Direction finding during mouse renal development

Abstract

The adult kidney consists of hundreds of thousands of fine epithelial tubules as functional units called nephrons. Nephrons have U-shaped tubules: loops of Henle that descend from the cortex to the medulla. This radial arrangement is critical to maintain water homeostasis in the kidney. Although Henle’s loops are crucial to renal physiology, the cue(s) they uses to navigate to the medulla are not understood. In this thesis, I investigate how the loop of Henle elongates during mouse renal development and show that it is probably guided to the medulla by diffusible, heparin-binding molecules. I used immumohistochemistry (IHC) on cryosections of embryonic kidneys to study the natural anatomy of the Henle’s loop. I used a low-volume culture system to allow embryonic kidneys (both natural and tissue-engineered) to form loops of Henle ex vivo and manipulated their direction of growth. Time-lapse imaging of Lgr-5 EGFP embryonic kidneys demonstrated the movement of the apex of the loop which suggested the idea of guidance cue(s) acting on the loop of Henle. Cut-and-paste experiments showed that loops appeared to be attracted to maturing collecting duct. Co-culture with an exogenous tubule inducer suggested the embryonic spinal cord as another source to attract the loops. Using raTAL (rat thick ascending loop of Henle) and 6TA2 (embryonic collecting duct cells) cell lines, I designed and performed a cell migration assay to test whether raTAL was attracted to 6TA2 cells. raTAL cells were notably attracted to 6TA2 cells compared to other cell lines. raTAL cells were also attracted to 6TA2-conditioned medium, which indicated that raTAL cells were attracted by secreted molecule(s). To begin to characterise those secreted molecule(s), heparin-binding protein-coated beads were used in the cell migration system and showed that at least one critical guidance factor is heparin-binding. From this study, I found that the apex of the Henle’s loop does move and loops are attracted by secreted molecule(s) possibly from the collecting duct. Although target molecule(s) were unidentified, this study provides the first mechanistic information about the guidance of the loop of Henle. Moreover, this was the first study of guidance of epithelial tubule shafts (rather than tips) adding to our understanding of general tubule morphogenesis.