Identification and Characterization of Foxj1-induced Genes in Motile Cilia
Supervisor: Sudipto Roy
Motile cilia beat rhythmically to direct fluid flow over epithelia and drive cellular locomotion. Dysfunctional cilia lead to a variety of human diseases, collectively called ciliopathies. Our group previously discovered that in vertebrates, the forkhead domain containing transcription factor Foxj1 is the master regulator of motile cilia differentiation. Given that there are currently no genome-wide organism based screens for ciliary genes, we carried out the first such large-scale screen for ciliary genes using the zebrafish as the model system.
We first identified the targets of Foxj1 using microarray analysis. By using several validation tools, we showed that the identified list of direct and indirect targets of Foxj1, called as Foxj1-induced genes (FIGs), contain many novel motile ciliary genes. Further, I knocked down 50 randomly selected FIGs using morpholinos, and found that this led to the development of cilia-dysfunction phenotypes in 31 out of the 50 selected candidates, thereby yielding a hit rate of 62% based on discernable phenotypes in zebrafish embryos. On extrapolating these findings, we predict that the list of FIGs contain 252 to 383 novel cilia genes at the 95% confidence interval.
Additionally, I have reported the characterization of the first leucine-rich repeat encoding gene – lrrc9, whose knockdown led to shortening and axonemal disorganization of motile cilia. Thus, our collection of FIGs contains several novel ciliary genes, and is a valuable resource for advancing our understanding of ciliary biology and aiding in the quest for genes whose mutations may lead to ciliary disorders.
Figure legend: Loss of lrrc9 leads to significant reduction in ciliary length.
In the wild-type zebrafish embryo, ciliary axonemes within the kidney duct, labeled by anti-acetylated tubulin antibody (green), appear long and continuous. Loss of Lrrc9 leads to a significant reduction in ciliary length. Basal bodies are marked by gamma tubulin antibodies (red). DAPI marks nuclei (blue). Scale bar = 5 μm.
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