Established by the British Society for Developmental Biology (BSDB) in 2014, The Gurdon/The Company of Biologists Summer Studentship scheme provides financial support to allow highly motivated undergraduate students an opportunity to engage in practical research during their summer vacation. Each year, ten successful applicants spend eight weeks in the research laboratories of their choices, and the feedback we receive is outstanding.
Our third report from the 2016 class comes from Iona Imrie, who undertook her studentship with Jamie Davies in Edinburgh.
Development of the vascular system in the mouse mesonephros
During the summer of 2016, I was fortunate enough to be awarded a BSDB Gurdon Studentship. The funding enabled me to undertake a research project in the lab of Jamie Davies at the University of Edinburgh. Under the supervision of a PhD student in the lab, David Munro, I studied the vascularization of a primitive and transient murine kidney- the mesonephros.
During mammalian embryogenesis, 3 paired renal organs develop sequentially in a cranio-caudal direction. The most primitive kidney is the pronephros, followed by the mesonephros and then the permanent kidney, the metanephros. Both the pronephros and mesonephros are temporary, with the pronephros being nonfunctional and the mesonephros being functional in some species. The mouse mesonephros comprises 18-26 pairs of tubules. The most cranial tubules connect to the nephric duct, while the caudal tubules do not. The mesonephros first appears at embryonic day 9 (E9), before regressing completely in females. In males, the cranial tubules do not degenerate, instead forming the epididymis. Given that, (a) the fate of the mesonephros is sexually dimorphic and (b) the mesonephric tubules are not uniformly connected to the nephric duct, there could be differences in vascular development that run in parallel with these differences in morphology. During my project, I observed vascular development in order to identify where blood vessels came from and if there were differences in vascular development between sexes and cranial/caudal tubules of the mesonephros.
My project began with dissecting embryos at E11.5. The mesonephroi obtained by dissection were stained with antibodies against CD31, Laminin and Pan-cytokeratin. These antibodies stained endothelia, basement membrane and nephric duct/tubules respectively.
I set out to create a timeline of mesonephric development starting at E10.5 through to E15.5 (Figures 1-4). Dissecting at E10.5 proved difficult due to the small size of the embryo. The gonad develops adjacent to the mesonephros, and I left itattached to the mesonephros in order to observe interactions of vasculature between the two.
Having dissected and stained mesonephroi from various ages, I made the following observations:
E10.5– Aorta branches into mesonephros (Figure 1).
E11.5– Tubules elongate and the mesonephros vascular plexus (mvp) begins to form (Figure 2).
E13.5– In females, branching of mvp into mesonephros and gonad occurs. In males, branching into the mesonephros occurs but branching into the gonad is not as visible as in females.
E14.5– Formation of the coelomic vessel on male gonad. Continued branching of mvp into mesonephros/gonad in females (Figure 3).
E15.5– Branching of coelomic vessel into gonad, and joining of the gonadal artery to this vessel (Figure 4).
Overall, the vasculature of the mesonephros seems to branch from the mvp and form a network of vessels around the nephric duct. The vessels around the tubules do not look particularly organized. However, confocal slices at 40x objective show vessels wrapping around the tubules. I did not observe glomeruli in the tubules at any age- these structures are rarely found in mouse mesonephroi.
As an adjunct to the main project of vascularization, I tested the functionality of the mesonephric tubules using an assay developed by one of Prof. Davies post doctoral researchers, Dr. Melanie Lawrence. The assay tested the functionality of the tubules to uptake fluorescent anions and cations. The ability of cells in these tubules to transport organic anions and cations would suggest the tubules are functioning as a primitive kidney. It has been assumed that the murine mesonephros has this role, but this has never been proven. Melanie had been using her assay to answer this question, using mesonephroi from different gestational ages. The unpublished data that Melanie has collected shows that these tubules do have the ability to transport organic anions and cations. I was able to help with assaying at some of the gestational time points.
I cultured mesonephroi using the Sebinger culture method, and then assayed the uptake of 6-carboxyfluorescin (anion) through organic anion transporters (OAT) in the basolateral membrane of tubule cells, and uptake of the DAPI (cation) through organic cation transporters (OCT), also in the basolateral membrane. As a control, inhibitors of OAT (Probenecid) and OCT (cimetidine and metformin) were added to mesonephric cultures to show that any fluorescence seen in the tubules was due to uptake via these transporters and not by another process.
The Gurdon Studentship has been an invaluable opportunity. I cannot stress enough how important it is for medical sciences students like myself to spend time in a research lab. As a second year, I didn’t really know what being a scientist would be like. From spending time in the Davies Lab, I have been able to understand and practice techniques that were briefly introduced in lectures, while developing a scientific mind, improving my time management skills and working alongside scientists who are extremely dedicated to their research. I would especially like to thank Jamie Davies for giving me this opportunity, and David Munro and Chris Mills for their guidance throughout my time in the lab.