RESUMO
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.
Assuntos
Perfilação da Expressão Gênica/métodos , Imagem Óptica/métodos , Proteína Wnt1/metabolismo , Aciltransferases/metabolismo , Animais , Embrião de Galinha , Galinhas/metabolismo , Ectoderma/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipoilação , Proteínas de Membrana/metabolismo , Camundongos , Crista Neural/metabolismo , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Proteína Wnt1/fisiologia , Proteína Wnt3A/metabolismo , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: WNTLESS (WLS) is a multi-transmembrane protein that transports Wnt ligands from the Golgi to the cell surface. Although WLS loss-of-function experiments in the developing central nervous system reveal phenotypes consistent with defects in WNT1 and WNT3A signaling, data from complementary gain-of-function experiments have not yet been reported. Here, we report the phenotypic consequences of WLS overexpression in cultured cells and in the developing chick spinal cord. RESULTS: Overexpression of small amounts of WLS along with either WNT1 or WNT3A promotes the Wnt/ß-catenin pathway in HEK293T cells, while overexpression of higher levels of WLS inhibits the Wnt/ß-catenin pathway in these cells. Similarly, overexpressed WLS inhibits the Wnt/ß-catenin pathway in the developing spinal cord, as assessed by cell proliferation and specification. These effects appear to be Wnt-specific as overexpression of WLS inhibits the expression of FZD10, a target of ß-catenin-dependent transcription. CONCLUSIONS: Our results show that overexpression of WLS inhibits Wnt/ß-catenin signaling in the spinal cord. As the activation of the Wnt/ß-catenin pathway in the spinal cord requires WNT1 or WNT3A, our results are consistent with a model in which the relative concentration of WLS to Wnt regulates WNT1/3A signaling in the developing spinal cord.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Animais , Proliferação de Células , Embrião de Galinha , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Medula Espinal/metabolismoRESUMO
BACKGROUND: WNT1 and WNT3A drive a dorsal to ventral gradient of ß-catenin-dependent Wnt signaling in the developing spinal cord. However, the identity of the receptors mediating downstream functions remains poorly understood. RESULTS: In this report, we show that the spatiotemporal expression patterns of FZD10 and WNT1/WNT3A are highly correlated. We further show that in the presence of LRP6, FZD10 promotes WNT1 and WNT3A signaling using an 8xSuperTopFlash reporter assay. Consistent with a functional role for FZD10, we demonstrate that FZD10 is required for proliferation in the spinal cord. Finally, by using an in situ proximity ligation assay, we observe an interaction between FZD10 and WNT1 and WNT3A proteins. CONCLUSIONS: Together, our results identify FZD10 as a receptor for WNT1 and WNT3A in the developing chick spinal cord.