Genetic regulators of ventral midbrain gene expression and nigrostriatal circuit integrity

Complex traits are a fundamental feature of diverse organisms. Understanding the genetic architecture of a complex trait is arduous but paramount because heterogeneity is prevalent in populations and often disease-related. Genome-wide association studies have identified many genetic variants associated with complex human traits, but they can only explain a small portion of the expected heritability. This is partially because human genomes are highly diverse with large inter-personal difference. It has been estimated that every human differs from each other by at least 5 million variants. Moreover, many common variants with small effect can contribute to complex traits, but they cannot survive from stringent statistical cutoff given the currently available sample size. Mice are an ideal substitute. They are maintained in a controlled condition to minimize the variation introduced by environment. Each mouse of an inbred strain is genetically identical, but different strains bear innate genetic heterogeneity between each other, mimicking human diversity. Hence, in this work we used inbred mouse strains to study the genetic variation of complex traits. We focused on ventral midbrain, the brain region controlling motor functions and behaviors such as anxiety and fear learning that differ profoundly between inbred mouse strains. Such phenotypic diversity is directed by differences in gene expression that is controlled by cis- and trans-acting regulatory variants. Profound understanding on the genetic variation of ventral midbrain and its related phenotypic differences could pave the way to apprehend the whole genetic makeup of its associated disease phenotypes such as Parkinson’s disease and schizophrenia. Therefore, we set out to investigate the cis- and trans-acting variants affecting mouse ventral midbrain by coupling tissue-level and cell type-specific transcriptomic and epigenomic data. Transcriptomic comparison on ventral midbrains of C57BL/6J, A/J and DBA/2J, three inbred strains segregated by ~ 6 million genetic variants, pinpointed PTTG1 was the only transcription factor significantly altered at transcriptional level between the three strains. Pttg1 ablation on C57BL/6J background led to midbrain transcriptome to shift closer to A/J and DBA/2J during aging, suggesting Pttg1 is a novel regulator for ventral midbrain transcriptome. As ventral midbrain is a mixture of cells, tissue level transcriptome cannot always reveal cell type-specific regulatory variation. Therefore, we set out to generate single nuclei chromatin accessibility profiles on ¬ventral midbrains of C57BL/6J and A/J, providing a rich resource to study the transcriptional control of cellular identity and genetic diversity in this brain region. Data integration with existing single cell transcriptomes predicted the key transcription factors controlling cell identity. Putative regulatory variants showed differential accessibility across cell types, indicating genetic variation can direct cell type-specific gene expression. Comparing chromatin accessibility between mice revealed potential trans-acting variation that can affect strain-specific gene expression in a given cell type. The diverse transcriptome profiles in ventral midbrain can lead to phenotypic variation. Nigrostriatal circuit, bridging from ventral midbrain to dorsal striatum by dopaminergic neurons, is an important pathway controlling motor activity. To search for phenotypes related to dopaminergic neurons, we measured the dopamine concentration in dorsal striatum of eight inbred mouse strains. Interestingly, dopamine levels were varied among stains, suggesting it is a complex trait linked to genetic variation in ventral midbrain. To understand the genetic variation contributing to dopamine level differences, we conducted quantitative trait locus (QTL) mapping with 32 CC strains and found a QTL significantly associated with the trait on chromosome X. As expression changes are likely to be underlying the phenotypic variation, we leveraged our previous transcriptomic data from C57BL/6J and A/J to search for genes differentially expressed in the QTL locus. Col4a6 is the most likely QTL gene because of its 9-fold expression difference between C57BL/6J and A/J. Indeed, COL4A6 has been shown to regulate axogenesis during brain development. This coincides with our observation that A/J had less axon branching in dorsal striatum than C57BL/6J, prompting us to propose that Col4a6 can regulate the axon formation of dopaminergic neurons in embryonic stages. Our study provides a comprehensive overview on cis- and trans-regulatory variants affecting expression phenotypes in ventral midbrain, and how they could possibly introduce phenotypic difference associated with this brain region. In addition, our single nuclei chromatin landscapes of ventral midbrain are a rich resource for analysis on gene regulation and cell identity. Our work paves the way to apprehend full genetic makeup on the gene expression control of ventral midbrain, the result of which is important to understand the genetic background of midbrain associated phenotypes