To accompany our paper “Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary” I have been asked by staff at the Node to discuss the path we took when developing a successful imaging protocol.
Germline follicle formation in the Drosophila ovary is a very dynamic process – involving the coordinated migration and division of many different cells and cell types. I was trying to study how two of these cell populations – somatic escort cells and germline stem cells together build germline cysts, the 1st stage of follicle development. Whilst lineage data had suggested that germline cysts are generated by the coordinated division of one germline stem cell and two escort cells, actually finding examples of escort cells dividing in vivo was proving difficult, whereas dividing germline stem cells are found frequently. Escort cells are quite extraordinary cells; their nuclei are jammed in little cracks between large germ cells yet they have incredibly long, thin cytoplasmic processes that wrap the germ cells. These characteristics make escort cells pretty hard to see and therefore to study. I decided that the only way I was going to be able to make any progress in unraveling the escort cell/germline stem cell coordination mystery was to be able to watch both cell populations live in their native environment whilst they built a germline cyst. Given that cyst formation involves highly dynamic cellular behaviours and takes around 12 hours this was going to be quite a challenge.
Upon trying to image live germaria (the structure within the ovary that builds germline follicles) I immediately encountered a big problem – movement. The ovaries of Drosophila are subdivided into strings of developing follicles (called ovarioles) with the germaria at one end. Each of these ovariolar strings is surrounded by a sheath of muscle. Upon dissection the muscle contracts so that regular, peristaltic movements pass down the ovarioles causing them to flap around in the culture dish. The only method I found to prevent these contractions was to manually remove the muscle layer. Even then, I still had a movement problem; given that germaria are attached to strings of follicles some of which are an order of magnitude larger than a germarium, any little rocking or rolling movements of the large follicle would result in the attached germarium flying out of the field of view, and this appeared to happen frequently. I partially fixed this problem by dissecting flies that have just emerged from the pupal case, whose ovaries do not contain the more mature, larger follicles. Additionally, programming the microscope stage to move slowly and smoothly between each position during imaging helped. However, some movement remains despite trying many different methods to immobilize the tissue (including coating the dish with extra-cellular matrix components, draping the tissue with membranes and placing it in gels). Given that follicle formation is such a dynamic process, this is not entirely surprising. By re-focusing the microscope at regular intervals during imaging up to half of the germaria can be kept in focus for 12 hours.
Although overcoming tissue movement difficulties was a lengthy process, once achieved the path towards imaging success went quickly. This was thanks to the previous development by the Montell lab of culture medium designed to nurture more mature follicle stages that I found also supported follicle formation. Germaria could be cultured and imaged for greater than one entire cycle of follicle formation (14 hours). Once the method was developed and the first movies made the answer to the escort cell/germline stem cell coordination problem was almost immediately evident: Escort cells do not divide with gemline stem cells to generate a germline cyst which then migrates as one unit down the germarium, instead escort cells remain in one place, dividing rarely, and simply ‘hand-over’ the germ cells from one escort cell to the next. Demonstrating the ease with which dynamic processes can be studied when viewed unfolding in living tissue.
Morris, L., & Spradling, A. (2011). Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary Development, 138 (11), 2207-2215 DOI: 10.1242/dev.065508