Spotlight on UCSC Stem Cell Scholar Anne Royou

Spotlight on UCSC Stem Cell Scholar Anne Royou
Spotlight on UCSC Stem Cell Scholar Anne Royou
Friday, January 12, 2007

The UCSC Training Program in Systems Biology of Stem Cells is sponsored by the California Institute for Regenerative Medicine (CIRM), established in early 2005 with the passage of Proposition 71, the California Stem Cell Research and Cures Initiative. The California Institute for Quantitative Biomedical Research (QB3) has augmented the UCSC program by funding an additional scholar. The first set of scholars chosen for the UCSC program started in July 2006. This story begins a series of spotlights on the research conducted by these scholars.

The UCSC CIRM fellowship opportunity came just at the right time for postdoctoral scholar Anne Royou, who works in the laboratory of molecular, cell, and developmental biologist William Sullivan.

Royou's research in the fertility of fruit flies, drosophila, had just begun to touch on both germ line and somatic stem cells. This dovetails with the purpose of the UCSC stem cell training program, which focuses on the basic biological systems operating in the processes of self-renewal and differentiation of stem cells.

Royou was studying the role of the gene Grapes (grp) in regulating the progression of mitosis'when a cell divides into two daughter cells. She had discovered that when DNA is damaged, the grp gene delays the onset of anaphase'the stage in cell division when the chromosomes move to opposite sides of the dividing cell. 'This delay allows the cell to repair and then continue dividing,' she said. The grp gene has a homolog in humans, the conserved cell cycle checkpoint (Chk1).

After publishing this finding in Current Biology, she concentrated her efforts on identifying the mechanism by which the grp gene controls mitosis. To do this, she needed to collect eggs from homozygotic grp-mutant females'females who received the grp mutation from both parents. 'That is when I realized that the grp-mutant females were laying way fewer eggs than the wild type strain.'

Out of curiosity, she began dissecting the ovaries to try to understand why the grp-mutant females were under-producing eggs, 'and I immediately saw the problem these females were having making eggs. The egg production is fairly normal when the females are young, but then it gets dramatically worse when they are older.'

She delved further into the details of the oogenesis process, staining the ovaries from wild type and grp-mutant females at different ages with immunofluorescent dyes. 'That's when I discovered that a type of cells important to the process'somatic follicle cells'were depleted in the older females. This leads to the defect in oocyte production in the grp mutants,' she said.

The literature on the subject helped Royou identify the phenotype as a problem in somatic stem cell proliferation and self-renewal.

The process whereby drosophila produce mature egg cells parallels that in humans, so studying it offers clues to factors that influence human fertility. Sullivan offered this perspective: 'Identifying and characterizing this gene will answer two key questions: Is this gene present in humans' and Does it have a similar function in humans' Our bet is the answers will be yes and yes.'

Drosophila egg formation depends on the function of a structure called the germarium, which contains germ cells at various stages of development. Each germarium possesses 1 to 3 germ line stem cells (GSC) localized at the anterior tip, one of the defect sites apparent in the mutant strain.

The GSCs divide asymmetrically to give rise to another GSC and a cystoblast. The former stays associated with the cap cells that form the niche, and the latter undergoes 4 rounds of incomplete division to form a cyst of 16 interconnected germ cells. In a normal cyst, one germ cell goes on to differentiate into an egg cell, and 15 become nurse cells, providing nutrition to the developing egg. These cysts progress towards the posterior of the germarium, where they are enveloped by a layer of somatic follicle cells and pinch off into an egg chamber, where the egg further develops.

Dissecting the ovaries of the mutant flies reveals a striking defect in the mid part of the germarium, where somatic stem cells divide asymmetrically to provide the continuous supply of follicle cells. These follicle cells normally envelop new cysts, allowing the egg to survive. As the flies age, the stem cells produce fewer of the follicular cells.

Royou postulates that as the mutant flies age, they are losing stem cells as they differentiate, or else the stem cells are no longer dividing properly'either way causing a lag in the number of follicular cells available in the germarium.

'That's when I applied for this grant,' Royou said. Thinking that she may have identified a new role for the grp gene in adult stem cell proliferation, she wrote a proposal to study the pathway.

At the beginning of her study under the stem cell fellowship, Royou ran controls to confirm that a mutation in the grp gene was responsible for the defect she had been seeing. These studies convinced her that a second mutation on the same chromosome as the grp gene (chromosome 2) was contributing to the stem cell phenotype.

Royou immediately redirected her research to identify this second gene mutation. Once she finds it, she can use the same basic plan to determine a previously unexpected pathway involved in cell proliferation.

'So far, I have identified the new phenotype, the defect, and the fact that the defect changes with age. To solve this question, I need to identify the gene responsible for this new phenotype.' Royou said. She is now mapping the gene to identify the mutation. 'This will take longer than I expected, but it is exciting!'

To search for the responsible mutation, Royou began mapping the gene by looking for the defect in flies lacking huge pieces of chromosome 2. Using this trial-and-error approach that allowed her to test hundreds of gene mutations at a time, she quickly ruled out 70% of the genes on chromosome 2. To find out where the mutation lies in the remaining 30%, she has adopted a more precise and time-consuming technique: mapping by recombination.

She extols the virtue of drosophila as a model organism. 'All of the genes are identified. The access to genomic information for drosophila is fantastic.' And with flies, she does not need to culture stem cells, she can just dissect individuals and view the stem cells directly. She spends her days counting egg cells under a dissecting microscope, 'I look at young females, old females, mutant females, and wild type females.'

Once she identifies the responsible gene and perhaps additional genes involved in somatic stem cell renewal, Royou will resume the initial aim of her study: to define the pathway involved in stem cell proliferation.

Royou hopes this training will lead to a career in academia, and it has started off well. 'This gives me the opportunity to build a project that I can bring with me to start my own lab,' she said.

Royou has enjoyed her participation so far in the new stem cell training program. She participated in the stem cell journal club, which began last fall, and has taken the first stem cell class offered on campus: Introduction to Stem Cell Biology. 'The class brought in really good speakers and it was very alive and interactive,' she said. ' When I started the stem cell program, I didn't know much about stem cells at all. Now I can see the potential.'

'Having Anne in the lab enabled us to go from spectators and fans of stem cell research to active participants," Sullivan said. "This has been a wonderful opportunity.'