RESUMEN
Wnt signaling is a crucial developmental pathway involved in early development as well as stem-cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic ß-catenin, and Nkd1 functions to inhibit the nuclear localization of ß-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2gh1/gh1, nkd1gh2/gh2 single mutants and axin2gh1/gh1;nkd1gh2/gh2 double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2gh1/gh1;nkd1gh2/gh2 double mutants. Further, Wnt target gene expression by qRT-PCR and RNA-seq, and protein expression by mass spectrometry demonstrated that the double axin2gh1/gh1;nkd1gh2/gh2 mutant resembled the nkd1gh2/gh2 phenotype demonstrating that Nkd1 functions downstream of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of ß-catenin-dependent transcription having novel readouts of Wnt activity compared with nkd1gh2/gh2 or the axin2gh1/gh1;nkd1gh2/gh2 double mutant. We also investigated the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/ß-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.
Asunto(s)
Proteína Axina , Vía de Señalización Wnt , Proteínas de Pez Cebra , Pez Cebra , beta Catenina , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína Axina/metabolismo , Proteína Axina/genética , beta Catenina/metabolismo , Proteínas Portadoras , Mutación/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Wnt signaling is a major player during development and its misregulation often leads to disease, especially cancer. The negative feedback Wnt regulator homologs, Nkd1 and Nkd2, have been shown to inhibit Wnt signaling during development, and current evidence suggests that Nkds degrade Dvl proteins to antagonize Wnt signaling. Here, we demonstrate that during early zebrafish development Nkd1 does not alter either endogenous or exogenous levels of Dvl2. Furthermore, Dvl2 does not affect the levels of Nkd1. Cumulatively, these results demonstrate that Dvl2 is a ubiquitous and stable protein and that Nkds may not always function to degrade Dvl proteins as a method of inhibiting Wnt signaling.