Transcriptional mechanisms governing serotonin neuron axon patterning

Serotonin (5-hydroxytryptamine, 5-HT) is a major neuromodulator that influences CNS circuitry for numerous behavioral and physiological processes. The ubiquitous influence of 5-HT is achieved through an expansive and highly collateralized axonal architecture that innervates nearly all of the brain and spinal cord. Thus, it is not surprising, given such extensive reach throughout the CNS, that disruption of serotonergic signaling is linked to a wide range of neuropsychiatric disorders. 5-HT innervation of the forebrain arises largely from 5-HT neuron cell bodies located in the midbrain/pons raphe nuclei (B4-B9) while 5-HT projections to the spinal cord originate mostly from medullary raphe nuclei (B1-B3 nuclei). However virtually nothing is known about the intrinsic regulatory factors that control 5-HT axonal growth and guidance. The LIM-homeodomain transcription factor, Lmx1b, plays an essential role in 5-HT neuron terminal differentiation through its activation of 5-HT neuron transmitter identity genes. Does Lmx1b solely regulate the network of genes required to maintain identity or does it have a broader role to control further features of serotonin neurons such as axonal projections? To investigate whether Lmx1b regulates features of serotonin neurons such as axonal projections, we generated an Lmx1b floxed mouse line that expresses Cre recombinase specifically in newborn 5-HT neurons while simultaneously labeling 5-HT axons with Td-Tomato (Lmx1bfl/fl;ePet-Cre;Ai9). Using this mouse model, we can visualize axons, dendrites, and the 5-HT mutant cell bodies in Lmx1bcKO animals and assess any differences in these features compared to control animals. Further, we have performed RNA sequencing (RNAseq) of flow sorted TdTomato+ 5-HT mutant (Lmx1bcKO) and control cells to assess gene regulation differences.



The mechanisms by which transcription factors (TFs) regulate early neuronal development and differentiation is fairly well established, however very little is understood about the exact role of these TFs in controlling postmitotic neuron function.  Many TFs that are critical to the fate specification and neurotransmitter identity of neurons continue to be expressed throughout life, but what their “late functions” are remain largely unidentified. Recently, cell-type specific genetic tools have been developed that allowed us to study the importance of continued expression of these traditionally “developmental TFs”. Recent studies have suggested that the continued expression of some of these developmental TFs is required in postnatal life to maintain neuron identity.  Importantly, findings have also suggested that deficiencies in these TFs in later life can cause neuronal dysfunction and abnormal behavioral phenotypes such as anxiety and neurodegenerative locomotor defects.   The maintenance of the serotonin (5-hydroxytryptamine, 5-HT) neurotransmitter identity is of particular interest because of its role in modulating an enormous array of behaviors including sleeping feeding, learning, mood and aggression.  Additionally, 5-HT system  dysfunction has been implicated in several neurodevelopmental psychiatric disorders such as major depression, schizophrenia, and anxiety. Recent studies on 5-HT neuron differentiation factors, Pet-1 and Lmx1b show that these developmentally crucial TFs continue to function throughout the life of 5-HT neurons.  However, the exact role these factors play in postnatal 5-HT neurons remains unclear.  We aim to investigate these functions using genetic tools that allow for the conditional knock out Pet-1 and Lmx1b in adulthood. 


investigation of adult brain serotonin deficiency

Serotonin (5-HT) is a crucial neuromodulator linked to many psychiatric disorders. However, after more than 60 years of study, its role in behavior remains poorly understood, in part because of a lack of methods to target 5-HT synthesis specifically in the adult brain. We have developed a genetic approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system by stereotaxic injection of an adeno-associated virus expressing Cre recombinase (AAV-Cre) into the midbrain/pons of mice carrying a loxP-conditional tryptophan hydroxylase 2 (Tph2) allele. We investigated the behavioral effects of deficient brain 5-HT synthesis and discovered a unique composite phenotype. Surprisingly, adult 5-HT deficiency did not affect anxiety-like behavior, but resulted in a robust hyperactivity phenotype in novel and home cage environments. Moreover, loss of 5-HT led to an altered pattern of circadian behavior characterized by an advance in the onset and a delay in the offset of daily activity, thus revealing a requirement for adult 5-HT in the control of daily activity patterns. Notably, after normalizing for hyperactivity,wefound that the normal prolonged break in nocturnal activity (siesta), a period of rapid eye movement (REM) and non-REM sleep, was absent in all animals in which 5-HT deficiency was verified. Our findings identify adult 5-HT as a requirement for siestas, implicate adult 5-HT in sleep–wake homeostasis, and highlight the importance of our adult-specific 5-HT-synthesis-targeting approach in understanding 5-HT’s role in controlling behavior.


Transcriptional Target Switching Postnatally Regulates Maturation of Serotonin Neurons

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.