logo SBA


Digital archive of theses discussed at the University of Pisa


Thesis etd-02072008-195300

Thesis type
Tesi di dottorato di ricerca
Thesis title
Generation of a Tph2/EGFP knockin mouse line for the study of the role of serotonin during the central nervous system development
Academic discipline
Course of study
Relatore Prof. Pasqualetti, Massimo
  • Behavioural analysis
  • Central nervous system development
  • Embryonic stem cells
  • Gene targeting
  • Homologous recombination
  • Hypothalamic-pituitary axis
  • Raphe nuclei
  • Serotonin (5-HT)
  • Tryptopan hydroxylase 2 (Tph2)
Graduation session start date
Release date
Serotonergic neurons constitute one of the most widely distributed neuronal systems in the mammalian brain. These neurons, that are among the earliest to develop during embryogenesis, collectively form the raphe system and they cluster as two main groups: the caudal group projecting to the spinal cord, and the rostral providing widespread serotonergic innervation to the anterior brain. Serotonin (5-hydroxytryptamine, 5-HT) is synthesized in two steps with tryptophan hydroxylases (Tphs) as the rate-limiting enzymes. In mammals, two isoformes have been identified, Tph1 and Tph2. Tph1 expression is confined to the periphery, whereas, Tph2 is selectively expressed in the serotonergic neurons of the central nervous system (CNS).
Accordingly to their broad innervation, serotonergic neurons have been implicated in the modulation of numerous physiological processes, such as the control of appetite, sleep, memory, mood, stress and sexual behaviour. The synthesis of 5-HT and the dynamic expression of its receptors early in embryonic development, as well as the early availability of maternal 5-HT, has led to the hypothesis that this neurotransmitter could behave as growth regulator during specific developmental events. In mammals, the hypothesis for a role of 5-HT in the CNS development is supported by the appearance of serotonergic innervation from the raphe nuclei in the cortical anlage of the embryonic telencephalon by the time in which neuroepithelial cell proliferation, migration and neuronal differentiation take place. A further support to a role for 5-HT in brain development comes from a large amount of data suggesting that dysregulation of serotonergic signalling may be at the origin of those disorders thought to have developmental bases, such as schizophrenia, affective disorders, anxiety, autism and mental retardation. In order to address the role of 5-HT during CNS development it will be crucial to generate appropriate genetic models devoid of 5-HT.
In this context, the aim of my doctorate research project was to generate a suitable genetic tool for shedding light on the role of 5-HT during development of CNS. To this purpose I generated, by means of gene targeting via homologous recombination in mouse embryonic stem (ES) cells, a knockin Tph2EGFP(FRT-neo-FRT) mouse line, in which the rate-limiting enzyme for the synthesis of 5-HT, Tph2, was targeted. First, I generated a Tph2EGFP(FRT-neo-FRT) ES cells line in which the first exon of the Tph2 gene is replaced by the coding region of the Enhanced Green Fluorescent Protein (EGFP) reporter gene and the neo cassette. Tph2EGFP(FRT-neo-FRT) ES cells were injected into murine blastocysts to obtain chimeras. Tph2EGFP(FRT-neo-FRT) heterozygous animals obtained after germ line transmission from chimeras were viable up to adulthood and fertile. EGFP transcriptional activation within the Tph2 expression domain allowed the possibility to directly visualize development of serotonergic neurons and their projections as highlighted by green fluorescence.
Recently, I started the characterization of the Tph2EGFP(FRT-neo-FRT) mutant knockin mouse line both in a pure SV129/Ola and mixed genetic background. Tph2EGFP(FRT-neo-FRT)-/- animals having SV129/Ola pure genetic background show early post-natal lethal phenotype whereas Tph2EGFP(FRT-neo-FRT)-/- animals with mixed genetic background are viable and reach adulthood showing that the genetic background plays a crucial role on the penetrance of the phenotype for the Tph2 mutation. Inactivation of Tph2 activity resulted in an impairment of the growth rate that was analysed within the first month of life and that is likely due to a down-regulation of GH mRNA expression level. In parallel, several other aspects were analyzed such as the evaluation of 5-HT brain levels in Tph2 mutant as compared to wild-type animals. Moreover, expression profiling of genes involved in 5-HT metabolism was performed. Also, in order to evaluate 5-HT-related emotional states in rodents I used a battery of behavioural tests on wild type, Tph2EGFP(FRT-neo-FRT) )+/- and Tph2EGFP(FRT-neo-FRT)-/- animals. Finally, I analysed the consequences due to the absence of Tph2 activity of distinct brain structures such as the cerebral cortex.