Development and function of monoaminergic systems in the brain of zebrafish

Abstract

This thesis explores the development of monoaminergic systems in the central nervous system (CNS) of zebrafish. The serotonergic cells of the hypothalamus pose the main focus of the present work. Most vertebrates except for mammals possess serotonin (5-HT) synthesising cells in more than one region of the CNS. In zebrafish such regions are, e.g. the hypothalamus, the raphe nuclei and the spinal cord. Serotonin functions as a neurotransmitter and neuromodulator in the CNS. Presumably due to its neuromodulatory tasks hypothalamic serotonergic cells are in contact with the cerebrospinal fluid (CSF), which expands the field of potential serotonergic targets tremendously. This highlights that serotonergic CSF-contacting (CSF-c) cells are vital for the execution of many functions and behaviours. Further, the hypothalamic serotonergic clusters constitute the largest population of serotonergic cells in the CNS of zebrafish. Together, these facts emphasise the need to understand the development and function of serotonergic CSF-c cells in the hypothalamus. Few studies have dealt with this subject, hence, information about the development of these cells is scarce. The zinc-finger transcription factor fezf2, and Fibroblast growth factor (Fgf)-signalling via the ETS-domain transcription factor etv5b are known to regulate serotonergic cell development in the hypothalamus (Bosco et al., 2013; Rink and Guo, 2004). However, the main Fgf ligand responsible for this mediation has not been determined prior to this work. The present thesis identifies Fgf3 as a crucial Fgf ligand. To achieve this result three independent strategies to impair Fgf3 activity have been applied to zebrafish embryos: the fgf3t24152 mutant, an fgf3 morpholino-based knock-down and the CRISPR/Cas9 technique. The investigations show that Fgf3 regulates the development of monoaminergic CSF-c cells in the hypothalamus. Additionally, Fgf3 impacts on cells expressing the peptide hormone arginine vasopressin (avp). Most interestingly, the requirement for Fgf3 by these cells follows a caudo-rostral gradient with a higher dependence on Fgf3 by caudal cells. This also seems to be the case for dopaminergic CSF-c cells in the hypothalamus (Koch et al., 2014). Moreover, etv5b a downstream target of Fgf-signalling is demonstrated to be under the control of Fgf3. With regard to serotonergic CSF-c cell development, it is shown that fgf3 is expressed several hours before tph1a and 5-HT (Bellipanni et al., 2002; Bosco et al., 2013). Together with the result that the hypothalamus is already smaller before mature serotonergic CSF-c cells appear, this argues for an early impact of Fgf3 on serotonergic specification. This hypothesis is supported by several findings in this study: the universal decrease of proliferating cells in the hypothalamus and simultaneous increase of cell death after fgf3 impairment. Complementary cell fate experiments confirm that proliferating serotonergic progenitors need Fgf3 to commit serotonergic specification. Further, these results corroborate findings of an earlier study stating that hypothalamic serotonergic progenitors require Fgf-signalling via etv5b to maintain the progenitor pool (Bosco et al., 2013). Additionally, the transcriptome of the hypothalamus has been analysed and 13 previously overlooked transcripts of Fgf ligands are expressed at developmental stages. The transcriptome analysis provides evidence for a self-compensatory mechanism of fgf3 since expression of fgf3 is upregulated as a consequence of its own impairment. Moreover, the Fgf-signalling pathway appears to be mildly affected by fgf3 manipulation. Together, Fgf-signalling and especially Fgf3 are established to be of critical importance during hypothalamic development with effects on serotonergic, dopaminergic CSF-c and avp expressing cells. Furthermore, this thesis provides two strategies to impair the tph1a gene. Both strategies will facilitate investigations regarding the function of hypothalamic serotonergic CSF-c cells. Finally, the presented findings in this study provide insights into the emergence of the posterior recess region of the hypothalamus, thereby, contributing to the understanding of the evolution of the vertebrate hypothalamus.