New findings suggest intrinsic gene regulatory programs are needed throughout life for the maturation and health of postmitotic neurons. Although tremendous progress has been made in elucidating the regulatory mechanisms that control early neuronal specification far less is known about the regulatory programs that act later in life in specific neuron-types. In many instances transcription factors (TFs) that play crucial early roles in neuronal differentiation, i.e. in the acquisition of transmitter identity, continue to be expressed in the mature neurons they helped to generate. This project is aimed at investigating the regulatory factors that act across the lifespan to build and sustain the serotonin (5-HT) neurotransmitter system. Serotonergic regulatory mechanisms are of particular interest as 5-HT has wide-ranging modulatory effects on central neural circuitry and altered 5-HT gene expression has been implicated in several neuropsychiatric disorders. We will use our validated conditional targeting approaches to test specific hypotheses about the structure and function of regulatory networks that are continuously active in 5-HT neurons. Our working hypothesis is that 5-HT neuron TFs, Pet-1 and Lmx1b, which are directly involved in acquisition of 5-HT transmitter identity, continue to perform critical regulatory functions that build 5-HT neuron connectivity and sustain their identity.
In aim 1, we will test the hypothesis that continuous Pet-1 expression is required to build 5-HT connectivity and prevent progressive decay of 5-HT gene regulatory networks and 5-HT neuron cell state identity. These studies will be performed with Pet-1 targeted mice using our constitutive 5-HT specific cre line, ePet-Cre, floxed Pet-1 allele and the Ai9 (RCL-tdT) reporter to mark 5-HT neurons with Td-tomato and enable sorting of postnatal neurons for RNA-seq studies at different life stage.
In aim 2, we will investigate the importance of Pet-1 and Lmx1b in sustaining the health of adult 5-HT neurons. We will individually target these TFs with our tamoxifen-inducible 5-HT neuron-type approaches to eliminate expression in adulthood. We will determine whether adult loss of these TFs causes progressive decay in 5-HT neuron identity, function, connectivity, and 5-HT modulated behaviors. We will also investigate whether these two TFs perform distinct functions and whether their targets switch later in life.
In aim 3, we will investigate two mechanistic hypotheses about how continuously expressed TFs control postmitotic neuron gene expression. In the first set of experiments we will study how Pet-1 temporally switches targets and test the hypothesis Pet-1 occupancy in 5-HT chromatin is dynamic and changes at specific genes as 5-HT neurons progress through postmitotic life. The second set of experiments will test the hypothesis that Pet-1 is a regulatory master of a postmitotic TF network that performs specific regulatory functions in 5-HT neurons. We will investigate expression and function of Pet-1 controlled TFs in 5- HT neurons. We will focus initially on the glucocorticoid receptor gene, Nr3c1, to determine its function in 5-HT neurons and how Pet-1 transcriptionally integrates 5-HT neurons with the HPA axis and stress responses.
This research project will elucidate the importance of continuously expressed neuronal transcription factors in the life long maintenance of neuronal health. We will focus our studies on Pet-1 and Lmx1b, two regulatory factors that initially perform critical roles in the acquisition of serotonin transmitter identity, by determining the respective functions of these factors in sustaining 5-HT gene regulatory networks and 5-HT neuron cell state identity. We will investigate their importance in building and maintaining 5-HT neuron connectivity and in sustaining psychiatric disease-relevant emotional behaviors.
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