Differential requirements of tubulin genes in mammalian forebrain development

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases. Author summary Tubulin proteins are assembled into microtubules to provide essential cellular cytoskeletal elements. Microtubules are especially crucial for neuronal development. Multiple studies demonstrate that human malformations of cortical development are caused by genetic variants in several tubulin genes. Interestingly, most of these developmental phenotypes are the result of de novo, heterozygous variants likely to act as dominant negatives interfering with normal tubulin function(s). The roles of individual tubulin genes and the mechanism(s) leading to these malformations remain unclear. Genome sequencing efforts revealed high sequence similarity between many tubulin genes, raising the possibility of functional compensation. The requirements for many of these tubulin genes have not been previously addressed in loss of function experiments. We have generated novel deletions of several tubulin genes known to cause human disease to assess if they are required for brain development, or if the human variants act to alter function of the tubulin proteins leading to the pathogenesis. Surprisingly, our results show that some individual tubulin genes are absolutely required for survival while others are not and have much more benign cortical malformations.