Shared and Distinct Functional Effects of Patient-Specific <i>Tbr1</i> Mutations on Cortical Development

Abstract

T-Box Brain Transcription Factor 1 (TBR1) plays essential roles in brain development, mediating neuronal migration, fate specification, and axon tract formation. While heterozygous loss-of-function and missense <i>TBR1</i> mutations are associated with neurodevelopmental conditions, the effects of these heterogeneous mutations on brain development have yet to be fully explored. We characterized multiple mouse lines carrying <i>Tbr1</i> mutations differing by type and exonic location, including the previously generated <i>Tbr1</i> exon 2-3 knock-out (KO) line, and we analyzed male and female mice at neonatal and adult stages. The frameshift patient mutation A136PfsX80 (A136fs) caused reduced TBR1 protein in cortex similar to <i>Tbr1</i> KO, while the missense patient mutation K228E caused significant TBR1 upregulation. Analysis of cortical layer formation found similar defects between KO and A136fs homozygotes in their CUX1⁺ and CTIP2⁺ layer positions, while K228E homozygosity produced layering defects distinct from these mutants. Meanwhile, the examination of cortical apoptosis found extensive cell death in KO homozygotes but limited cell death in A136fs or K228E homozygotes. Despite their discordant cortical phenotypes, these <i>Tbr1</i> mutations produced several congruent phenotypes, including anterior commissure reduction in heterozygotes, which was previously observed in humans with <i>TBR1</i> mutations. These results indicate that patient-specific <i>Tbr1</i> mutant mice will be valuable translational models for pinpointing shared and distinct etiologies among patients with <i>TBR1</i>-related developmental conditions.<b>SIGNIFICANCE STATEMENT</b> Mutations of the <i>TBR1</i> gene increase the likelihood of neurodevelopmental conditions such as intellectual disability and autism. Therefore, the study of <i>TBR1</i> can offer insights into the biological mechanisms underlying these conditions, which affect millions worldwide. To improve the modeling of <i>TBR1</i>-related conditions over current <i>Tbr1</i> knock-out mice, we created mouse lines carrying <i>Tbr1</i> mutations identical to those found in human patients. Mice with one mutant <i>Tbr1</i> copy show reduced amygdalar connections regardless of mutation type, suggesting a core biomarker for <i>TBR1</i>-related disorders. In mice with two mutant <i>Tbr1</i> copies, brain phenotypes diverge by mutation type, suggesting differences in <i>Tbr1</i> gene functionality in different patients. These mouse models will serve as valuable tools for understanding genotype-phenotype relationships among patients with neurodevelopmental conditions.