The cilium has emerged as a critical cellular organelle in the pathology of kidney cystic disease, retin degeneration, hydrocephalus, and left-right asymmetry defects. Now that a large number of proteins have been identified as part of the """"""""cilia proteome"""""""", we propose as the central hypothesis of this application that a significant number of human disease genes will be linked to novel genes that are currently represented in the cilia proteome but are yet uncharacterized. We have exploited the zebrafish to identify novel genes involved in cilia formation and to determine the physiological roles of cilia in kidney, brain, and the generation of left- right asymmetry. Zebrafish are particularly well-suited for high-throughput analysis of the cilia proteome because 1) cilia function can be studied in living embryos in multiple organ systems, 2) both sensory and motile cilia can be studied, 3) phenotypes related to cilia defects have been well characterized, and 4) forward and reverse genetic approaches to gene function are well established.
In Aim 1 we plan to address the lack of useful phenotyping tools for high-throughput analysis of the cilia;proteome by generating new epitope-tagged cilia protein-expressing transgenic lines of zebrafish to image cilia, basal bodies and the localization of IFT proteins in living embryos. We will also make transgenics that report that activity of sensory cilia (calcium indicator transgenics). Proteomic and genomic approaches have identified hundreds of proteins associated with cilia and each o these is a candidate human disease gene.
In Aim 2 we will conduct a systematic analysis of cilia proteome genes that are highly conserved but completely uncharacterized. Zebrafish homologs of novel cilia genes will be targeted with antisense morpholino oligos and morphant embryos subjected to a battery of assays including live imaging, confocal immunofluorescence of cilia and basal body markers, and cilia sensory signaling.
In Aim 3 we will positionally clone the schmalhans gene, a novel gene associated with cilia motility and a candidate gene for human primary ciliary dyskinesia. The overall goal of these studies is to 1) identify novel ciliogenic genes 2) generate new tools to study cilia formation and 3) categorize the large number of cilia associated proteins in terms of their function(s).

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053093-14
Application #
8055576
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Hoshizaki, Deborah K
Project Start
1997-08-15
Project End
2013-03-31
Budget Start
2011-04-01
Budget End
2013-03-31
Support Year
14
Fiscal Year
2011
Total Cost
$344,579
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Bergboer, Judith G M; Wyatt, Cameron; Austin-Tse, Christina et al. (2018) Assaying sensory ciliopathies using calcium biosensor expression in zebrafish ciliated olfactory neurons. Cilia 7:2
Noonan, Haley R; Metelo, Ana M; Kamei, Caramai N et al. (2016) Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma. Dis Model Mech 9:873-84
Oltrabella, Francesca; Pietka, Grzegorz; Ramirez, Irene Barinaga-Rementeria et al. (2015) The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule. PLoS Genet 11:e1005058
Bizet, Albane A; Becker-Heck, Anita; Ryan, Rebecca et al. (2015) Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization. Nat Commun 6:8666
Jin, Daqing; Ni, Terri T; Sun, Jianjian et al. (2014) Prostaglandin signalling regulates ciliogenesis by modulating intraflagellar transport. Nat Cell Biol 16:841-51
Pathak, Narendra; Austin-Tse, Christina A; Liu, Yan et al. (2014) Cytoplasmic carboxypeptidase 5 regulates tubulin glutamylation and zebrafish cilia formation and function. Mol Biol Cell 25:1836-44
Thomas, Sophie; Wright, Kevin J; Le Corre, Stéphanie et al. (2014) A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium. Hum Mutat 35:137-46
Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M et al. (2014) Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis. Am J Physiol Renal Physiol 307:F1334-41
Palmyre, Aurélien; Lee, Jeongeun; Ryklin, Gennadiy et al. (2014) Collective epithelial migration drives kidney repair after acute injury. PLoS One 9:e101304
Slaats, Gisela G; Ghosh, Amiya K; Falke, Lucas L et al. (2014) Nephronophthisis-associated CEP164 regulates cell cycle progression, apoptosis and epithelial-to-mesenchymal transition. PLoS Genet 10:e1004594

Showing the most recent 10 out of 54 publications