A mouse molecular genetics approach to study the development and function of the serotonergic system

In mammals, neurons producing serotonin are generated during early embryonic development in the ventral region of the hindbrain where they form distinct nuclei called raphe nuclei and project to the whole CNS, from the anterior brain to the spinal cord. The evidence that the timing of appearance of this monoaminergic system in the embryonic telencephalon overlaps with proliferation, migration and neuronal differentiation, has prompted speculation long ago that 5-HT, before acting as neurotransmitter, may play a role in neural development. Consistently, several lines of evidence associate an altered serotonergic signalling with neuropsychiatric disorders in humans. Serotonergic neurons specification is controlled by a combination of transcription factors and secreted molecules such as Shh, Fgf4/8, Nkx2.2, Lmx1b and Pet1. Among these, Pet1 is a transcription factor known to be selectively expressed in serotonergic neuron precursors. In the mouse, Pet1 mRNA expression appears between 10 and 11 dpc (days post coitum), about one day before serotonin is synthesized, and it persists in serotonergic neurons up to adulthood, where it promotes the expression of the key enzyme for the serotonin synthesis in the CNS, Triptophan hydroxylase 2 (Tph2). The use of suitable genetic tools in mouse such as the Pet1/Cre transgenic mouse line and Tph2::eGFP knockin mouse line are crucial to address questions about the development of the serotonergic system and the role of 5-HT in development. Pet1/Cre mouse line allows the genetically fate mapp Pet1 expressing cells, by expressing Cre recombinase under the transcriptional control of a 210 Kb promoter region of the Pet1 gene. Tph2::eGFP knock-in mouse line, in which the eGFP replaces the Tph2 coding region, allows to study the effects of lack of serotonin on development. Moreover, thanks to the presence of eGFP, Tph2::eGFP mouse line permits to trace serotonergic neurons and their projections in Tph2 mutant mice lacking serotonin. Eventually, a floxed-Tph2 knock-in mouse line, once generated, will allow a conditional, time and space-controlled, inactivation of serotonin synthesis within the brain. The use of such models allowed to uncover that Pet1 gene expression is not restricted to the serotonergic neurons as previously described but, rather, it is expressed within the central nervous system in a non-serotonergic population of neurons, and, in periphery, Pet1 is activated early in the developing pancreas and kidney. Analysis of Tph2::eGFP mouse line showed that lack of central serotonin results in severe abnormalities in the serotonergic circuitry formation with a brain region-specific effect. Further molecular analysis showed that serotonin modulates specific factors to control the formation of its own circuitry. This results allowed us to put forward the hypothesis that serotonin acts on the serotonergic system development via the combination of both a cell- and non-cell-autonomous mechanism.