Modulating expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics

Abstract

The boundaries of brain regions are associated with the tissuespecific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we 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 is secreted 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 Porcn inhibitor Wnt-C59 (C59), which, when 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.

Modeling of Wnt-mediated tissue patterning in vertebrate embryogenesis

“…With the cytoneme-based transport mechanism, more cells adapted their final fate and hence the tissue established the proper proportions of the pattern much earlier in development than with the diffusion-based transport. For instance, 75% of all cells adopt their final fate after 55 minutes with the cytoneme-based transport, whereas the diffusive transport is almost two times slower in reaching this threshold (t = 80minutes), when choosing the diffusion constant to be D = 0.1 μm 2 /s similar to the measured value of Wnt3-GFP [ 55 ]. Even for a ligand which diffuses orders of a magnitude faster patterning occurs at a similar time point or later (cf.…”

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments

“…These transgenic lines were used as reporters of Wnt/β‐catenin signaling activity. Double transgenic embryos from in‐cross of Tg(‐4.0 wnt3 :Wnt3EGFP) F2 and Tg(‐4.0 wnt3 :Wnt3EGFP) F3 , which express functional fusion protein Wnt3‐EGFP in the brain, were screened to obtain appropriate Wnt3‐EGFP expression for SPIM‐FCS studies .…”

Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions

“…Our group showed that in zebrafish embryos, Wnt3 associates with domains on the membrane ( Azbazdar et al, 2019 ; Ng et al, 2016 ; Sezgin et al, 2017 ). Blocking the activity of Porc and thus reducing Wnt acylation resulted in reduced domain confinement and defective brain development in zebrafish embryos ( Ng et al, 2016 ; Teh et al, 2015 ). The understanding of the Wnt3 action mechanism in zebrafish brain development, therefore, requires identifying its source regions, determining its mode of transport, demarcating receiving target tissues, and measuring Wnt3-receptor interactions.…”