Direct visualization of the Wntless-induced redistribution of WNT1 in developing chick embryos

Developmental biology

Frederick Santana^1¹  Lisa M. Galli^1² 

[1] Department of Biology, Holy Names University, 3500 Mountain Boulevard, Oakland, CA 94619, USA^2;Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA^1

关键词: Palmitoylation;    Live imaging;    WNT1;    WNT3A;    WNT7A;    PORCN;    WLS;    Chick;    Zebrafish;    Neural crest;    Ectoderm;    Filopodia;    eGFP;    moxGFP;    mCherry;    Membrane trafficking;   

DOI  :  10.1016/j.ydbio.2018.04.025

学科分类：生物科学（综合）

来源: Academic Press

【 摘 要 】

Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.

【 授权许可】

CC BY