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Live Imaging of Epithelial Development in Mutant Drosophila Embryos

Deborah Andrew, Cell Biology and Biomedical Engineering, Johns Hopkins School of Medicine.
Funding: R01DE12873 (01/02/99-06/30/09) R01DE13899 (02/01/01 – 04/31/11).

Morphogenesis requires simultaneous force generation and cell deformation. Although the transcriptional control of force generation has been studied extensively, the concurrent modulations of cellular material properties that enable deformation remain unknown. In this project we use the salivary gland and trachea of Drosophila melanogaster as model systems in an attempt to gain a better understanding of how certain highly conserved components of the apical membrane and sub-membranous cytoskeleton affect the material properties of cells involved in tube morphogenesis. Both of these organs begin as simple sheets of polarized epithelial cells on the embryo surface that, through a combination of cell shape changes and cell migration, transition into tubular organs over the course of several hours. In this collaboration we use MPM imaging to track cell shape, nuclear position and localization of cytoskeletal molecules during formation of these organs in live wild-type and mutant embryos.

Using a nuclearly-localized version of DsRed, the Andrew lab recombined this line with fly lines expressing various GFP fusion molecules and developed protocols for live imaging. These experiments involve weak GFP signals to avoid large perturbations of cellular physiology, a weak DsRed signal due to the relatively long maturation time. We need to follow these organs as they develop in a benign manner that avoids photodamage. After MPM system optimization to maximize sensitivity, these initial studies were remarkably successful (Cheshire et al, 2008).  Using mutant flies, this work investigates the BTB domain transcription factor RIBBON and its relation apical stiffness and viscosity in elongating tubes in Drosophila embryos. Live imaging of ribbon mutants indicated slowed and incomplete lumenal morphogenesis, and coupled with information from mechanical models of lumenal extension and cell morphology we believe that ribbon epithelia fail primarily in cell extension due to approximately a 2-fold increased apical stiffness and 1.6-fold increased apical viscosity. We conclude that RIBBON functions as a novel morphogenic cassette regulating material properties of the apical membrane during tube elongation.

References

Cheshire, AM, Kerman, BE, Zipfel, WR,  and DJ Andrew. 2008.   Transcriptional Control of Apical Membrane Mechanics during Tube Morphogenesis. Dev Dyn 237(10): 2874-88

 

 

 

 

 



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