Gene regulatory mechanisms controlling development of serotonin neuron subtypes

Although significant progress has been made in understanding the genetic origins of neurodevelopmental disorders, it remains unclear what specific molecular steps are disrupted and in which specific neurons types they are dysfunctional or inoperable. One potential reason for this lack of understanding is that neurons, originally classified according the type of transmitter produced, are now well known to possess substantial molecular, cellular, and functional heterogeneity. It seems plausible that neurodevelopmental disorders may arise not only from developmentally altered identities of an entire population of one particular neuron type but also from altered development of one of its specific molecular or functional subtype(s). There has been a decades-long intense interest in the regulatory mechanisms controlling 5-HT neurons as 5-HT has wide-ranging modulatory effects on central neural circuitry and dysfunction of the serotonergic system has been implicated in several neuropsychiatric diseases including depression, stress-related anxiety disorders, autism, intellectual disability, OCD, and schizophrenia. 5-HT neurons possess tremendous molecular, morphological, and electrophysiological heterogeneity. However, developmental trajectories of 5-HT neuron subtypes are currently unknown as are the regulatory mechanisms that govern their development. The objective of the proposed research is to use single cell RNA-seq and single cell ATAC-seq to comprehensively define the spatiotemporal developmental trajectories of 5-HT neuron subtype transcriptomes and chromatin accessibility. We will combine recent advances in single-cell genomics methods together with our well- established serotonergic transgenic tools, our extensive experience in flow sorting mouse 5-HT neurons, and our bioinformatics expertise to investigate the development of single-cell 5-HT neuron transcriptomes and chromatin accessibility throughout fetal to early postnatal maturation. We will also investigate at the single cell level, the hypothesis that the two disease-associated terminal selectors in 5-HT neurons, Pet1 and Lmx1b, function to determine postmitotic 5-HT neuron subtypes through differential regulation of subtype-specific gene expression, subtype-specific chromatin accessibility and control of downstream subtype-specific transcription factor codes. Pet1 is of special interest as homozygous knockout mutations in FEV, the human ortholog of Pet1, were recently reported in two brothers with Intellectual Disability and Autism Spectrum Disorder. In Aim 1, we will define the developmental trajectories of 5-HT neuron subtypes. In Aim 2, we will investigate the control of 5-HT neuron subtype transcriptomes by Pet1 and Lmx1b. In Aim 3, we will determine the chromatin mechanisms involved in the generation of 5-HT neuron subtypes. The completion of our proposed aims will lead to a greater understanding of the subtype-specific gene regulatory networks that generate 5-HT neuron subtypes, which may help illuminate specific neurogenetic pathways that are disrupted in neurodevelopmental disorders such as ASD/ID.