system 33 34 2 Summary statement 35 36 This study highlights Fgf3 in a novel context where it is being part of a signalling 37 pathway of critical importance for development of hypothalamic monoaminergic cells 38 in zebrafish. Abstract 42 43 In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal 44 fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the 45 hindbrain serotonergic neurons, little is known about the development and function of 46 these cells. Here, we identify Fibroblast growth factor (Fgf)3 as the main Fgf ligand 47 controlling the ontogeny of serotonergic CSF-c cells. We show that fgf3 positively 48 regulates the number of serotonergic CSF-c cells, as well as a subset of 49 dopaminergic and neuroendocrine cells in the posterior hypothalamus. Further, 50 expression of the ETS-domain transcription factor etv5b is downregulated after fgf3 51 impairment. Previous findings identified etv5b as critical for the proliferation of 52 serotonergic progenitors in the hypothalamus, and therefore we now suggest that 53 Fgf3 acts via etv5b during early development to ultimately control the number of 54 mature serotonergic CSF-c cells. Moreover, our analysis of the developing 55 hypothalamic transcriptome shows that the expression of fgf3 is upregulated upon 56 fgf3 loss-of-function, suggesting activation of a self-compensatory mechanism. 57 Together, these results highlight Fgf3 in a novel context as part of a signalling 58 pathway of critical importance for hypothalamic development.59 60 61 62Serotonin (5-hydroxytryptamine, 5-HT) is an ancient signalling molecule present in 63 the nervous system of animals from cnidarian and bilaterian lineages (Hay-Schmidt,