PhD Graduate: Shang Yew TAY
Thesis Title: Hedgehog Signaling in the Zebrafish Embryo: Role of Kif7 and DZIP1
Signaling by the Hedgehog (Hh) family of lipid-modified morphogens has a profound role in controlling cell fate specification and pattern formation in most metazoans. Given this central role of the Hh pathway in normal development and physiology, it is unsurprising that aberrations in Hh signal transduction lie at the heart of the most common forms of human cancers. Orthologues of nearly all of the core components of the Hh pathway, defined originally through genetic analysis in Drosophila, have now been discovered in vertebrates and shown to have highly conserved functions. The one striking exception to this rule has been the kinesin-like protein Costal2 (Cos2), which plays a pivotal role in controlling the activity of the zinc-finger-containing transcriptional regulator, Cubitus interruptus/Gli, that modulates all Hh-dependent target gene expression. The first part of this thesis reports the cloning of a kinesin-related gene from the zebrafish that in structure as well as function, represents the first vertebrate orthologue of Cos2 (also called Kinesin family member 7 (Kif7)). Using a combination of genetic and biochemical analysis, we provide evidence that as in Drosophila, zebrafish Cos2/Kif7 acts principally as an intracellular repressor of signal transduction, in conjunction with Suppressor of Fused, another protein that negatively regulates signaling in Hh-responsive cells. The second part of the thesis embodies work on the zebrafish iguana (igu) gene. The igu locus encodes a zinc finger and coiled-coil containing protein, DZIP1, that was previously implicated in Hh signaling. It is now argued that aberrant Hh activity in Igu/DZIP1-deficient zebrafish arises from their severe lack of primary cilia. In the absence of Igu/DZIP1 function, basal bodies can migrate to the cell surface and appear to engage with the apical membrane. However, formation of ciliary pits and axonemal outgrowth is completely inhibited. Igu/DZIP1 localizes to the base of primary and motile cilia, in the immediate vicinity or closely associated with the basal bodies. These findings identify the Igu/DZIP1 protein as a novel and critical component of ciliogenesis.
Figure Legend: (A) Muscle cells in a wild-type zebrafish embryo stained with antibodies to the homeodomain proteins Prox1 (green) and Engrailed (red). Muscle nuclei expressing both proteins appear yellow. Formation of these cells requires Hh signaling. (B) Prox1 and Engrailed expressing muscle cells are produced in over-abundance in embryos deficient in Cos2/Kif7, signifying ectopic Hh signaling. (C) TEM through the neural tube of a wild-type embryo showing a primary cilium. The basal body (long arrow), axoneme (short arrow) and apical membrane of the ciliary pit (arrowhead) are indicated. (D) Similar section of an igu mutant embryo showing an apically docked basal body (long arrow), but complete absence of axoneme (short arrow) and ciliary pit (arrowhead).
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