the behavioral phenotype

(73). The Tuba1aND/ND mouse was first found by looking for movement defects. These mice were unable to breathe at birth and died not long after. When the mice were examined embryonically, their brains showed layer defects in the cerebral cortex, hindbrain, hippocampus, and corpus callosum. The research did not focus on the brain, instead, it focused on the innervation of muscles that would cause the movement defects. Research found that neurons were not making it to their target muscles and thus not innervating that muscle. The conclusion was that the N102D mutation caused microtubule instability which in turn caused neurons not to reach their target muscles (74).

Tubb2b Mouse

To date, there has only been one published Tubb2b mutation in a mouse. The

Tubb2bbrdp/brdp mouse was found through an ENU screen. The Tubb2bbrdp mouse’s most striking phenotype was ventriculomegaly, but there was also a size reduction in multiple parts of the brain.

This phenotype was observed in embryos as early as E11.5, leading to the conclusion that the mutation had an effect on neurogenesis. Staining for proliferating cells with a pHH3 antibody against phospho-histone H3, showed an increase in the number of proliferating cells at the VZ, but also away from the VZ and into the IZ. TuJ1 antibody staining, which binds to Tubb3 in differentiated neurons, was able to show a decrease in the number of differentiated cells. When compared to a wild-type animal, the TuJ1 positive cells occupied more of the cortex in the

Tubb2bbrdp mutant. TUNEL staining for cell death shows increased cell death in mutants when compared to wild-type animals. BrdU pulse-chase experiments to study migration of neurons found minimal migration defects, leading to the conclusion that the phenotype is largely caused by increased cell death (75).

A second paper with the Tubb2bbrdp/+ mice probed the behavioral hyperactivity phenotype.

Through multiple behavioral tests, it was determined that Tubb2bbrdp/+ mice were less anxious, and had trouble learning and remembering how to get to a platform in a water maze. Closer inspection of the hippocampus found that the structure was disorganized. The conclusion drawn from this research was that the hippocampal disorganization was the main cause of the behavioral defects

(76).

Tubg Mouse

There have been only two mouse models made with mutations in gamma-tubulins. These were both a null allele for each gamma-tubulin gene. Mouse matings were able to recover Tubg1+/-

, but never Tubg1-/- live animals. Homozygous embryos were not able to be recovered after the blastocyst stage. Closer examination of the 8-cell and blastocyst stages determined that Tubg1-/- embryos had mitotic spindle disorganization leading to mitotic arrest, inhibiting the embryo’s ability to implant and grow. The conclusion was that the embryo was able to use the maternal

Tubg1 to reach an 8-cell or blastocyst stage, once that was depleted, there was no embryonic Tubg1 to continue the process. The Tubg2-/- mouse was born in normal Mendelian ratios and did not appear to have any brain defects. It was reported that there might have been some behavioral defects, but there was no data published (5).

A 2012 paper gave a better understanding of the Tubg null phenotype. Using U20S cells, it was determined that Tubg2 could substitute for Tubg1. Multiple experiments, in which Tubg1 was knocked out, were able to recapitulate the mitotic spindle defects and mitotic arrest in U20S cells. Following experiments used mouse Tubg2 or human TUBG2 with a FLAG epitope tag to rescue the Tubg1 knockout cells. The rescued cells had correct spindle orientation and were able to go through mitosis. In vitro results concluded that Tubg2 can take the place of Tubg1, but this does not occur in Tubg1-/- mice. Tubg2 was more prevalent in the brain than the rest of the body in adult mice, but expression in the early embryo was unknown. RT-PCR was able to determine that by the time an embryo reaches the blastocyst stage, Tubg2 was severely decreased while Tubg1 was only slightly lower than at the oocyte stage. Since RT-PCR only looks at the amount of mRNA, a 2D-PAGE gel was used to see if the gamma-tubulins could be separated, and to see if there was a difference in protein levels. 2D-PAGE gel separation was able to differentiate Tubg1

23 from Tubg2, finding that in a blastocyst there were minimal amounts of Tubg2 protein. This lead to the conclusion that even though Tubg2 can functionally substitute for Tubg1, there is not enough of Tubg2 protein in the blastocyst to allow for the embryo to survive and develop (4).

Together these mouse models suggest that tubulin mutations tend to lead to migration defects, which are potentially caused by straighter or more rigid microtubules. It can be said that

Tubb2b is expressed in the developing mouse brain and it is highly likely that Tuba1a is also expressed in the developing mouse brain. It is unclear whether other alpha- or beta-tubulins are expressed in the brain. All the mice presented, with the exception of the Tubg* mice, have single nucleotide polymorphism (SNP) mutations. Several questions arise: are amino acid changes in tubulin proteins not recognized by cells and are the cells unable to express a homolog to avoid causing a phenotype? Tuba1a has two homologs: could they substitute for Tuba1a when the protein is damaged? Reliable data is currently not available to determine where individual tubulin proteins are expressed in the mouse. Even the Tubb2b-eGFP mouse does not definitively show that Tubb2b is incorporated into the microtubules in cells that have GFP expression. In addition, the exact composition of tubulins in the microtubules is largely unknown. To date, there are no studies that indicate if Tuba1* all incorporate into microtubules, or if they are all incorporated within the same cell. The possibility exists that cells in the mouse embryo may use one Tuba1, a combination of two Tuba1, or all three Tuba1. This can also be said for Tubb2*. These genes present a lot of questions that are just waiting to be answered.

Chapter II. Not All Tubulins are Created Equal: Tuba1a is Required for Proper Brain Development

INTRODUCTION

What is a Tubulin?

Tubulins are the building blocks that form microtubules. Specifically, within the central nervous system, microtubules are important for neurite extensions and the migration of neurons.

Tubulins form microtubules by alternating alpha and beta monomers to form a heterodimer. Within the heterodimer is one molecule of GTP that lies between the alpha- and beta-tubulin. There is a second GTP that binds to the beta-tubulin and is hydrolyzed to GDP to allow extension of the filament. The heterodimers form long chains, which together form a cylindrical structure. This cylindrical structure can have one end that will be polymerizing and growing, while on the other end it is depolymerizing, allowing the microtubule to shrink. The ability of the microtubule to grow slowly and shrink quickly is called dynamic instability. This is the process used by neurite extensions. Microtubule-associated proteins (MAPs) also play an important role in assisting microtubules in transport and neuronal maturation. While a lot of research has been done with

MAPs, very little has been done on how mutations in the tubulin genes actually affect the microtubular structure and the impact it has on neurons (77).

TUBA1A, TUBB2A, TUBB2B Phenotypes

To date, there have been 49 TUBA1A (10-32), 5 TUBB2A (36-39), and 22 TUBB2B de novo single nucleotide polymorphism (SNP) patient mutations published (11, 12, 23, 40, 41, 43-47).

Along with these SNP mutations, there are three other TUBB2B mutations that have been published; one has been linked to Uner Tan Syndrome (78), one is a mosaic mother with two daughters that have mutations, and a de novo Leu361_Lys362delinsHISLeuGln mutation (27).

There is a wide range of phenotypes between the different tubulins, but also within each gene.

TUBA1A mutations have most often been associated with lissencephaly, but they are also associated with pachygyria, cerebellar hypoplasia, microcephaly, polymicrogyria, cortical dysgenesis, and ventricle dilation. All these phenotypes often present with learning disabilities, epilepsy, or prenatal death (10-32). TUBB2A mutations are associated with cortical dysplasia, pachygyria, and polymicrogyria (36-39). TUBB2B mutations have most often been associated with polymicrogyria, but also with microcephaly, pachygyria, dysmorphic basal ganglia, and enlarged ventricles (40-48). Other tubulins involved in brain abnormalities include TUBB3, TUBB4A, and

TUBB5. A recent paper reported that TUBA8 was associated with polymicrogyria with optic nerve hypoplasia, but a mouse model and reevaluation of the exome data lead to the implication of a different gene (79, 80). Tubulin mutations have also been associated with other diseases. TUBA4A has been implicated in ALS (35), TUBB1 has been associated with macrothrombocytopenia (64-

67), and TUBB8 has been associated with an oocyte maturation defect (62, 63).

Gene Similarities

TUBA1A, TUBB2A, and TUBB2B are of particular interest because of the striking phenotypes produced by mutations in these genes. TUBA1A has two homologs that are almost identical at an amino acid level. TUBA1B is 99.6% identical (449/451 aa) to TUBA1A in its amino acid sequence, while TUBA1C has a 98.4% identical (444/451 aa) amino acid sequence to

TUBA1A. TUBB2A and TUBB2B are 99.6% identical (443/445 aa) in their amino acid sequences.

Even the RNA sequences for TUBA1* and TUBB2* are highly homologous. They are not as homologous as the proteins, but there are large stretches of nucleotides which have no differences between sequences. Not only do they have very similar sequences, but TUBA1A, TUBA1B, and

TUBA1C are adjacent to each other on chromosome 12 and TUBB2A and TUBB2B are adjacent to each other on chromosome 6. This is also true in the mouse genome. Tuba1a, Tuba1b, and Tuba1c

27 are adjacent to each other on chromosome 15. Tubb2a and Tubb2b are adjacent to each other on chromosome 13 with a 50kb intergenic region separating them.

No Null Alleles Published

Considering the large number of patient mutations in tubulin genes with a spectrum of phenotypes published, relatively few mouse models address these phenotypes. There are no null tubulin mouse alleles published for Tuba1a, Tuba1b, Tuba1c, Tubb2a, or Tubb2b. Three mice, created through ENU screens, model Tuba1a phenotypes (69, 73, 74). There are no mouse models for Tubb2a and only one mouse model for Tubb2b, the Tubb2bbrdp/+ mouse (75). None of the mutations in these mice are the same variants seen in human genetics, but they do give some insight into how neurons are perturbed when one of the tubulin genes is mutated. The Tubb2b-eGFP mouse, made as a BAC transgenic mouse, gave the first reliable data as to where Tubb2b is expressed in the developing mouse (68). The homology between these tubulin genes is so high that it has made raising specific antibodies against them impossible. There have been previous studies which document tubulin expression levels in the mouse, these were done before it was understood that there were different homologs within the tubulin family. Therefore, it is hard to confirm that the current expression data is solely for one tubulin.

Hypothesis

All the cortical malformation patients with reported tubulin mutations, except for two, have been de novo SNP mutations and the patients are heterozygous for their mutation. It is interesting that even though TUBA1A has two homologs that are almost exactly identical, and that TUBB2A and TUBB2B are almost identical homologs of each other, a SNP in one of them will cause such severe phenotypes in humans and in the mouse. CRISPR-CAS9 technology to make germline edits

28 to the mouse genome was used to investigate the question: How does the deletion of individual tubulin genes affect the developing mouse brain? Two alternative hypotheses can be proposed.

The first is that deletion of Tuba1a, Tubb2a, or Tubb2b individually will not have any morphological change in the developing mouse brain because their homologs compensate for the deletion. Therefore, individual tubulins are not specific and all tubulins are not required for proper brain development. The contrasting hypothesis is that deletion of Tuba1a, Tubb2a, or Tubb2b individually will have a significant morphological impact on the developing mouse brain.

Therefore, tubulins are each unique and required for proper brain development. This second hypothesis has been posed as the multi-tubulin hypothesis that states that different isotypes of tubulins perform specific functions in different cells (2).

MATERIALS & METHODS Mouse Husbandry

CRISPR-CAS9

CRISPR guides (Table 1) were created and validated in the lab by a former post-doc, Ryan

Liegel. Injections were done by the Transgenic Animal and Genome Editing Core at Cincinnati

Children’s Hospital Medical Center (CCHMC). Deletions were detected by PCR and founder mice from the Transgenic Core were outcrossed to C57BL6/J (Jackson Labs) mice. All tubulin deletion mice were created and maintained on a C57BL6/J (Jackson Labs) background. Once mutations were determined via PCR and Sanger sequencing, the colony was maintained through intercross and outcrossing of mice. Copulation plug checks determined matings and were recorded as embryonic day (E) 0.5 at noon the following day. It was established that noon was time point

E0.5. Mice were maintained following IACUC and NIH guidelines.

ENU Mutagenesis

The initial ENU injections and screening was done by a Research Assistant III in the lab,

Chelsea Menke. Mutagenized C57BL6/J (Jackson Labs) males were outcrossed to FVB (Jackson

Labs) females. The initial phenotype was characterized by Chelsea Menke. The exome sequencing to find the mutation was done by Chelsea Menke and Dr. Rolf Stottmann. The line was maintained on an FVB background and genotyped using Sanger sequencing.

Tuba8em1J

Tuba8em1J/wt mice were purchased from Jackson Labs (jax.org). They were intercrossed to produce homozygous mice. The line was maintained by intercrosses and genotyped through PCR.

Table 1: CRISPR sgRNA guide, PCR primer, and sequencing primer sequences.

CRISPR Guides Sequence Tuba1a 5’UTR #1** CAAAGTCTACGGATGCTAGG GGG Tuba1a 5’UTR #2 AAAAGTAGCAGAAGATACGG GGG Tuba1a 3’UTR #1** AATAAATACCACGACCTCAG AGG Tuba1a 3’UTR #2 TACTGTCTGTCCATCAAGCG GGG Tubb2a 5’UTR #1** GCAGGACTTGAACTGCAGCC CGG Tubb2a 5’UTR #2 GGGGGCGTAATAACCCTAGG AGG Tubb2a 3’UTR #1** ACTAAGCAGAAGTCCCATGA TGG Tubb2a 3’UTR #2 TCTGAAATAGAAACCATCAT GGG Tubb2b 5’UTR #1** GGGCTTGTGGCCAATCAGCG CGG Tubb2b 5’UTR #2 ACGTAATGCTCTGAGCCCCA GGG Tubb2b 3’UTR #1** TTAGGGGTCCAGTCACCTAT AGG Tubb2b 3’UTR #2 GATAATGCTATTCTTCAGGG AGG

PCR/Sequencing Primers Tuba1a Deletion F* ATTAGTGTGGGAAGGTTGGGTC Tuba1a Deletion R ACTAAGGCCAGTTAAGATGACCT Tuba1a WT F GCCTCCATCACACTAGTCTGT Tuba1a WT R TTGACAGAGACCCAAGCTGC Tubb2a Deletion F* TGGGAAACATTTGTAGTGTTCCT Tubb2a Deletion R ATTAGCTACCTCCACTTCCCTC Tubb2a Intron 1 F CCTGAAAGCCGACCAACTTG Tubb2a Intron 1 R CCCAAAGACACGCCTCTGTA Tubb2a D4223 WT F TCTTCCCTGCAATGTGACTCT Tubb2a D4223 WT R AATTATGGTGCTCAGTTTGCC Tubb2b Deletion F* GCCCTCCTTTTAAAGCCGTG Tubb2b Deletion R* ACTCAGCTTGCCCTTCTCAA Tubb2b WT R AGCTTTAGGGGTCCAGTCAC Tubb2b Intron 1 F GCGCGGCTCTAGGTAAATAC Tubb2b Intron 1 R CGAGGAAGCTGTAGGGTGAT Tuba8 WT F CTTCAGTGAGACTGGCAACG Tuba8 Mut F* TGACCTTGATGATCTATTTCTGC Tuba8 Common R CCCTAACTAGCTCCCCAAGG * Primers used for sequencing ** Validated CRISPR guides

PCR and Sanger Sequencing

Primers were created to amplify the region spanning the whole gene of Tuba1a, Tubb2a, or Tubb2b (Table 1). Tubb2a deletion primers create a 440bp and 699bp band for the larger and

31 smaller deletions. Tubb2b deletion primers create a band at 196bp. Tuba1a deletion primers create a 281bp band for the larger deletion and a 372bp band for the smaller deletion. The original primers only amplified for the deletion, and not the wild-type band because the PCR amplification product would be over 4kb and does not amplify under our standard conditions. Tubb2a primers were created specifically for wild-type animals (Table 1). The smaller deletion required the wild- type primers to be run separately from the deletion primers and created a 275bp band. For the larger of the two Tubb2a deletions, a three primer reaction was created which produced a 498bp band in wild-type and heterozygous animals. The heterozygous animals produce two bands; one for wild-type allele and one for the deletion allele. It was also possible to use a three primer reaction for Tubb2b where the heterozygotes produced two bands; one at a 208bp for the deletion and one at 159bp for the wild-type allele. This reaction had to be run at a 58oC annealing temperature.

Tuba1a wild-type primers produced a 363bp band, only seen in wild-type and heterozygous animals. These primers could be run on a standard program with a 56oC annealing temperature and a 30 second extension time. All initial founder deletion PCR products were sent for Sanger sequencing with 100ng/μL of DNA and either the forward or reverse primer. Sanger sequencing was done by the DNA core at CCHMC. Sequencing results were analyzed using Benchling

(benchling.com).

Tuba8em1J primers (Table 1) were ordered based on the recommendation from Jackson

Laboratory. These were used in a three primer PCR reaction which showed two different sized bands. A smaller band at 216bp that corresponds to the wild-type allele and a larger band at 285bp that corresponds to Tuba8em1J/em1J. This amplification used a step-down program for 10 cycles at

65oC and then amplification at 60oC for 28 cycles.

Mouse Weights

Using an empty cage to zero the scale, weights of mice were taken at P28-P31. Mice were placed in the cage and the weight was recorded. Weights were statistically analyzed using

GraphPad Prism software with a t-test to look for a difference in overall weight of the mouse when comparing homozygotes to wild-type mice.

Adult Brain Harvesting and Histological Staining

At P28-P31, mice were euthanized using 0.1mL sodium pentobarbital, then transcardially perfused with heparinized PBS and formalin (SIGMA), and their brains harvested. The brains were dissected out, cut in half sagittally, and fixed in formalin. After fixation, each half was washed in

1X PBS and stored in 70% ethanol until being embedded in paraffin. Embedded brains were sectioned at a 5µm thickness from paraffin blocks. Brains were stained with hematoxylin

(SIGMA-ALDRICH) and eosin Y Stain 1% (Azer Scientific) following established protocols.

Nissl staining was also done using cresyl violet (ACROS #405760025) following standard protocols. All images were taken on a Zeiss AxioImage at the same magnification. All samples were done in triplicate.

Embryo Harvesting and Staining

Embryos used for histology were harvested at E14.5 and E16.5 according to copulation plug dates. Embryos were imaged and then fixed in Bouin’s solution (SIGMA). After fixation, embryos were washed and stored in 70% ethanol. They were then embedded in paraffin, sectioned at a 10µm thickness and stained with hematoxylin and eosin following standard protocols. Images were taken on a Zeiss AxioImage. All sectioning and staining was done with littermate controls.

Immunohistochemistry (IHC) Staining

Embryos collected for IHC staining were collected at E14.5 based on copulation plugs.

Embryos were fixed for 2 hours in 4% paraformaldehyde (PFA), dehydrated in 30% sucrose, and then embedded in OCT compound (Tissue-Tek). Embryo heads were cryosectioned at a 10µm thickness. IHC staining was done using citrate buffer antigen retrieval, slides were incubated in boiling citrate buffer (1.0M sodium citrate, 1.0M citric acid pH 6.0) and allowed to cool to room temperature. Slides were blocked in 4% normal goat serum in PBS-Triton X and incubated with primary antibody overnight at 4oC. Sections were stained with secondary antibody for 1 hour and

DAPI for 15 minutes at room temperature. Staining for proliferating cells was done with pHH3

(Sigma, 1:500). The secondary antibody for pHH3 was goat anti-rabbit (Alexa Fluor 488, 1:500).

Post-mitotic neurons were identified by staining with TuJ1 (Sigma, 1:500). Secondary antibody for TuJ1 was goat anti-mouse (Alexa Fluor 488, 1:500). Images were taken on a Nikon Eclipse Ti confocal microscope. All sectioning and staining was done with littermate controls.

Cell Counting

Quantification of pHH3 positive cells was done using Imaris Software (Bitplane). Images were analyzed by drawing an area around the VZ that began at the apical surface of the ventricle and included five rows of cells. The number of pHH3 positives cells were tallied and the number of positive cells per area was calculated. Images were all at the same magnification. The area was calculated in m2. The cells per area quantity was then plotted using GraphPad Prism software.

Quantities were then analyzed using an unpaired t-test, also through GraphPad Prism software

(p≤0.05), to compare the amount of proliferating cells at the VZ in homozygotes to wild-type mice.

RESULTS

Null Alleles of Tuba1a, Tubb2a, and Tubb2b, and Tuba8 via CRISPR-CAS9 Genome Editing

We used CRISPR-CAS9 genome editing in the mouse to create deletions to remove

Tuba1a, Tubb2a, or Tubb2b individually and generate stable lines. CRISPR-CAS9 guides were generated to guide the endonuclease to two intergenic regions flanking each tubulin gene (Table

1). We attempted to multiplex the CRISPR-CAS9 editing for Tubb2a and Tubb2b with the goal of creating each single deletion as well as a simultaneous deletion of both. A total of four guides were created for each gene, two for each side of each gene (Fig.6A-C). We recovered two alleles of

Tubb2a and one of Tubb2b. Tubb2aem1Rstot (Tubb2ad3964; chr13: 34,074,221 - 34,078,184) and

Tubb2aem2Rstot (Tubb2ad4223; chr13: 34,074,224 - 34,078, 447) are two independent deletion alleles which excise the entire Tubb2a open reading frame. Tubb2bem1Rstot (Tubb2bd4183; chr13:

34,126,376 - 34,130,558) is also a complete deletion of the gene (Fig.6E-F, I-J, 7A,C). Four guides were used to delete Tuba1a via blastocyst injection and subsequent transfer to pseudo-pregnant females (Fig.6C). Surviving offspring were PCR genotyped for the presence of a CRISPR-CAS9 mediated deletion. We designed primers flanking each gene which would only amplify if the intervening sequence was deleting. We recovered two independent alleles for Tuba1a (Fig.6C,G

7B). Tuba1aem1Rstot (Tuba1ad4353; chr15:98,949,678 - 98,954,031) and Tuba1aem2Rstot (Tuba1ad4262; chr15: 98,949,770 - 98,954,031) are two independent alleles with complete excision of the Tuba1a gene (Fig. 6K, 7D). Tuba8em1J/wt (chr6:121,220,250 - 121,220,729) was obtained commercially and has a deletion of exon 2 (Fig. 6D,H,L).

Figure 6. PCR and Sanger sequencing confirm successful deletion of Tubb2a, Tubb2b, Tuba1a and exon 2 of Tuba8. Schematics of CRISPR guides and genotyping primers for (A) Tubb2a, (B) Tubb2b, (C) Tuba1a, and (D) Tuba8. Deletion of (E,I) Tubb2a, (F,J) Tubb2b, and (G,K) Tuba1a shown through PCR and Sanger sequencing. PCR used deletion primers (top row) and wild-type primers (bottom row) to distinguish between heterozygous and homozygous animals. Tuba8 exon deletion was shown through (H) PCR and (L) Sanger sequencing.

Figure 7. Second alleles of Tubb2a and Tuba1a deletions was confirmed through PCR and Sanger sequencing. PCR gel and Sanger sequencing of the second (A,C) Tubb2a and (B,D) Tuba1a alleles creased through CRISPR-CAS9.

Tubb2a, Tubb2b, and Tuba8 are not Required for Normal Brain Morphogenesis.

Successful mating of CRISPR mosaic founders to B6 animals produced heterozygotes that were viable and fertile. We hypothesized the deletion alleles may yield recessive phenotypes so we intercrossed heterozygous carriers for each of the six alleles. We found no reduction from

Mendelian expectations in the number of live animals at weaning for any allele of Tubb2a, Tubb2b, or Tuba8 (Table 2). Homozygote animals did not appear smaller and body weight at P28 did not show any failure to thrive (Fig. 8A-C). Also, brains were collected for both whole mount analysis and histological examination from P28-P31 to assess any possible structural abnormalities resulting from loss of these tubulin genes. Analysis of the adult brains did not show any morphological differences (Fig. 9A-D). H&E (data not shown) and Nissl staining of sections did not show any morphological differences (Fig. 9F-I). A conditional knockout Tuba8 mouse was created by another group and was reported to have no brain phenotype (80). The Tuba8em1J/em1J mice are viable and fertile. Their brains were also collected from P28-P31, they had no distinct gross morphological differences. H&E (data not shown) and Nissl staining also did not show any morphological differences when compared to wild-type animals (Fig. 9E, J).

Table 2. Survival at weaning of mice with deletions of Tubb2a, Tubb2b, Tuba8, and Tuba1a.

wild-type heterozygous homozygous total p Tubb2ad3964/wt x Tubb2ad3964/wt 31 53 30 115 0.75 Tubb2ad4223/wt x Tubb2a d4223/wt 8 21 13 43 0.55

Tubb2bd4183/wt x Tubb2bd4183/wt 42 74 28 144 0.24

Tuba8em1J/wt x Tuba8em1J/wt 5 7 6 18 0.61

Tuba1ad4262/wt x Tuba1ad4262/wt 27 30 0 57 3e-06 Tuba1ad4353/wt x Tuba1ad4353/wt 27 45 0 72 4e-06 Tuba1a combined 54 75 0 129 3e-11

Figure 8. Homozygous deletion of Tubb2a or Tubb2b has no significant difference on the weight of adult mice. Weights of adult mice at P28 with (A, B) Tubb2a deletion and (C) Tubb2b deletion when compared to wild-type and heterozygous littermates.

Figure 9. Homozygous deletion of Tubb2a, Tubb2b, and exon 2 of Tuba8 has no morphological or histological effect on the adult mouse brain. No gross morphological (A-E) differences are seen in animals homozygous for deletions when compared to wild-type. Histological analysis (F-J) showed normal patterning of neurons in the cortex of the adult mouse brain. Black boxes are the indicated regions seen in F’-J’.

Tuba1a is Required for Survival and Brain Development

In stark contrast to our findings with Tubb2a, Tubb2b, and Tuba8, Tuba1a is absolutely required for survival to weaning as we have not recovered any homozygotes for either Tuba1a deletion allele to date (n=129 total; Table 2). While an analysis of the Tuba1ad4262 allele suggests an effect on heterozygote survival, a combined analysis of both deletion alleles is not consistent with this conclusion. Given that these deletions are very similar and we find no meaningful annotations to the differing 100bp, we do not conclude the heterozygote loss is biologically significant and do not ascribe this to Tuba1a loss of function.

To begin to understand the reason for the lethality and to assess brain development in the homozygotes, we collected embryos at late organogenesis stages. A combined analysis of both

Tuba1a deletion alleles in embryos from E14.5-E18.5 does not reveal a significant lethality during embryonic stages. We did note that in the Tuba1ad4353/wt intercrosses, the Tuba1ad4353/wt heterozygotes appear to be overrepresented. Similar to the adult analysis, however, when we combine the very similar alleles, we see no significant loss of any genotype (Table 3).

Whole mount images of Tuba1ad4353/d4353 and Tuba1ad4262/d4262 animals showed very distinct and consistent phenotypes. We first noticed edema (excess fluid) around the neck, which extended down the spinal cord. There was also significant hemorrhaging/bruising around the neck and shoulders of the homozygous animals (Fig. 10A-C). We also noted subtle changes in the angle of the snout of mutant animals indicating potential skeletal defects. H&E staining revealed an even more striking phenotype. The first noticeable morphological differences are, the presence of

41 ventriculomegaly (enlargement of ventricles), a wider base of the third ventricle, and hypoplastic basal ganglia. Closer inspection of the cortex showed the intermediate zone is significantly decreased or almost completely missing. The cortical plate is also decreased and the ventricular zone is wider when compared to control animals (Fig. 10D-I). We also noted a partial penetrance of cleft palate in homozygous animals. A cleft palate presents when the two palatal shelves of the roof of the mouth fail to fuse. In Tuba1ad4353/d4353 animals, there was a 27% incidence of cleft palate (3/11 animals). In the Tuba1ad4262/d4262 animals, there was a 21% incidence of cleft palate

(3/14 animals). One embryo out of 52 heterozygous animals had a cleft palate, but this was likely secondary to developmental delay. Animals with a cleft palate are unable to survive for long after birth. A cleft palate creates the inability of a pup to simultaneously drink milk and breathe, causing the pup to suffocate. The partial incidence of cleft palate in homozygous mice could be the cause of death for the portion of pups that have it, but it is not a complete explanation of the lethality of all tubulin mouse phenotypes.

Table 3. Embryonic survival of Tuba1a deletion mutants (E14.5-E18.5).

wild-type heterozygous homozygous total p Tuba1ad4262/wt x Tuba1ad4262/wt 7 18 11 36 0.64 Tuba1ad4353/wt x Tuba1ad4353/wt 22 64 25 111 0.25 Tuba1a combined 29 82 36 147 NOT SIG

Figure 10. Loss of Tuba1a causes edema, hemorrhaging, enlarged ventricles and layering defects. Tuba1a deletion mutants have significant gross morphological phenotypes when compared to wild-type (A-C) making them distinguishable upon initial embryo harvesting. Histological analysis (D-I) shows enlarged ventricles with loss of IZ. Black boxes show enlarged area seen in D’-I’. J is an enlargement of E showing the proper elevation of palatal shelves. A fraction of mutants have a cleft palate (K) from a failure of the palatal shelf to elevate and fuse as marked by the arrowheads.

Complementation Analysis Confirms Quas is an Allele of Tuba1a

A previous ENU screen conducted in the lab found a mutant with a SNP mutation in

Tuba1a. The SNP, a thymine to an adenine, created a nonsense mutation and early stop at amino acid 215 (R215*). This line was named the Quasimodo mutant because of the abnormal curvature of the thoracic region seen in these mutants (Fig. 11A-B). Heterozygous crosses failed to produce live homozygous mice at weaning. Histological analysis of homozygous mutants showed a brain phenotype mainly consisting of ventriculomegaly, with a loss of the IZ and a wide third ventricle

(Fig. 11C-F). To confirm that the Tuba1a R215* variant is actually the causative mutation, we crossed Tuba1aquas/+ with Tuba1ad4353/+ animals. Having the Tuba1ad4353 null allele in our colony allowed us to easily do a complementation test confirming the Tuba1aquas mutation. Out of three litters with 22 live pups, there were no Tuba1ad4353/quas animals alive at weaning (Table 4).

Embryonically, in a litter of 10 pups, only one Tuba1ad4353/quas was recovered. The mutant had an exaggerated curvature of the thoracic region, as previously seen in Tuba1aquas/quas mice (Fig. 12A-

B). Histologically at E17.5, H&E staining showed ventriculomegaly with a loss of the intermediate zone, hypoplastic basal ganglia, and a cleft palate (Fig. 12C-F). Without any

Tuba1ad4353/quas live and a histological phenotype similar to Tuba1aquas/quas, we were able to conclude that the R215* variant found in Tuba1a was causative.

Table 4. Survival stats of Tuba1ad4353/wt x Tuba1aquas/wt at weaning and embryonically (E17.5).

wild-type quas/wt d4353/wt d4353/quas total p Tuba1ad4533/wt x Tuba1aquas/wt 7 6 9 0 22 0.04 weaning

Tuba1ad4353/wt x Tuba1aquas/wt 3 3 3 1 10 0.86 embryos

Figure 11. Tuba1aquas/quas mutants show significant cortical malformations as early as E14.5. Mutants can be seen with edema when compared to wild-types (A-B). Histological analysis of mutants shows enlarged ventricles with decreased IZ (C-F). Black boxes are an indication of the areas that have been enlarged in C’-F’.

Figure 12. Quas allele confirmed as a causative allele of Tuba1a through a complementation test. Tuba1ad4353/quas mutants have gross morphological (A-B) similar to Tuba1aquas mutants. Histological analysis (C-F) revealed a similar phenotype to Tuba1aquas mutants with the enlarged ventricles and loss of IZ. Black boxes are an indication of the areas that have been enlarged in C’- F’.

Tuba1ad4353 and Tuba1aquas have Abnormal Proliferation and Increased Differentiation

To better understand the phenotypes of these Tuba1a mutations, we first tested the hypothesis that mutations in Tuba1a cause a decrease in proliferation and abnormal migration using immunohistochemistry staining. The first stain we used was pHH3, this binds to phospho- histone H3 which is found in proliferating cells. Preliminary data suggests Tuba1aquas/quas mutants have an increase in the number of proliferating cells at the VZ when compared to wild-type littermate controls (Fig. 13A-B). The number of pHH3 positive cells was counted using Imaris software and compared to the area of the VZ. Statistical data has confirmed that there is a significant increase in the number of pHH3 positive cells in mutants when compared to wild-type or heterozygous animals (p=0.02, Fig. 13E). Looking at neuron differentiation, we used the TuJ1 antibody which binds Tubb3. Cells positive for TuJ1 appear to be more expanded in the cortex of mutant animals than in those of littermate wild-type controls suggesting an increase in neuron differentiation (Fig. 13C-D).

We then turned to the Tuba1ad4353 animals, which have very similar phenotypes but may have slightly different molecular mechanisms. Proliferating cell differences between wild-type and mutant animals were not as distinct as the differences seen in Tuba1aquas animals. It appears there may be a decrease in the number of pHH3 positive cells in mutants, although quantification has not been completed (Fig. 14A-B). Differentiated neurons were measured by staining for TuJ1.

When comparing the Tuba1ad4353/d4353 mouse to wild-type littermate controls, preliminary data suggest an increase in neuronal differentiation (Fig. 14C-D). The possible differences between these mutants could be due to the Tuba1aquas mutant still making Tuba1a protein, which could be incorporating into the microtubule structure causing structural abnormalities. The Tuba1a deletion mutants have no Tuba1a protein available for incorporation into microtubules.

Figure 13. Tuba1aquas mutants show an increase in proliferation and increased differentiation. pHH3 antibody staining (A,B) showed an increase in proliferating cells of mutants when compared to wild-type and heterozygous animals (E). TuJ1 antibody staining (C,D) of differentiated neurons shows increased differentiation in mutants when compared to littermate controls. (*p<0.05)

Figure 14. Tuba1ad4353 mutants show a possible decrease in proliferation and an increase in differentiation. pHH3 antibody staining (A,B) showed an increase in proliferating cells of mutants when compared to wild-type and heterozygous animals. TuJ1 antibody staining (C,D) of differentiated neurons shows increased differentiation in mutants when compared to littermate controls.

DISCUSSION

Deletion Alleles Phenotypes

Here we describe the successful creation of three novel tubulin deletion mice. Using the

CRISPR-CAS9 genome editing technology, we created deletions of Tubb2a, Tubb2b, and Tuba1a.

We recovered two alleles of Tubb2a, one allele of Tubb2b, and two alleles of Tuba1a. Each of these alleles completely deleted the gene and was confirmed by Sanger sequencing. The fourth mouse described was the Tuba8em1J/wt mouse, which was commercially acquired. Tubb2a, Tubb2b, and Tuba8 deletion mice were all viable and fertile. In comparison, the Tuba1a deletion was embryonic lethal in the homozygous state while heterozygous animals were viable and fertile.

Homozygotes had a distinct phenotype that included edema, bruising, ventriculomegaly, a wider base of the third ventricles, hypoplastic basal ganglia, and a decreased intermediate zone. Close to a quarter of homozygous animals had a cleft palate, which could contribute to the lethality.

Consequences of Tubulin Deletions

The results presented show that the multi-tubulin hypothesis is correct for certain genes of the tubulin family but incorrect for others. The data suggests Tubb2*, individually, are not required for proper brain development. Tuba1* are different in that Tuba1a is required for a viable embryo and proper brain development. Proper brain development in Tubb2a and Tubb2b mice may be happening because the loss of one homolog is being compensated for by the remaining homolog.

While Tuba1a has two other homologs that are almost identical in their amino acid sequence, the data suggests that there is something unique about Tuba1a that is required for proper brain development. These results also suggest that a cell may not be able to detect mutated proteins and use a homolog to compensate for the mutation. Of the 5 TUBB2A (36-39) and 22 TUBB2B de

50 novo patient mutations published (11, 12, 23, 40, 41, 43-47), all have brain phenotypes from SNP mutations. In these patients, the variant allele is being incorporated into the microtubule and causing their phenotype. A possible mechanism for the phenotype is that the mutant tubulin is causing the microtubule to become more rigid and less flexible, similar to what was seen in the

Tuba1aJna mutant where neurons migrated improperly and caused migration defects (72). If neurons are unable to properly migrate in the human brain, then it would be logical that we would see the phenotypes associated with these genes. If the homologous tubulin had been compensating for the mutation, like what we see in our deletion alleles, then these patient brains would be normal.

Complement Test

Having the Tuba1a deletion in our colony provided the opportunity to perform a complementation test between Tuba1ad4353 and Tuba1aquas. We confirmed that the Tuba1a R215* variant, found during an ENU screen, was causative. A strong indication that the Tuba1a R215* variant was the causative mutation was the absence of live pups with both the SNP mutation and the deletion allele, in three litters. Embryonically, one litter at E17.5 had one mutant, with the exaggerated curvature of the thoracic region that we associated with the Tuba1aquas mutant.

Histologically, the one Tuba1aquas/D4353 mutant had the characteristic ventriculomegaly, loss of the intermediate zone, hypoplastic basal ganglia, and a cleft palate. The Tuba1aquas mutant also had ventriculomegaly, diminished or lost intermediate zone, hypoplastic basal ganglia, but we have not done sectioning of heads to be able to determine if they also have a cleft palate. Not recovering any double mutants live, and having the same phenotype as a Tuba1aquas mutant, concludes that the Tuba1a R215* variant in the quas line is causative.

Preliminary Molecular Work

A previous tubulin mutant in our colony, the Tubb2bbrdp mutant, had an increase in proliferating cells and apoptotic cells (75). Using the work done with the Tubb2bbrdp mutant as a template for the Tuba1a mutations we have obtained preliminary data for pHH3 and TuJ1 staining.

For the Tuba1aquas mutation, preliminary data appeared to show that there was increased proliferation, this was confirmed by using Imaris counting software and GraphPad Prism software for the unpaired t-test. TuJ1 staining for post-mitotic neurons appears to have an increased area in mutants, suggesting a migration defect. The increase in proliferation does not help to explain the phenotype unless we also find an increase in basal progenitors or apoptotic cells. The migration defect, seen in Tuba1aquas mutants, does make sense from previous research on Tuba1a mutations, which all suggest that neurons have trouble migrating (71, 72, 74). In the Tuba1ad4353 mutants, pHH3 positive cells will need to be quantified, but mutant animals appear to have a decrease in the number of proliferating cells. The preliminary evidence for increased differentiation caused by a loss of Tuba1a is not as strong but is still evident as seen by TuJ1 staining. We cannot confirm a decrease in proliferating cells at this time, but if it is accurate then it could be that these cells are not able to correctly divide, which leads to fewer cells to compose the cortex and leads to ventriculomegaly. Increased differentiation, suggesting a migration defect, as stated before is not unusual for mice with Tuba1a mutations and is almost expected in this mouse. Preliminary data of TuJ1 staining suggests increased differentiation, but more replicates will need to be done to confirm. An increase in differentiation can explain why we have a decrease in the CP and an increased VZ. The cells are dividing, but are not able to migrate out to the CP.

Continuing Experiments

With neither Tubb2a nor Tubb2b deletions individually causing a phenotype, future studies with both genes deleted will be needed to ascertain if the genes compensate for each other or if a different beta-tubulin is replacing the missing gene. The fact that deletion of Tuba1a is not compensated for by either Tuba1b or Tuba1c is very intriguing and requires further study. For all three genes here, there needs to be a better understanding of where they are expressed, and if some of these genes are co-expressed in the same cells. The Tubb2b-GFP mouse shows that Tubb2b is expressed in the brain (68), but what about Tubb2a? Are they expressed together in the same cells?

For the alpha-tubulins, is Tuba1b or Tuba1c even expressed in the brain? If they are not, then there could be a very good reason as to why the deletion of Tuba1a gives such a striking phenotype.

Compensation could be addressed through epitope-tagged alleles that would allow us to use

Western blots to determine if there is an increase in protein levels. If we are unable to make epitope tags, then we could try using RNA for RNA in situ hybridization. Having the deletion alleles in our colony, we could possibly make probes for each tubulin gene and then test them on our deletion homozygous mice. If the probes are specific to only one tubulin gene, then they will not bind anything in a mouse homozygous for the deletion.

There also needs to be further molecular analysis of the Tuba1a brain phenotype to determine mechanism. Patients with certain TUBA1A mutations resemble the phenotype that is seen in the Tuba1a deletion mouse. Further study of this mouse, or mice modeling patient mutations, could lead to a better understanding of what happens to these cells during development.

We also saw edema in our mutants. Edema is a buildup of excess fluid which could be caused by leaky vessels. It could also be caused by a heart valve defect. The Tuba1aND/ND mouse had problems with innervation of muscles because nerves were unable to get to their target muscle

(74). There could be a similar problem in our Tuba1a deletion mice. If the nerves are unable to get to their target muscles, in this case the heart, then the heart would not be able to beat properly or with enough force to push all the fluid out of it. This would cause the fluid to back up into the heart and can lead to the edema that we are seeing in our mice. We also need to look at the skeletons of our mutants to better investigate the possible skeletal defects that we saw. Skeletal preps of our mutants would be able to tell us what kinds of defects we are looking for. This would give us another indication of where Tuba1a is critical for development.

Chapter III. Future Directions

Continued Characterization of the Tuba1aD4353/D4353 and Tuba1aquas/quas Phenotype.

All of the immunohistochemistry that was described in chapter 2 will need to be replicated, some several times, but all done in triplicate. In addition, we need to stain with CC3 to look at apoptotic cells, Tbr2 to look at basal progenitors, and NeuN staining to look at cell differentiation.

Previous work in the Tubb2bbrdp mouse showed that mutants had an increase in apoptotic cells

(75). Preliminary data of IHC staining has suggested that Tuba1aquas/quas animals have an increase in proliferation, as seen with pHH3, and possible problems in differentiation, as seen with TuJ1.

Tuba1aD435/D4353 preliminary IHC staining of pHH3 will need to be quantified before any conclusions can be drawn and TuJ1 has suggested an increase in neuronal differentiation.

Imaris software will be used to count pHH3 cells both at the VZ and away from the VZ.

This comparison is important to determine where proliferating neurons are in the cortex because in the Tubb2bbrdp/brdp mice there was an increase in proliferating cells away from the VZ (76). We would also like to use the Imaris software to count CC3 positive cells. Again, this is in comparison to the Tubb2bbrdp/brdp mice which showed an increase in apoptotic cells that compensated for the increase in proliferating cells (76). This technology could also be used to compare the area of

TuJ1 and NeuN cells to see if these areas are increased in the mutant mice when compared to wild- type. The increase in an area of post-mitotic neurons can give evidence towards a migration defect.

The number of positive Tbr2 cells should be counted to determine if there is a difference in the number of basal progenitors.

Generate Knock-Outs of Tuba1b and Tuba1c using CRISPR-CAS9

Tuba1b and Tuba1c are the two homologs of Tuba1a. At 99.6% and 95.8% homology,

Tuba1b and Tuba1c are the two most likely candidates to be able to compensate for a loss of

Tuba1a. Since the Tuba1a null allele is lethal, are the other two genes also as critical to brain formation? Deletions of these genes will produce very interesting results. If there is a phenotype in either deletion or both, it will support the hypothesis that each of the Tuba1* has very distinct roles in brain development. The absence of a phenotype would suggest that only Tuba1a is absolutely required for proper development. It would be more curious if Tuba1b is deleted and there is no phenotype because the protein of Tuba1b is almost identical to Tuba1a. Would this mean that Tuba1a is able to compensate for any loss of Tuba1b? Does it not need to compensate for Tuba1b because it is not expressed in the brain? Another possibility is that Tuba1b and Tuba1c are expressed in the brain, but at a later stage in development than Tuba1a.

Expression of Tubb2a, Tubb2b, Tuba1a, Tuba1c, Tuba1b

It has been shown that Tubb2b is expressed in the brain and central nervous system by a

Tubb2b-eGFP mouse (68). As previously discussed, it is impossible to determine where these genes are expressed because of the protein homology between them. There is also a high homology between mRNA sequences that would make it difficult to confirm any probe specificity to one tubulin. To better understand why Tubb2a and Tubb2b knockout mice do not have a phenotype, we should create epitope-tagged alleles that will allow us to determine where these genes are expressed. We would also need to do this for Tuba1a, Tuba1b, and Tuba1c.

Understanding where all of these genes are expressed can help explain the phenotypes that we have seen in all knockout mice. If Tubb2a is also expressed in the brain and central nervous system like Tubb2b then it gives more evidence to the fact that Tubb2a or Tubb2b can substitute for each other. Since the Tuba1a knockout mouse has a phenotype, it suggests Tuba1a has a special role in the developing mouse brain. Epitope-tagged Tuba1b and Tuba1c can allow for expression data that would tell us if either of them is expressed in the brain. If they are not, then it is possible that

57 they are not able to substitute for Tuba1a because they are not actually there. If they are expressed in the brain, then we need to look deeper into Tuba1a to determine what exactly about it is absolutely required for brain development.

We can also use these tags to look at the structure of microtubules to determine which percentage of them are Tubb2a, Tubb2b, Tuba1a, Tuba1b, or Tuba1c. Using epitope tag mice we can make cell lines that would allow us to use fluorescent antibodies against each of the tags to determine the composition of microtubules. A cell line may be more effective when trying to determine the concentration of different tubulins because we would be able to look more closely at the cells. Neurons are too tightly packed in the cortex to effectively see microtubule composition. Antibodies to each tag are available and protocols are in place for both fluorescence staining and Western blot. Western blots can also be used for some expression data because we are able to quantify protein levels in certain areas based on our dissections. We have already collaborated with the CCHMC Transgenic Core to help design and inject CRISPR guides into a mouse to create FLAG, HA, and myc epitope tags.

Using CRISPR-CAS9 to Create Patient Mutations in Mice

Of the different tubulin mouse lines published for Tuba1a and Tubb2b, none of them model patient mutations. These mice were found through ENU screens, which present very interesting phenotypes. While similar to the patient phenotypes they do not accurately represent the patient mutations. Using CRISPR-CAS9 we can target specific nucleotides to create different mutations in the mouse. Exact patient mutations can be modeled in the mouse because the protein sequences of TUBA1A, TUBB2A, and TUBB2B are identical between the human and the mouse. The published human mutations in any of these genes have not been restricted to a specific area of the gene and have caused vastly different phenotypes. By making a variety of mutations we can gain

58 a better understanding of the different phenotypes. We can also gain a better understanding of how different parts of the protein are important in the microtubule structure. The mouse mutants would be evaluated using the same methods that are described in this paper. Besides just the IHC staining to understand the molecular mechanism, it would also be interesting to be able to use an epitope tag on these mutated alleles to look at microtubule structure. We can use CRISPR-CAS9 to create a mouse with both the mutation and the epitope tag. There are two possible strategies we could use to make our mouse model. The first is we would need to make one large donor that would include both the patient mutation and the tag. The second is we would need to breed mice to homozygosity for the epitope tag and then do a second round of CRISPR injections to get the patient mutation. The lab’s previous results with CRISPR-CAS9 mutated mice has shown that our collaborations with the Transgenic Core have been successful. There is still a lot of work to be done with this family of genes, but the lab is in an optimal position with our resources to be able to investigate.

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