Serotoninergic Regulation of Body Axis Morphology

Informations projet

SeRBoMo

N° de convention de subvention: 101106749

DOI

10.3030/101106749

Projet clôturé

Date de signature de la CE 26 Avril 2023

Date de début 21 Juin 2023

Date de fin 20 Juin 2025

Financé au titre de

• Marie Skłodowska-Curie Actions (MSCA)

Coût total

Aucune donnée

Contribution de l’UE

€ 195 914,88

Coordonné par

INSTITUT DU CERVEAU ET DE LA MOELLE EPINIERE
France

Periodic Reporting for period 1 - SeRBoMo (Serotoninergic Regulation of Body Axis Morphology)

Période du rapport: 2023-06-21 au 2025-06-20

Résumé du contexte et des objectifs généraux du projet

Idiopatic scoliosis (IS) is a human pathology that affects 1-4% of adolescents worldwide, who suffer from spine deformities that arise during puberty. At present, evidence for genetic causes of IS exist, but the aetiology of this pathology is still not understood.

As of now, no effective treatment is available for IS. This is partly due to the impossibility of early diagnosis, as IS can be detected only when spine curvatures are already visible in patients. This leads to painful treatment and life conditions, which are often not improved after bracing, as well as costly surgery and expensive orthopaedic equipment. The combination of these two factors induces only ~3K patients/year in Europe to undergo surgery, out of a total number of ~20K severe cases who would need treatment. For these reasons, understanding the fundamental caused of IS will help informing clinical research, ultimately ameliorating the life condition of patients.

The longterm goal of this research project is to gain insights into novel physiological mechanisms leading to a defective body axis alignment, which are also present in IS patients.

To this end, zebrafish has proven to be a powerful organism to model human pathologies that entail body axis misalignment as, by swimming in a viscous liquid, its spine is subject to forces similar to the ones acting on the spine in bipedal position. Surprisingly, a scoliotic-like phenotype arises in the tph2ct817/817 mutant zebrafish. Tph2 is one of the rate-limiting enzymes for serotonin synthesis, so that the mutant fish are genetically depleted of serotonin in some neurons of the CNS (Central Nervous System). The overarching aim of this research project is to unravel how the neuronal activity of serotoninergic neurons can control the maintenance of a straight longitudinal body axis during the zebrafish post embryonic life.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

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.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

In the tph2ct817/ct817 mutant fish, spine misalignments appear around three weeks post fertilisation, a time when the larval fish grow quickly in size, which is comparable to the growth spurt occurring in humans during adolescence. These spinal malformations are not paired to vertebral defects, and spinal curvatures worsen with the age of the fish; these are also two features shared with the human pathology. Overall, these findings indicate that the tph2 mutant fish can serve as an appropriate model for studying AIS (adolescence idiopathic scoliosis). Representative images are reported in panel A and B of the figure uploaded.

By two weeks post fertilisation, the trunk of tph2 mutant fish is completely devoid of serotoninergic cell bodies and projections. In contrast, wild type and heterozygous fish show an increase in the number of serotoninergic neurons in the trunk as the animal grow. These results show that the depletion of serotoninergic transmission in the entire trunk is completed before the onset of spinal misalignment. Representative images are reported in panel C and D of the figure uploaded.


The Reissner fibre, which is necessary to develop a straight axis during embryonic development, is not defective in tph2 mutant fish. Representative images are reported in panel E of the figure uploaded.

Among the serotoninergic trunk populations that are depleted of serotonin in the tph2 mutant fish, are three sensory types of neurons, namely Rohon Beard neurons, DRG neurons and lateral line afferent neurons. These results point to the possibility that a defective proprioception, which is sustained during the entire life of the fish, can lead to spine misalignment. One representative image of the whole trunk of a 6dpf larvae is reported in panel F of the figure uploaded.

representative figure for the main findings of the project