TUBA1A Causes Corpus Callosum Agenesis: A Zebrafish Model
The Center for Rare Childhood Diseases (C4RCD) identified a patient with rare brain malformations. The patient exhibited agenesis of the corpus callosum without lissencephaly. After whole exome sequencing, a heterozygous de novo substitution mutation (c.344 T>C) was found in TUBA1A, resulting in a missense amino acid substitution (I155T). Normally, patients with similar mutations exhibit lissencephaly with other brain malformations. The C4RCD patient’s phenotype is hypothesized to be caused by the mutation in TUBA1A, because mutations in this gene are known to cause tubulinopathies. The TUBA1A gene encodes α-tubulin, which forms and organizes microtubules by heterodimerizing with β-tubulin. Microtubules are rigid hollow fibers that form the largest fiber in the cytoskeleton and play an important role in cell division and migration of neuronal cells in the developing brain. We hypothesized that microinjection of mutant mRNA would cause corpus callosum agenesis in the zebrafish. The zebrafish model is used due to many factors: ease of genetic mutation, high genetic homology with humans, similar central nervous system (CNS), external fertilization and development. To study the role of α-tubulin in corpus callosum agenesis, mutant and wildtype mRNA will be injected into zebrafish eggs in varying concentrations. After incubation, observations will be conducted at 48 and 72 hours. The observations will include: brain morphology, embryo phenotype and behavior. The mRNA was obtained through transcription of pBluescript II KS (+) 3.0 kb with either mutant or wildtype mRNA, after restriction, with BamH1 and Hind3, and purification. Purified in vitro, translated mRNA was microinjected into the zebrafish single cell embryos and observations were conducted, staining and confocal microscopy were used to obtain images of the axonal tracts in the zebrafish midbrain at 72 hours post-fertilization. The embryos injected with wildtype mRNA expressed continuous uninterrupted axons. The embryos injected with mutant mRNA expressed discontinuous axons, possibly due to aggregation of the α-tubulin. We expect that our future experiments in the zebrafish model will help us understand the pathological role of the TUBA1A mutation that causes brain malformation in the patient and ultimately confirm the genetic diagnosis of patient disease.