Gomes Da Silva Martinho, Renato; (2019) Genetic and functional analyses of the developing asymmetric zebrafish habenula. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The vertebrate brain develops anatomical and functional left-right asymmetries in localised regions, without affecting the laterality of the surrounding structures. To understand how brain laterality develops, we studied the development of robust habenular asymmetries in larval zebrafish. The left and right dorsal habenulae (dHb) have different sizes, asymmetric patterns of gene expression and establish distinct afferent and efferent connections. Although we are beginning to understand the molecular pathways that establish these asymmetries, the pathways underlying formation of the habenular progenitors remains largely elusive. Also, despite several habenular gene expression patterns being broadly asymmetric, there are no habenular markers for smaller habenular neuronal subpopulations in 4 dpf zebrafish. Lastly, we still need to investigate the importance of habenular asymmetry for its correct function and normal behaviour. This thesis aims to tackle these three gaps in habenular asymmetry research. To do so, we first characterised the A66u757 mutant, which develops a smaller and symmetric habenula. The causative mutation is in the rerea gene, which encodes a co-regulator of nuclear receptors that modulates the expression of fgf8. In line with this, the pattern of fgf8 expression is expanded in the diencephalon of mutants, and the parapineal is malformed, as observed in other FGF signalling mutants. Lastly, we show that the habenular phenotype of these mutants is concomitant with a delay in the formation of habenular progenitors. Second, to understand which neuronal subpopulations compose the zebrafish habenula at 4 dpf, we developed a protocol to obtain habenular single cells for RNA sequencing. Cells collected from the left and right nuclei still express habenular genes in an asymmetric fashion. However, to increase the number of collected cells, we harvested GFP-positive cells from a transgenic line that expresses GFP in the dHb and in the olfactory organ. Consequently, we sequenced 586 cells, which were separated into dHb, ventral habenula and olfactory organ clusters. However, due to the relatively small number of sequenced cells, we did not manage to discriminate subpopulations of the dHb. We propose optimisation steps that will allow us to finish this work. Lastly, to study the role of habenular asymmetry in behaviour, we tested two habenular mutants in an operant learning paradigm, through the ROAST assay. We show that wild-type and left-isomerised dHb mutant larvae learn to terminate an aversive stimulus by changing the direction of a stereotypical aversive-heat-response. Despite not finding differences between these mutants and wild-types, we did not exclude the role of the habenula in this assay and propose future research to further test this function.

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