Semil P. Choksi1,‡, Deepak Babu1,2, Doreen Lau1, Xianwen Yu1,* and Sudipto Roy1,2,3,4,‡
1 - Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673.
2 - NUS Graduate School of Integrative Sciences and Engineering, Centre for Life Sciences, 28 Medical Drive, Singapore 117456.
3 - Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.
4 - Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent Ridge Road, Singapore 119288.
*Present address: Department of Biological Sciences, Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.
‡Authors for correspondence (firstname.lastname@example.org; email@example.com)
Published in Development, 19 August 2014.
Please click here for the A*STAR Press Release.
Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30%of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans.
(A) We and others previously showed that the transcription factor Foxj1 is the master regulator of motile cilia differentiation (reviewed in Choksi et al., 2014a, Yu et al., 2008, Stubbs et al., 2008). In this work, we identify the genes which are regulated by Foxj1 to understand which genes are needed to make motile cilia.
(B) Using gene expression microarrays, we compared the transcriptomes of wild type zebrafish embryos to embryos overexpressing Foxj1. We identified nearly 600 genes upregulated by Foxj1 (Choksi et al., 2014b). Many of these genes (83) are known ciliary genes, however, the vast majority have never been associated with cilia before. In order to characterize this set of novel genes, we selected 50 of the genes at random and subjected them to a systematic functional screen.
(C) First, we examined the localization of the gene products of the 50 genes. This entailed expressing GFP-tagged versions of the proteins in the zebrafish (green) and simultaneously labelling the motile cilia with antibodies raised against acetylated tubulin (red). Overall, 30% of the proteins were found to localize to motile cilia.
(D) Next, we looked at the effect of knocking down each of the genes on zebrafish development. We injected morpholinos targeting each gene into the embryo, then screened for five morphological defects related to cilia dysfunction. An example phenotype is shown: hydrocephalus, or swelling of the brain ventricles.
This is often the result of an accumulation of cerebrospinal fluid in the brain ventricles due to defective motile cilia of the central nervous system. Overall, 60% of the novel genes showed at least two cilia-associated phenotypes. Finally, we examined the structure and motility of cilia in morphant embryos. Labelling the cilia of the embryonic kidney with antibodies recognizing Arl13b (green) and gamma tubulin (red), we see malformed cilia in seven different gene knockdowns. A particular morpholino-injected embryo exhibiting severely shortened cilia is shown. The knockdown of an additional nine genes significantly reduced ciliary motility.
This is the first comprehensive, functional screen for motile cilia genes performed in any vertebrate organism to date. The collection of motile cilia genes will be invaluable for our understanding of how cilia are made, and importantly, will greatly facilitate the diagnosis of cilia-based disorders in humans.
Choksi SP, Lauter G, Swoboda P, Roy S. Switching on cilia: transcriptional networks regulating ciliogenesis. Development. 2014a.
Choksi SP, Babu D, Lau D, Yu X, Roy S. Systematic discovery of novel ciliary genes through functional genomics in the zebrafish. Development. 2014b.
Stubbs JL, Oishi I, Izpisúa Belmonte JC, Kintner C. The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. Nature Genetics. 2008.
Yu X, Ng CP, Habacher H, Roy S. Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nature Genetics. 2008.
For more information on Sudipto ROY's lab, please click here.