Follicle Stem Cell Behaviour

When I started in the Kalderon Lab five years ago, I had no idea that our work would lead us to drastically revise an entire model system of adult stem cell behavior. I joined the lab straight out of college, setting forth with a wide-eyed passion for stem cell research. The lab was a perfect fit, specializing in the behavior of somatic stem cells found in the fruit fly ovary. Our cells of interest, called Follicle Stem Cells (FSCs), closely mimic the behavior of various somatic adult stem cells in mammals, including those found in the stomach and intestine.

FSCs were only discovered some 30 years prior, isolated by their primary function of producing epithelia for passing germline cysts. These newly coated cysts will eventually become mature oocytes, and the continuous creation of cysts ensures constant activity from the FSCs. The literature told us that there were solely two FSCs, and that their sole function was to produce differentiated epithelial daughter cells, called Follicle Cells (FCs). However, as the techniques to study FSC behavior became more refined, we observed clear inaccuracies in the description of the system.

The creation of germline cysts and their subsequent epithelial coating takes place in an organ called the germarium. Germline Stem Cells (GSCs), found at the anterior of the germarium, are responsible for the initial cyst production. Their asymmetric division creates a germline daughter cell, which in turn divides four times to create a 16-cell cyst. The development of these nascent germline cysts is guided and protected by the long cellular processes of quiescent somatic cells called Escort Cells. The continuous generation of new cysts drives the overall cyst movement in the posterior direction. Halfway through the germarium is where the FSCs come into play, as these cells produce the FCs that will coat the cyst in an epithelium. The coated cyst then buds off from the posterior of the germarium and undergoes a series of developmental stages en route to becoming a mature oocyte.

The germarium as a whole is shaped like a peanut, where the larger end is the anterior, and the middle, concave-in section is where the FSCs are located. The literature suggested that the two FSCs were always in the same position across from one another, but the radial symmetry of the germarium called this into question. How could the two FSCs always appear across from one another if the germarium was round in shape? When we looked more closely at the system, we found an entire ring of cells in the middle region that had FSC morphology. A ring-like structure could explain how the two observed FSCs would always appear on opposite sides, but raised a lot more questions about FSC behavior in general.

Our new FSC ring hypothesis needed proof, and perhaps our strongest evidence for more-than-two FSCs came from a multicolor lineage analysis experiment. For this, we genetically introduced three uniquely colored markers: GFP, RFP and LacZ, which can be stained in blue. With our setup, FSCs could be labeled in six different color combinations: Green only, Blue only, Green + Blue, Blue + Red, Green + Red, and Green + Blue + Red. If there were only two FSCs, then only two lineages would be observed in the FC epithelia. However, the ovaries that we examined contained an average of four incorporated lineages, with some containing all six! It seemed that there was some validity to our new description of FSCs after all. 

In these multicolor analyses, we routinely observed that the color combinations of the ECs corresponded to that of the FSCs in the same germarium. With closer experimentation, we were able to prove that FSCs were also responsible for the production of ECs, in addition to their known role in producing differentiated epithelial daughter cells. The origin of ECs had previously been debated; this study thus provided another important conclusion for our understanding of FSC behavior.

Through careful analysis and mathematical modeling, we determined that there are roughly 14-16 FSCs in the germaria in total. Our next question was, of course, how could that many FSCs coordinate their behavior to properly function in daughter cell production? The answer was a principle called Population Asymmetry, where the behavior of the stem cells is regulated not through individual cell interactions but through the population as a whole. At the fundamental level, this means that, when an FSC divides, the cell fate of the two daughter cells is not immediately determined by that division. “Division-independent differentiation” is a cornerstone of the Population Asymmetry model. An FSC doesn’t produce an FC or an EC; it actually becomes one.

Since FSC behavior is best explained at the population level, it follows that external morphogenic signaling pathways would be strong influencers on this system. A great example of how this works is the Wnt signaling pathway. The Wnt ligand is emitted from a specialized cell type in the anterior of the germarium, and the pathway decreases in strength in the posterior direction. The Wnt gradient reaches the FSCs, but not beyond them. When we induce a mutation that mimics constitutive Wnt activation in FSCs, we see that almost all of the cells become ECs, where the Wnt gradient is high. When we induce a mutation that deactivates Wnt in FSCs, we see EC production is completely shut off. Thus, it is likely that the level of Wnt signaling is precisely regulated in FSCs, and is responsible for controlling the FSC-EC cell fate decision. Currently, we are examining the JAK–STAT and Hedgehog pathways, the interaction between these pathways, and additional factors known to affect stem cell behavior.

Much like FSCs, adult stem cells in the mammalian intestine were recently shown to create daughter cells in two directions, with the Wnt pathway playing a role in regulating that cell fate decision. Through our research, we have not only discovered similarities with these two systems, but we have opened the door for the study of population-based stem cell systems in general through our elucidation of this new FSC model. It’s been an absolute privilege to turn my passion for stem cell research into a new frontier for developmental biology.