The experimental work carried out has been focused on investigating the correlation between the genetic depletion of serotonin and the appearance of a scoliotic phenotype in zebrafish.
In order to describe the onset and development of the scoliotic phenotype, whole-organism morphological analysis have been performed. Specifically, for larval stages (i.e. 6, 14 and 21 dpf -days post fertilisation-) live imaging of body morphology was carried out, followed by analysis of the body axis both from dorsal and lateral view. For adult fish, (namely 3, 8 and 18 months post fertilisation) skeletal preparations on fixed fish were carried out. Briefly, samples were fixed, cleared and stained with a bone-specific dye. After imaging, local vertebral displacement was measured to quantify the severity of body axis misalignment. These experiments showed that spine misalignments appear around three weeks post fertilisation in the tph2 mutant fish, and the scoliotic phenotype worsens as the fish age.
The genetic depletion of serotonin has been assessed by quantifying the number of neurons labelled by serotonin in the tph2 mutant fish. By means of immunohistochemistry, spinal serotoninergic neurons have been stained and quantified in whole-mounted wild type, heterozygous and mutant tph2 larvae at 6, 10, 14 and 21 dpf. These experiments show an initial decrease in the number of serotoninergic neurons in tph2 mutants, which becomes a complete depletion by 14 dpf.
In order to identify the serotoninergic neuronal populations that are lost in the tph2 mutants, live imaging of the tph2-tagged transgenic line Tg(tph2:GAL4;UAS-NTR:mCherry) has been performed at 6, 10 and 14 dpf. Thanks to this transgenic line, the cells expressing tph2 are visible in vivo and their morphology can be characterised. Different serotoninergic neuronal populations have been found to labelled by the transgene, namely serotoninergic interneurons and sensory neurons, among which are Rohon Beard, Dorsal Root Ganglia (DRG) neurons and lateral line afferent neurons.
The Reissner fibre, a proteinous thread that in fish runs in the central canal of the spinal cord, is necessary to maintain a straight body axis during embryonic development. To assess that the spine misalignment described is not correlated with a defective Reissner fibre, live imaging of wild type, heterozygous and mutant tph2 fish in the Tg(sspo:sspo-GFP) transgenic line was carried out. Thanks to the sspo:sspo-GFP transgene, the Reissner fibre can be visualised in vivo, and it was found to be intact in tph2 mutant fish both at 6 and 20 dpf.
The spine undergoes a segmentation process during development, which if defective can lead to body axis deformities. In order to assess this, tph2 wild type, heterozygous and mutant larvae in the Tg(col9a2:GFP-caax;entpd5:pkRed) double transgenic line have been imaged in vivo at 6 and 20 dpf. Thanks to the double transgene the developing vertebrae are visible in red and intervertebral discs in green. These experiments show no difference in the segmentation pattern of tph2 mutant fish compared to heterozygous and wild type.
