In HEK-293 cells, knockdown of ITCH significantly increased Wnt-induced TOPflash activity and the accumulation of free b-catenin induced by Wnt3a. Wnt3a-mediated induction of Wnt target genes AXIN2 and NKD1 was also potentiated, suggesting that ITCH negatively PF-4217903 regulates the canonical Wnt pathway. Given this implication of Itch in the two major signaling pathways, Cxcr4 and Wnt, involved in pLL primordium migration, we investigated the effects of Itch depletion in lateral line formation in zebrafish embryos. Our study presents the first direct demonstration of the implication of the ubiquitin-ligase Itch in the regulation of signal transduction in a living organism. This asymmetric distribution directs primordium migration along the myoseptum in response to Sdf1 secretion and disturbing this equilibrium results in slowed primordium migration speed or stalling of the primordium. Wnt signaling occurs mainly in the leading region of the pLL primordium and activates Fgf signaling in the medial and trailing region. Fgf signaling organizes the primordium precursor cells in rosettes that will become the neuromasts and restricts Wnt signaling to the leading cells. The expression of cxcr4b is regulated at the transcriptional level by Wnt and oestrogen signaling, as response elements for Lef1 and Esr1 are present in the upstream control region of the cxcr4b gene. lef1 morphants and mutants demonstrate truncated pLL, as the migrating primordium collapses before it reaches the end of the tail caused by decreased cell proliferation and lack of progenitors. Lef1 depletion alone has no effect on cxcr4b or cxcr7b expression, and pLL primordium migration and differentiation appears to be normal. However, increasing Wnt signaling, as occurs in apc mutants, strongly inhibits primordium migration while increasing cxcr4b expression domain and excluding cxcr7b from the primordium. itchb morphants similarly exhibited displacement of the cxcr4b and cxcr7b expression domains, consistent with increased cxcr4b and Wnt signaling. In cultured mammalian cell lines, Itch has been shown to increase both Wnt and Cxcr4 signaling. Itch can regulate the Wnt signaling pathway by recognizing and ubiquitylating phosphorylated Dvl. Dvl is recruited to the activated Wnt receptor complex and activates both the canonical and noncanonical signaling pathways. Upon activation by Wnt, Dvl become hyperphosphorylated, and this phosphorylation is essential to fully activate b-catenin stabilization. Inactivation of Itch stabilizes phosphorylated Dvl, increasing Wnt signaling. In zebrafish, Dvl degradation has been shown to be implicated in Wnt signaling regulation. On the other hand, Dvl expression has also been shown to be stable in zebrafish embryos during primordium migration. It must be stressed that Itch specifically targets phosphorylated Dvl and promotes its proteasomal degradation. Consequently, Itch depletion could increase Wnt signaling and expression of Wnt signaling.