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Regulatory Mechanisms Controlling Maturation of Serotonin Neuron Identity and Function.

ABSTRACT: The brain serotonin (5-hydroxytryptamine; 5-HT) system has been extensively studied for its role in normal physiology and behavior, as well as, neuropsychiatric disorders. The broad influence of 5-HT on brain function, is in part due to the vast connectivity pattern of 5-HT-producing neurons throughout the CNS. 5-HT neurons are born and terminally specified midway through embryogenesis, then enter a protracted period of maturation, where they functionally integrate into CNS circuitry and then are maintained throughout life. The transcriptional regulatory networks controlling progenitor cell generation and terminal specification of 5-HT neurons are relatively well-understood, yet the factors controlling 5-HT neuron maturation are only recently coming to light. In this review, we first provide an update on the regulatory network controlling 5-HT neuron development, then delve deeper into the properties and regulatory strategies governing 5-HT neuron maturation. In particular, we discuss the role of the 5-HT neuron terminal selector transcription factor (TF) Pet-1 as a key regulator of 5-HT neuron maturation. Pet-1 was originally shown to positively regulate genes needed for 5-HT synthesis, reuptake and vesicular transport, hence 5-HT neuron-type transmitter identity. It has now been shown to regulate, both positively and negatively, many other categories of genes in 5-HT neurons including ion channels, GPCRs, transporters, neuropeptides, and other transcription factors. Its function as a terminal selector results in the maturation of 5-HT neuron excitability, firing characteristics, and synaptic modulation by several neurotransmitters. Furthermore, there is a temporal requirement for Pet-1 in the control of postmitotic gene expression trajectories thus indicating a direct role in 5-HT neuron maturation. Proper regulation of the maturation of cellular identity is critical for normal neuronal functioning and perturbations in the gene regulatory networks controlling these processes may result in long-lasting changes in brain function in adulthood. Further study of 5-HT neuron gene regulatory networks is likely to provide additional insight into how neurons acquire their mature identities and how terminal selector-type TFs function in postmitotic vertebrate neurons.

SUBMITTER: Spencer WC

PROVIDER: S-EPMC5515867 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:Transcriptional programming of cell identity promises to open up new frontiers in regenerative medicine by enabling the efficient production of clinically relevant cell types. We examine if such cellular programming is accomplished by transcription factors that each have an independent and additive effect on cellular identity, or if programming factors synergize to produce an effect that is not independently obtainable. The combinations of Ngn2-Isl1-Lhx3 and Ngn2-Isl1-Phox2a transcription factors program embryonic stem cells to express a spinal or cranial motor neuron identity respectively. The two alternate expression programs are determined by recruitment of Isl1/Lhx3 and Isl1/Phox2a pairs to distinct genomic locations characterized by two alternative dimeric homeobox motifs. These results suggest that the function of programming modules relies on synergistic interactions among transcription factors and thus cannot be extrapolated from the study of individual transcription factors in a different cellular context. In this study, we functionally characterize induced motor neurons that have been directly generated from ES cells via the forced expression of two different combinations of three transcription factors. Spinal motor neurons are induced via the expression of Ngn2, Isl1, and Lhx3 (iNIL), while cortical motor neurons are induced via the expression of Ngn2, Isl1, and Phox2a (iNIP). Here we profile the gene expression patterns of both types of induced motor neurons, directed differentiation motor neurons, and control cells. In all, 20 microarray experiments are provided in this submission, including 3 replicates of a control condition, 3 replicates of cells that have 24hrs induction of iNIL, 2 replicates of induced spinal motor neurons (induction of iNIL for 48hrs) that have been Hb9-GFP sorted, 3 replicates of induced spinal motor neurons exposed to retinoic acid that have been Hb9-GFP sorted, 3 replicates of motor neurons that have been differentiated in vitro using RA and Hh signalling, 3 replicates of induced cortical motor neurons (induction of iNIP for 48hrs), and 3 replicates of cells in which Isl1 in induced alone (induction of iI for 48hrs). For ChIP-Seq Samples: In this study, we functionally characterize induced motor neurons that have been directly generated from ES cells via the forced expression of two different combinations of three transcription factors. Spinal motor neurons are induced via the expression of Ngn2, Isl1, and Lhx3 (iNIL), while cortical motor neurons are induced via the expression of Ngn2, Isl1, and Phox2a (iNIP). The genome-wide binding of some of the programming factors is characterized here using ChIP-seq. We characterize the binding of Lhx3 and Isl1/2 in iNIL cells, Phox2a and Isl1/2 in iNIP cells, and Isl1/2 in cells in which Isl1 is induced alone (iI). There are 7 Illumina sequence datasets in this submission; one replicate for each of iLhx3-V5 and Isl1/2 in iNIL cells, two replicates for each of iPhox2a-V5 and Isl1/2 in iNIP cells, and one replicate for Isl1/2 in iI cells. An appropriate pseudo-IP control experiment is included.

Project description:UNLABELLED:Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1(-/-) neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. SIGNIFICANCE STATEMENT:The regulatory mechanisms that control functional maturation of neurons are poorly understood. We show that in addition to inducing brain serotonin (5-HT) synthesis and reuptake, the Pet-1 ETS (E26 transformation-specific) factor subsequently globally coordinates postmitotic expression trajectories of genes necessary for maturation of 5-HT neuron excitability. Further, Pet-1 switches its transcriptional targets as 5-HT neurons mature from 5-HT synthesis genes to G-protein-coupled receptors, which are necessary for afferent synaptic modulation of 5-HT neuron excitability. Our findings uncover gene-specific switching of downstream targets as a previously unrecognized regulatory strategy through which continuously expressed transcription factors control acquisition of neuronal identity at different stages of development.

Project description:Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known aboutthe regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1 / neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. 5-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturationthrough secondary postmitotic regulatoryfactors. The early postnatal switch in Pet-1targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. 5-HT neuron mRNA profiles of E11.5, E15.5, and postnatal (P1-P3) wild type (WT) and Pet-1-/- mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Myc-tagged Pet-1 ChIP-seq was performed on E12.5 to E14.5 hindbrains and sequencing using NextSeq 500.

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Regulatory Mechanisms Controlling Maturation of Serotonin Neuron Identity and Function.

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