In collaboration with the Kintner lab, Jacobs helped develop an assay that allowed postdoctoral researcher and first author Brian Mitchell, Ph.D., to determine the polarity of cilia by scoring the orientation of hundreds of basal feet in Xenopus larvae, whose skin is covered with multi-ciliated cells.
Analyzing skin explants taken during early embryonic development, Mitchell discovered that, early on, when ciliated cells begin to differentiate in the skin but have not yet generated a significant fluid flow, all cilia roughly point towards the back end of the frog larvae. Within a few hours the cilia refine their polarity and converge precisely on a common axis. If he removed the skin samples before the frontback axis was established in the larvae, ciliated cells were unable to decide on a common direction.
Mutations in the gene DNAI1, which render cilia immotile, account for about 10 percent of all human cases of PCD, while the loss of functional Spag6 and TEKT2A/B, both essential components of cilia, cause a PCD-like disease in mice. "When we shut down these genes in Xenopus larvae, we could mimic the clinical observations in PCD patients," says Mitchell.
"The polarity of the cilia is still biased towards the posterior, but without a detectable flow the process of refinement is disrupted and they can't reorient themselves properly," says Kintner and adds that, "this model explains why ciliary disorientation is so commonly associated with ciliary dysfunction in human PCD."
For the final and most compelling test, the Salk researchers teamed up with experts in the study of flow on cells from blood vessels. With the help of Julie Li, Ph.D., and professor Shu C
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Contact: Mauricio Minotta
minotta@salk.edu
858-453-4100, ext. 1371
Salk Institute
22-Apr-2007