Abstract
Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles.
Highlights
Hedgehog (Hh) proteins play a fundamental role in animal development, controlling cell type specification, proliferation and survival in a variety of contexts through a signaling pathway, the core components of which are shared across species [reviewed in 1,2,3]
Studies in mouse have implied that Kif7 functions principally in the primary cilium, an organelle required for Hh signaling in vertebrates but absent from most Drosophila cells, suggesting a divergence in the mechanisms of action between phyla
We find that Kif7 acts principally to restrain Gli1 activity and suggest that it functions to sequester the Gli transcription factors, similar to its Drosophila counterpart Cos2, which we show can partially substitute for Kif7 function in the zebrafish embryo
Summary
Hedgehog (Hh) proteins play a fundamental role in animal development, controlling cell type specification, proliferation and survival in a variety of contexts through a signaling pathway, the core components of which are shared across species [reviewed in 1,2,3]. Cos physically interacts with the Gli family protein Cubitus interruptus (Ci), restraining the transcriptional activating activity of its full length form both by anchoring it in the cytoplasm as well as by recruiting the serine-threonine kinases that prime it for processing into a truncated transcriptional repressor [10,11,12]. Consistent with these effects, loss of Cos activity results in the ectopic activation of Hh target genes, both in embryos and imaginal discs [13,14]. In addition to its negative regulatory role, Cos has been shown to potentiate Hh pathway activity by promoting the dissociation of Ci from Suppressor of Fused (Sufu) [15], another negative pathway regulator that acts to inhibit nuclear import of Ci [10,16]
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