Loss of Nkd1 is dominant over loss of Axin2 in regulating Wnt signaling.

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 β-catenin, and Nkd1 functions to inhibit the nuclear localization of β-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2-/-, nkd1-/- single mutants and axin2-/-;nkd1-/- 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 axin2-/-;nkd1-/- double mutants. Further, Wnt target gene expression by qRT-PCR, and RNA-seq analysis and protein expression by mass spec demonstrated that the double axin2-/-;nkd1-/- mutant resembled the nkd1-/- phenotype demonstrating that Axin functions upstream of Nkd1 and that loss of Nkd1 is dominant over the loss of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of β-catenin-dependent transcription having novel readouts of Wnt activity compared to nkd1-/- or the axin2-/-;nkd1-/- double mutant. We also tested 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/β-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. To investigate Axin2 and Nkd1 regulation of Wnt signaling, axin2-/- and nkd1-/- single knockout zebrafish were created using sgRNA/Cas9. These were crossed together to create axin2-/-;nkd1-/- double knockout zebrafish. The nkd1-/- zebrafish were made in the Tu genetic background and the axin2-/- were made in the TL genetic background. The RNA-seq samples were collected at 50% epiboly with biological replicates coming from the same parents but collected on different days. Gene expression analysis of the RNA-seq data was used to determine that the nkd1-/- single mutant was similar to axin2-/-;nkd1-/- double mutant. Furthermore, gene expression analysis was used to assess Wnt target gene expression in the mutants