Intravital Imaging and Function of Cilia in Response to Kidney Injury
We are using mouse lines with fluorescently tagged cilia and bioreporters, intravital fluorescence confocal microscopy, and a surgically implanted abdominal window approach to image cilia in intact nephrons in living mice. Cilia are typically deflected in the direction of tubule flow under normal conditions. However, when flow is impaired, cilia behavior changes and they begin to oscillate and can elongate or regress. Impaired tubule flow also occurs following injury. Thus, one objective of this project is to investigate which in vivo conditions induce changes in cilia morphology, length, and number, ciliary Ca2+ fluctuations, and changes in transcriptional activity in response to normal flow, following injury and during repair. Applying an intravital imaging strategy to address these questions is innovative and is needed to understand renal cilia function and how the cilia respond to and regulate injury and repair mechanisms associated with cyst development.
Ciliary Protein Functions in Development
We are using both null and conditional mouse mutant models to examine the function of various proteins required in cilia during mammalian development. A variety of techniques including contrast-enhanced uCT, histology, and immunofluorescence staining are being used to analyze how loss of novel proteins affects embryogenesis due to functions within cilia. We have identified proteins essential for normal cardiac development, neural tube patterning, and craniofacial development and have begun to investigate how these proteins function in relation to primary cilia to regulate tissue morphogenesis.
Roles for Ciliary Protein Complexes in Maintaining Ciliary Form and Function
We are using the invertebrate model organism, C. elegans, to examine how the MKS and NPHP complexes at the transition zone create a selectively permeable barrier to maintain the unique composition of the ciliary compartment. In addition, we are investigating how the BBSome interacts with the MKS and NPHP complexes to shuttle ciliary proteins across the transition zone. We have conducted two large-scale EMS mutagenesis screens on animals harboring mutations in critical transition zone genes to identify novel genetic interactions that are required for proper cilia formation. To further study these genetic interactions in the cilia, we employ techniques such as chemotaxis and osmosensing assays, electron microscopy, and live and static imaging of transgenic fluorescent reporter lines.