Direction finding during mouse renal development
View/ Open
Chang2014.docx (34.89Mb)
Date
28/11/2014Author
Chang, C-Hong
Metadata
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.