The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signalling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. We used light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics and demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish Wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP secretes from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porc inhibitor Wnt-C59 (C59), which applied early reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane localized and secreted Wnt3 during cerebellum development.