Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish
Article first published online: 4 OCT 2011
Copyright © 2011 Wiley-Liss, Inc.
Volume 240, Issue 11, pages 2452–2465, November 2011
How to Cite
Clark, B. S., Winter, M., Cohen, A. R. and Link, B. A. (2011), Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish. Dev. Dyn., 240: 2452–2465. doi: 10.1002/dvdy.22758
- Issue published online: 19 OCT 2011
- Article first published online: 4 OCT 2011
- Manuscript Accepted: 31 AUG 2011
- National Institutes of Health. Grant Numbers: T32EY014536, R01NS076709, R01EY014167
- National Eye Institute Core Facilities. Grant Number: P30EY001931
- E. Matilda Ziegler Foundation
- endosome dynamics; neuroepithelia; organelle polarization; zebrafish transgenic lines
The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant-negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions. Developmental Dynamics 240:2452–2465, 2011. © 2011 Wiley Periodicals, Inc.