Hedgehog Signaling in the zebrafish embryo: Role of Kif7 and DZIP1

Abstract

Hedgehog (Hh) signaling was first discovered in Drosophila melanogaster and mis-regulation of this pathway has been linked to a wide spectrum of congenital abnormalities and malignancies in human. Orthologs of nearly all the core components of the Hh pathway have now been discovered in vertebrates. Subsequent studies have shown that key components of the Hh pathway are conserved through to the vertebrates. However, amongst them, the kinesin-like protein Costal2 (Cos2), which plays an important role in controlling the transcriptional activity of the Hh pathway, remained elusive. I report the cloning of a kinesin-related gene from Danio rerio zebrafish and propose that it is the first vertebrate Cos2 and is annotated as kinesin family member 7 (Kif7) in the mouse and human genome databases. Genetic and molecular analysis revealed that, similar to its function in Drosophila, zebrafish Cos2/Kif7 acts as an intracellular repressor of the Hh pathway. Cos2 has additive inhibitory effect on Hh signaling when it works together with Suppressor of Fused (Su(fu)), which is another protein that negatively regulates signaling in Hh-responsive cells. Co-immunoprecipitation shows that both the zebrafish Cos2 and Su(fu) bind to the vertebrate Hh transcription factor, Gli1. This collaborative effect of Cos2 and Su(fu) in regulating Gli activity is a conserved event in the Hh signaling cascade.

Recently, it was discovered that the primary cilia play important roles in many developmental and physiological processes. I report that the zebrafish iguana (igu) gene is critical for the differentiation of primary cilia. Igu encodes DAZ-interacting protein 1 (Dzip1) with zinc finger and coiled-coil domains and was first thought to play a role in the Hh pathway. Further molecular analyses show that the aberrant Hh activity in zebrafish igu mutants can now be explained as a result of primary cilia loss. By contrast, motile cilia formation appears less affected by the loss of Igu. In the absence of igu, basal bodies can still migrate and dock properly to the apical membrane. However, axonemal outgrowth and ciliary pits are completely absent. Igu proteins localize to the base of all cilia, and in close proximity to the basal bodies. These findings identify Igu as a novel and critical component of ciliogenesis.

The importance of primary cilium in Hh signal transduction is absolute in mammals but not in Drosophila. I showed that this requirement is also essential in zebrafish by demonstrating the localization of a key Hh component, Smoothened (Smo) to the primary cilia in response to Hh activation. This localization can be abolished by mutating a conserved cilia localizing domain. Mis-localization of Smo results in mis-regulation of the Hh pathway. Therefore, the primary cilium is an important organelle that serves as a platform for Hh components to aggregate, resulting in proper transduction of signals.