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. 2015 Sep 3;201(1):13–22. doi: 10.1534/genetics.115.179911

Sorting Out Identities: An Educational Primer for Use with “Novel Tools for Genetic Manipulation of Follicle Stem Cells in the Drosophila Ovary Reveal an Integrin-Dependent Transition from Quiescence to Proliferation”

Diane Silva 1, Jennifer C Jemc 1,1
PMCID: PMC4566258  PMID: 26354974

Abstract

SUMMARY Organisms are made up of thousands of different cell types that must migrate, proliferate, and interact with each other to yield functional organ systems and ultimately a viable organism. A characteristic that distinguishes one cell type from another is the set of genes that it expresses. An article by Hartman et al. in the April 2015 issue of GENETICS identified methods to uniquely identify different cell populations during oogenesis, providing valuable tools for future studies. This Primer article provides background information on the Drosophila ovary as a system in which to study stem cell regulation, mechanisms for regulating gene expression, and the techniques used by Hartman et al. to identify specific cell populations and study their function.

Related article in GENETICS: Hartman, T. R., E. M. Ventresca, A. Hopkins, D. Zinshteyn, T. Singh et al., 2015 Novel Tools for Genetic Manipulation of Follicle Stem Cells in the Drosophila Ovary Reveal an Integrin-Dependent Transition from Quiescence to Proliferation. Genetics 199: 935–957.

Keywords: follicle stem cell, ovary, transcriptional regulatory elements, integrin, Drosophila melanogaster

Background

Proper regulation of cell survival, division, and differentiation to a specific fate is critical throughout the lifetime of an organism. In the developing embryo, a single cell eventually gives rise to all the cells composing the adult. However, not all cells are created equal—if they were, the body would not be able to function. This raises the fundamental question “What allows one cell to look and function differently from another?” Examination of cells of different origins reveals that specific cell types express different sets of genes, allowing them to assume diverse functions. Cells begin to assume different fates based on signals received from their extracellular environment, including the cells around them. Amazingly, the molecules that control these outcomes are highly conserved from organisms like the nematode worm, composed of <1000 cells, to more complex eukaryotes including fruit flies, mice, and humans. When signals diffuse from one cell to the next, they set into motion a series of events that frequently leads to changes in gene expression. Genes consist not only of the DNA sequence encoding a specific RNA or protein, but also of critical transcriptional regulatory elements, including promoters, enhancers, and silencers, that help determine when, where, and to what level genes are expressed.

Production of specific cell types via differential gene expression is by no means unique to the developing embryo. Throughout its lifetime, an organism must replace specific populations of cells, balancing cell death with cell proliferation, to maintain homeostasis. Stem cells of various types play a critical role in maintaining homeostasis. In the adult organism, tissue-specific stem cells have the ability to give rise to the cell types present in the tissue in which they reside; these cells are responsible for replacing cells lost due to damage or death. Examples include the hematopoietic stem cells that give rise to the cells found in the blood, including red blood cells and lymphocytes, and germline stem cells (GSCs) that are critical for the continued production of sperm or eggs.

Hartman et al. (2015) focus on a population of somatic, or nongermline, stem cells in the fruit fly ovary known as follicle stem cells (FSCs). FSCs produce follicle cells that will surround the germ cells throughout most of oogenesis (reviewed in Spradling 1993). As these cells perform a critical role in supporting germ cell development, it is important to understand how these cells function in the gonad. However, a significant limitation to these studies has been a lack of ways to effectively distinguish the FSCs from other somatic cell populations within the gonad and to manipulate gene expression within specific cell types. Hartman et al. (2015) set out to alleviate this difficulty by identifying genetic elements that regulate gene expression in different cell populations in the ovary, specifically the FSCs. They then can label and manipulate the FSCs and probe the role of specific genes in FSCs.

The System: Drosophila Ovary

The fruit fly Drosophila melanogaster has proven to be an excellent model organism for scientific research given its 10-day generation time, conservation of genes (nearly 75% of human disease-associated genes are conserved in flies), and abundance of tools available for genetic manipulation (reviewed in Roote and Prokop 2013). Hartman et al. (2015) utilized these tools to examine gene expression in the adult ovary. Drosophila have two ovaries, each composed of 15–20 ovarioles (Figure 1; reviewed in Spradling 1993). Each ovariole consists of a single germarium and a number of maturing egg chambers that are connected by stalk cells, appearing like beads on a string (reviewed in Spradling 1993). The germarium functions as the source for both germ cells, some of which will give rise to eggs, and somatic gonadal cells, which support the development of the germ cells (reviewed in Spradling 1993). To continue to reproduce, female flies must continue to produce eggs through a process known as oogenesis. Critical to this process are two populations of stem cells: the GSCs and the FSCs. These cell populations each exist in a specialized microenvironment called “the niche” that supplies essential factors specific for their maintenance (reviewed in Morrison and Spradling 2008).

Figure 1.

Figure 1

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The Drosophila ovary and oogenesis. The ovary is composed of 15–20 ovarioles. At the anterior end of each ovariole is a structure known as the germarium, which provides the germ cells and somatic gonadal cells that compose the subsequent egg chambers. Terminal filament cells (purple), cap cells (pink), germline stem cells (light pink), gonialblast and germline cysts (yellow), inner germarial sheath cells (light blue), follicle stem cells (dark blue), follicle cells (green), stalk cells (dark green), and oocyte (orange).

The GSC niche is located in the anterior-most region of the germarium, where five to seven terminal filament cells and three to four cap cells produce factors that regulate the proliferation and maintenance of two to three GSCs (reviewed in Spradling 1993). Upon GSC division, one cell remains in the niche, thereby self-renewing the GSC population, while the other cell exits the niche and begins differentiation to a gonialblast. This cell undergoes four rounds of synchronized cell divisions in region 1 of the germarium, generating 2-, 4-, 8-, and 16-cell germline cysts that remain interconnected by a structure known as the fusome (Figure 1; reviewed in Spradling 1993). During this time, a population of somatic cells known as the inner germarial sheath (IGS) cells, or escort cells, wraps around the germline cysts (Figure 1; King 1970; Schulz et al. 2002; Decotto and Spradling 2005; Morris and Spradling 2011). These cells pass the germline cysts toward the posterior of the germarium, where germline cysts will exchange their interactions with IGS cells for encapsulation by follicle cells as they transition from region 2A to 2B (Decotto and Spradling 2005; Kirilly et al. 2011; Morris and Spradling 2011). The cyst is surrounded by a single layer of follicle cells and will bud off to form an egg chamber. Of the 16 germ cells in the egg chamber, one of these cells, the oocyte, will continue through meiosis to become the egg, while the other 15 cells function as nurse cells to provide RNAs, proteins, and organelles for the oocyte (Spradling et al. 1997).

Similar to the continued production of germ cells, continued production of follicle cells depends on a population of FSCs present in the germarium. Two FSCs are located halfway down the germarium at the junction of regions 2A and 2B (Figure 1; Margolis and Spradling 1995; Nystul and Spradling 2007). Their proliferation depends on signals received from regions located both anterior and posterior to the FSCs (Sahai-Hernandez et al. 2012; Vied et al. 2012; Sahai-Hernandez and Nystul 2013). Similar to GSCs, FSCs divide asymmetrically, giving rise to one daughter cell that remains in the niche as a FSC, and a second daughter cell that exits the niche and begins to differentiate (Morrison and Spradling 2008). These differentiating daughter cells first give rise to precursor follicle cells, and it is their inward migration that separates germline cysts into the individual egg chambers (Morris and Spradling 2011). Subsequently, precursor follicle cells give rise to polar cells, stalk cells, and the epithelial follicle cells that will encapsulate the 16-cell germline cyst (Figure 1; Nystul and Spradling 2010). Studies of the characteristics and functions of FSCs have been hampered by a lack of methods for specifically marking these cells. Therefore, Hartman et al. (2015) set out to identify additional tools that can be used to distinguish FSCs from other cell populations within the ovary and to demonstrate how these tools can be used to study the function of specific genes in the FSCs.

Regulation of Gene Expression

As described above, gene expression is commonly used to distinguish different cell types. A gene that is being expressed is transcribed from DNA to RNA. Transcription requires the presence of transcriptional regulatory elements in the DNA region surrounding and within the gene and a number of proteins, known as transcription factors, that recruit RNA polymerase to the DNA. Transcriptional regulatory elements are composed of two distinct families: promoters and distal regulatory elements, including enhancers and silencers (Maston et al. 2006). These elements play a critical role in determining when, where, and to what level genes are expressed.

A promoter is a region of DNA located at or just upstream of the transcriptional start site of a gene. The core promoter includes the transcriptional start site and defines the direction of transcription. In addition, it can include the TATA box, Initiator element, Downstream Promoter Element, and the Transcription Factor IIB Recognition Element. These elements bind the general transcription factors (TFIIA, TFIIB, TFIID TFIIE, TFIIF, and TFIIH) and a multi-subunit complex known as Mediator, which together are responsible for recruiting RNA Polymerase II. These factors initiate a low level of transcription, and therefore another class of transcription factors, known as activators, is required to achieve high levels of gene transcription. Transcriptional activators can bind to transcription factor-binding sites within the proximal promoter, which is located <1 kb upstream of the core promoter and requires a specific orientation relative to the core promoter for proper function (Figure 2A; Maston et al. 2006).

Figure 2.

Figure 2

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Regulation of gene expression. (A) Transcriptional activation of a target gene is regulated by core promoter elements like the TATA box (orange) that bind to general transcription factors (blue). Binding of activator proteins (green) to transcription factor binding sites (purple) in the proximal promoter and to enhancer elements distally (light green) also regulates gene expression. The Mediator protein complex (red) helps to bridge the gap from enhancer-bound proteins to the general transcription factors. The complex of transcription factors and activators recruits RNA polymerase (RNA Pol; yellow) for transcription. (B) GAL4/UAS System. GAL4 is expressed in a specific cell type based on the enhancer/promoter element located near the GAL4 insertion site in the genome. Flies containing this insertion are mated to another fly line that contains a target gene downstream of the UAS element. The GAL4 protein binds to the UAS element to activate transcription of the target gene. The GAL80 protein functions as an inhibitor of GAL4. If GAL80 is present, transcription of the target gene will be repressed.

Enhancers are distal regulatory elements that contain multiple binding sites for transcriptional activators. However, enhancers differ from promoters in that they function independently of orientation, can be present both upstream and downstream of the gene, and are located at a greater distance from the core promoter (up to hundreds of kilobases away), resulting in looping to bring enhancer-bound transcriptional activators in close proximity to promoter elements (Figure 2A; Pennacchio et al. 2013). Enhancers have the capability of regulating multiple genes; however, enhancer activity is often restricted based on the cell type or age or the specific physiological, pathological, or environmental conditions of the cell (Pennacchio et al. 2013). Finally, silencers share many of the same characteristics of enhancer elements, but are bound by transcriptional repressors rather than transcriptional activators (Maston et al. 2006).

Methods for Labeling Cells

Hartman et al. (2015) utilized promoter and enhancer elements to manipulate expression of genes to mark specific cell types. In particular, they used a set of tools, collectively known as the GAL4/UAS system from baker’s yeast, Saccharomyces cerevisiae, to identify distinct cell types in the Drosophila ovary. GAL4 is a transcriptional regulator that functions by binding to a specific enhancer element, known as the Upstream Activating Sequence (UAS) element, to promote transcription of downstream genes (Figure 2B; reviewed in Duffy 2002). Previous studies generated Drosophila strains in which the GAL4 gene was inserted at sites throughout the genome (reviewed in Duffy 2002). As a result, GAL4 is expressed in specific cell types, reflecting control by nearby transcriptional regulatory elements, including promoters and enhancers. The lines are referred to as GAL4 drivers, as different regulatory elements promote or “drive” expression of the GAL4 gene. In addition, the UAS element has also been inserted upstream of genes of interest, reporter genes, and sequences encoding RNA hairpins, and these sequences have been integrated into the Drosophila genome. When the GAL4 gene is expressed, it binds and activates expression of the gene downstream of the UAS element (Brand and Perrimon 1993). By mating flies containing the GAL4 gene under the control of different enhancer/promoter elements with flies carrying a UAS element with a desired downstream gene, it is possible to express genes in a variety of different patterns (Figure 2B; reviewed in Duffy 2002). The GAL4/UAS system has played an important role in research using many model organisms. Hartman et al. (2015) utilized an extensive collection of fly lines from multiple sources with insertions of GAL4 throughout the genome and a UAS element upstream of the Green Fluorescent Protein (GFP) gene to label specific populations of cells within the germarium and visualized them using immunofluorescence microscopy, as described below.

While the GAL4/UAS system restricts gene expression to a subset of cells, it is often desirable to limit expression to just a couple of cells at a time. Imagine that you are studying the shape of cells in a given tissue and that you have labeled the membranes of those cells. A problem arises when you need to distinguish one specific cell from its neighbor; you need a way to label just a few cells within the tissue rather than all of them. One method that has proven particularly useful for labeling a few cells in a tissue is known as Mosaic Analysis with a Repressible Cell Marker (MARCM) (Figure 3; Lee and Luo 1999). This method allows for tighter control of the GAL4/UAS system with the introduction of an inhibitor of GAL4, known as GAL80. GAL80 binds to GAL4, preventing it from activating transcription of a gene, like GFP, downstream of the UAS element (Figure 2B; reviewed in Duffy 2002). Two additional elements derived from yeast were also incorporated to generate mosaically labeled tissue: an enzyme known as FLP recombinase and FLP recombinase target (FRT) sites (Golic and Lindquist 1989). FLP expression was under the control of a promoter from the heat-shock protein Hsp70. Therefore, this enzyme was produced only when flies were incubated at a temperature of 37°, known as heat shocking (Golic and Lindquist 1989). The production of FLP and the presence of FRT sites in the same position on both copies of homologous chromosomes allow for crossing-over events to occur at the FRT sites during mitosis (Golic and Lindquist 1989). This is unique, as it allows for the induction of mitotic recombination, a process normally limited to meiosis. In the MARCM system, the gene encoding the GAL80 repressor protein must be present on one of the chromosomes containing the FRT site (Lee and Luo 1999). Thus, following DNA replication, FLP promotes a recombination event at the FRT sites. Chromosome segregation during mitosis can result in cells with three different genotypes (Figure 3, C–E). The first cell has the same genotype as the starting cell, thus expression of the GFP marker is repressed due to the presence of GAL80 (Figure 3C; Lee and Luo 1999). The second cell inherits two copies of the chromosome containing the FRT site but lacking the GAL80 gene, thereby allowing GAL4 to activate transcription of the GFP reporter gene (Figure 3D; Lee and Luo 1999). The third cell inherits two copies of the chromosome containing the FRT site and the GAL80 gene, thereby repressing transcription of the GFP marker (Figure 3E; Lee and Luo 1999). In the MARCM system, the GAL4 gene and the UAS elements are integrated at varying locations in the genome, but cannot be on the same chromosome arm as the FRT site or the GAL80 gene. This method can also be used to analyze cells mutant for a gene of interest by incorporating a mutant allele for the gene of interest on the non-GAL80 FRT chromosome (Figure 3).

Figure 3.

Figure 3

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MARCM technique. (A) The cell carries a GAL4 insertion and a UAS-GFP insertion within the genome on a chromosome different from those pictured (not shown). The two copies of the chromosomes shown both contain a FRT site (blue arrowhead) near the centromere (black circle) on one chromosome arm. One of the FRT chromosomes also carries the GAL80 gene, while the other chromosome lacks the GAL80 gene and either is wild type or carries a genetic mutation. Even though GAL4 is produced in this cell, GFP transcription is inhibited by GAL80. (B) Following heat shock at 37°C, FLP recombinase is expressed and can induce recombination between the FRT sites. The continued presence of GAL80 results in repression of GFP transcription. The cell undergoes mitosis and cytokinesis. Depending on the ways in which chromosomes segregate, one can generate a cell of the genotype(s) (illustrated in C–E). (C) The resulting cell inherits one chromosome lacking the GAL80 gene, and one chromosome carrying the GAL80 gene, resulting in the repression of GFP transcription. (D) The resulting cell inherits two chromosomes lacking the GAL80 gene, allowing GAL4 to activate GFP transcription. (E) The resulting cell inherits two chromosomes carrying the GAL80 gene, resulting in repression of GFP transcription.

Although MARCM is useful for marking single cells, it also labels all daughter cells that arise from that single cell, thereby labeling a population of adjacent cells. As one of the authors’ goals was to examine the shape, or morphology, of cells, it was disadvantageous to have adjacent cells labeled as they become difficult to distinguish. Therefore, Hartman et al. (2015) further refined the MARCM technique to control when labeled daughter cells are generated. Following the induction of clones by heat shock, they allowed the labeled daughter cells to differentiate into follicle cells and exit the germarium. Then they cultured these flies using grape juice plates, which are a poor source of proteins and lipids, to arrest cell division in the specific cells of interest. These cells are said to be quiescent. Addition of nutrient-rich yeast paste to the plates resulted in a transition back to proliferation. Using this technique, it was possible to label a limited number of cells and to inhibit the generation of similarly labeled daughter cells, allowing for analysis of cell morphology by immunofluorescence microscopy.

Visualization of Gene Expression

To be able to see the GFP reporter and to identify the cell and tissue types expressing the reporter, immunofluorescence microscopy is used. This technique allows one to identify where a protein of interest is expressed with the help of additional markers for other cell types and specific cellular structures. Before tissues or cells can be used for immunofluorescence microscopy, they are fixed to preserve and stabilize the tissue structure. Following fixation, the sample is incubated with the desired primary antibodies. A primary antibody recognizes a specific antigen, like GFP, and is generated by injecting a protein, or a portion of that protein, into a host animal, typically rabbit, mouse, guinea pig, rat, or chicken. Hartman et al. (2015) used not only a GFP antibody generated in the chicken, but also a Fasciclin 3 (Fas 3) antibody generated in mouse and a Vasa antibody generated in rabbit to mark the germ cells. After washing out any primary antibody that does not bind to antigen, the sample is incubated with secondary antibodies. Secondary antibodies are typically generated in goat or donkey by injecting the animal with the common region of an antibody from rabbit, mouse, guinea pig, rat, or chicken. Therefore, the secondary antibody recognizes the conserved region of the primary antibody. It is also linked to a detectable marker like a fluorescent molecule, known as a fluorophore, which can be visualized following exposure to light of a specific wavelength and a photosensitive detector in a confocal microscope. When using multiple primary antibodies, it is critical that each of the primary antibodies be generated in a different animal and that each secondary antibody be conjugated to a different fluorophore, making it possible to distinguish each of the different proteins. Using immunofluorescence microscopy combined with the GAL4/UAS system or MARCM, Hartman et al. (2015) were able to label different cell types or examine cell morphology in the germarium.

Reducing Gene Expression with RNA Interference

In addition to developing tools for labeling specific cell populations, Hartman et al. (2015) were also interested in using these tools to manipulate gene expression in these cells. While gene expression is regulated at the level of transcription, it can also be regulated post-transcriptionally by controlling the availability of a given messenger RNA (mRNA) for translation. Studies in a variety of organisms have demonstrated the use of RNA interference (RNAi) to control gene expression levels (reviewed in Ipsaro and Joshua-Tor 2015). RNAi is a mechanism by which cells fine-tune the levels of available RNA using microRNAs (miRNAs) and short interfering RNAs (siRNAs). In both of these mechanisms, RNA is produced that has the ability to undergo complementary base pairing, forming a double-stranded RNA (dsRNA) hairpin (Figure 4; Ipsaro and Joshua-Tor 2015). An enzyme called Dicer cuts the dsRNA into a mature 21- to 25-nt dsRNA. This dsRNA is loaded into a complex called the RNA-Induced Silencing Complex (RISC), which contains the Argonaute (Ago) protein, and one of the RNA strands is discarded (Figure 4; Ipsaro and Joshua-Tor 2015). The remaining single stranded RNA undergoes complementary base pairing with its target mRNA, resulting in post-transcriptional gene silencing. While siRNAs typically undergo perfect base pairing with their targets, miRNAs often undergo perfect binding with a critical sequence known as the seed sequence and imperfect binding elsewhere (Ipsaro and Joshua-Tor 2015). This results in different mechanisms of regulation. siRNAs usually promote slicing of the target mRNA, while miRNAs lead to translational repression by removing the 5′-methylguanosine cap and/or Poly(A)-binding proteins, two critical factors for recruiting proteins needed for efficient RNA translation (Ipsaro and Joshua-Tor 2015). This mechanism has been harnessed for use in the lab. Injection of short dsRNAs can promote RNAi. In addition, one can design a gene that encodes RNA capable of undergoing hairpin formation and is complementary to the mRNA from a gene of interest. If this sequence is inserted downstream of the UAS element described earlier, one can specifically control when and where RNAi occurs. Thus, there are a variety of tools available for manipulating gene expression in Drosophila that can be harnessed to study protein function.

Figure 4.

Figure 4

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Mechanisms for RNAi. RNA forms a hairpin and is trimmed to a shortened length of 70 nucleotides in some cases. This dsRNA is exported from the nucleus and cleaved by Dicer into the mature 21- to 25-nt dsRNA. This dsRNA is loaded onto the Ago/RNAi RISC, and one of the strands is discarded. Loaded Ago/RISC binds to target mRNA (orange). If complementarity is perfect, the mRNA target is cleaved. If complementarity is imperfect, the target mRNA is often destabilized by removal of the 5′-methylguanosine cap or poly(A) tail-binding proteins, reducing its translation.

Generating Transgenic Flies

Many of the genetic elements described above are derived from other organisms, like yeast, or are created in the lab, like gene-specific RNAi. To utilize these reagents, it is critical to create stable fly lines containing these genetic elements. The use of transposable elements/transposons carrying these genetic elements and the transposase enzyme allows for the incorporation of modified genetic elements into the fly genome (Rubin and Spradling 1982; Bachmann 2008). Once integrated into the genome, they are treated as endogenous genes. To generate flies carrying the GAL4 element near different enhancers and promoters, researchers first inserted the GAL4 gene into the most commonly used transposable element in flies, the P element. While transposons normally encode an enzyme called transposase, which helps them hop around the genome, the P element was modified to no longer harbor transposase activity (reviewed in Bachmann 2008). Therefore, once the P element is inserted in the genome it maintains a relatively stable position. To generate a transgenic fly, the DNA containing the modified P element and a temporary source of transposase are injected into the fly embryo at the posterior end (reviewed in Bachmann 2008). This is done at a time before the embryo has formed distinct cells via the process of cellularization. The posterior end of the embryo is where the pole cells will form, which will later give rise to sperm or eggs. Thus, the embryo that is injected will carry the DNA only in a subset of cells, but, importantly, this includes the cells that will be used to generate gametes for reproduction. In this way, the injected fly can pass on the newly inserted DNA to its offspring, resulting in a fly that will have the modified DNA in every cell of its body.

Unpacking the Experiments

One of the challenges in studying stem cells is distinguishing stem cells from other populations of cells in the tissue. Hartman et al. (2015) were interested in exploring the morphology of FSCs and how FSCs are maintained in the germarium. To do so, they developed new tools to genetically manipulate and identify individual FSCs within the germarium, starting with the GAL4/UAS system. First, they screened flies with different GAL4 insertions for lines expressing the GFP reporter in subpopulations of somatic cells within the germarium. Once fly lines were identified that expressed the reporter in the FSCs, Hartman et al. (2015) used the MARCM system to label a subset of FSCs and to analyze their morphology. They analyzed the function of one integrin subunit, encoded by the myospheroid (mys) gene, in the FSCs using their newly developed techniques. Finally, RNAi was used as a genetic tool to reduce gene expression of mys in FSCs to determine if mys is required for FSCs to transition from quiescence to proliferation. Thus, Hartman et al. (2015) developed and utilized a variety of genetic tools followed by immunofluorescence microscopy to improve the accuracy of somatic cell identification in the ovary and to define the roles of genes required for FSC function.

Utilizing the GAL4/UAS System for Cell Identification

Hartman et al. (2015) took advantage of an extensive collection of fly lines from multiple sources with insertions of GAL4 throughout the genome. By combining these GAL4 insertions and a UAS-GFP reporter, Hartman et al. (2015) were able to identify GAL4 insertions that were expressed in specific populations of cells within the germarium. They focused on GAL4 insertion lines near genes previously found to be expressed or to function in somatic cell populations in the ovary. Using this candidate approach, they identified lines expressing GAL4 in terminal filament and cap cells, stalk and polar cells, follicle cells, and IGS cells using immunofluorescence microscopy (Hartman et al. 2015). In many cases, GAL4 expression was observed in multiple somatic cell types and at multiple stages of oogenesis (see figures 1–4 and tables 1 and 2 in Hartman et al. 2015). These studies not only identified new ways of marking subsets of somatic cells within the germarium, but also resulted in the identification of GAL4 insertions that can be used to activate expression of other genes of interest at varying expression levels downstream of a UAS, including genes whose ubiquitous expression is lethal.

The next step was to find a GAL4 fly lineage that would distinguish IGS cells from FSCs at the region 2A/2B border of the embryo to analyze the genetic mechanisms controlling their behavior. Previously used fly lines expressed GAL4 not only in IGS cells, but also in FSCs and their daughter cells. Hartman et al. (2015) identified 15 GAL4 lines capable of expression in the IGS cells. While many of these insertions were expressed in other somatic cell populations as well, two of the GAL4 insertions, one in the forked ends (fend) gene and the other in the engrailed (en) gene, are expressed primarily in IGS cells, with sporadic cap cell and FSC expression (see figure 3 in Hartman et al. 2015). These new GAL4 lines are useful for altering gene expression within a more limited range of somatic cells.

From Quiescent to Proliferating FSCs

Hartman and colleagues used GAL4 expression in FSCs to study their characteristics. Previously, the distinction of FSCs from their prefollicle daughters necessitated the use of features like location, morphology, and gene expression levels (reviewed in Sahai-Hernandez et al. 2012). In an earlier study, O’Reilly et al. (2008) could not definitively say that the defects observed upon integrin mutation affected the FSCs or their prefollicle daughters using these characteristics, demonstrating the need for additional ways of distinguishing cells. As Hartman et al. (2010) had previously observed expression of 109-30-GAL4 in FSCs and all their daughters through stage 3 of egg chamber development, this GAL4 line was a good candidate for labeling FSCs. Combining the 109-30-GAL4 with the MARCM system allowed them to label a smaller population of FSCs (see figure 5 in Hartman et al. 2015). While IGS cells were also labeled when recombination was induced during larval stages, recombination induced during adult stages labeled few IGS cells due to their decreased proliferation in the adult (see figure 5 in Hartman et al. 2015). The use of 109-30-GAL4 within the MARCM system allowed Hartman et al. (2015) to analyze the morphological characteristics of FSCs, resulting in the identification of a microtubule-based cytoplasmic extension that extends across the germarium (see figure 5, K–M, in Hartman et al. 2015).

Figure 5.

Figure 5

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Cell-autonomous and cell-nonautonomous regulation by integrin. (A) Normal function of the FSC and daughter cell in the presence of wild-type integrin. (B) Loss-of-function integrin mutation in FSC leads to abnormal FSC function, demonstrating a cell autonomous requirement for integrin. (C) Loss-of-function integrin mutation in the daughter cell leads to abnormal function of the FSC, demonstrating a cell-nonautonomous requirement for integrin.

While the MARCM approach with 109-30-GAL4 successfully labeled FSCs and their immediate daughters, it remained difficult to assess dynamics in a single FSC when its neighboring prefollicle daughter cells were also labeled. Using nutrient deprivation, Hartman et al. (2015) generated a smaller number of labeled cells, allowing them to assess the morphological changes of FSCs in the germarium during their transition from quiescent to proliferating cells (see figure 6 in Hartman et al. 2015). An examination of the region 2A/2B border of the germarium revealed that FSCs from nutrient-deprived flies remained close to the surface of the germarium with short, thick projections. Six hours after the return to a nutrient-rich diet, FSC projections were more elongated (see figure 7 in Hartman et al. 2015). They continued to perform immunofluorescence microscopy at different time points following a return to a nutrient-rich diet to examine FSC location during their transition from quiescence to proliferation and found that FSCs and their daughter cells remain localized at the surface of the germarium during this transition (see figure 7 in Hartman et al. 2015).

One continued challenge was the labeling of both FSCs and their daughters, as well as some IGS cells, using the 109-30-GAL4 line. However, Hartman et al. (2015) observed that many of the GAL4 drivers expressed in the IGS cells were also expressed in the FSCs, but not in the differentiating follicle cells (see figure 3 in Hartman et al. 2015). As IGS cells rarely undergo cell division in the adult, Hartman et al. (2015) proposed that this class of GAL4 insertions might be useful for labeling FSCs within the MARCM system. Following the generation of GFP-positive cells by MARCM, the authors found that they were able to successfully label FSCs (see figure 8 in Hartman et al. 2015). In the case of 109-30-GAL4, the authors had observed labeling of some IGS cells at the region 2A/2B border, while IGS cells labeled by fend-GAL4 were located more anteriorly, decreasing the possibility of mistakenly identifying an IGS cell as a FSC. Similar to observations with 109-30-GAL4, FSCs were observed to send out projections across the germarium when labeled using the fend-GAL4 line following the feeding-induced transition from quiescence to proliferation (see figure 8, G–J, in Hartman et al. 2015).

Role of Integrins in FSCs

Hartman et al. (2015) set out to further examine the function of a protein called integrin in the FSCs using their new tools. Integrins are cell-surface receptors composed of two subunits, an α-subunit and a β-subunit, that serve a variety of functions, including linking the extracellular matrix outside the cell to the actin cytoskeleton inside the cell. Previously, it was shown that integrins are necessary for FSC localization, morphology, and proliferation, as integrin mutant FSCs were displaced and had altered cell shape (O’Reilly et al. 2008). Mislocalization of FSCs carrying an integrin mutation made it difficult to distinguish them from daughter cells. Hartman et al. (2015) set out to more closely examine the function of integrin in the FSCs, using a mutant in the mys gene. mys encodes the βPS-integrin subunit, one of two β-subunits in flies. MARCM was used to generate GFP-positive FSCs that were also mutant for mys. Similar to previously published results, mys mutant FSCs had altered cell shape, reduced proliferation, and mislocalization in the germarium (see figure 9 in Hartman et al. 2015). In addition, cellular projections in mys mutant cells exhibited a more random orientation as compared to controls (see figure 9 in Hartman et al. 2015). These results suggest that the integrin mutant FSCs are less likely to be maintained in the niche and have entered a quiescent state (Hartman et al. 2015).

While mutation of integrin clearly affects FSC function, it is formally possible that this effect could be an indirect effect from loss of integrin in the differentiating daughter follicle cells (Hartman et al. 2015). Thus, the question arises, “Is integrin regulation of FSCs cell autonomous or nonautonomous?” A protein required in the same cell in which it is produced is said to function cell-autonomously (Figure 5B), while a protein required for the proper function of another cell is said to function cell-nonautonomously (Figure 5C). Previous studies demonstrated that daughter cells influence FSCs, supporting the possibility that integrins could function cell-nonautonomously to regulate FSC function (Vied et al. 2012). To examine if integrin functions within FSCs or within the daughter cells to influence the FSCs, Hartman et al. (2015) induced marked FSC clones and prevented further production of follicle daughter cells by nutrient restriction. Following a return to a nutrient-rich diet and FSC division, the daughters of integrin mutant FSCs showed dramatic differences in morphology and positioning within the germarium relative to wild-type controls. The displacement of daughter cells and changes in FSC morphology were also observed when integrin levels were reduced in FSCs using RNAi (see figure 10 in Hartman et al. 2015). Taken together, these results support previous observations that integrins regulate FSC function cell-autonomously to promote FSC proliferation and maintenance in the niche (Figure 5B; O’Reilly et al. 2008).

Suggestions for Classroom Use

Regulation of gene expression is a key topic that relates not only to genetics, but also cell, molecular, and developmental biology. Organ development and function requires the cooperation of multiple cell types that perform diverse roles. The expression of different genes is one characteristic that distinguishes one cell type from another, causing it to assume a specific shape and function. Gene expression is regulated by transcription factors that bind regulatory elements found both proximal (promoters) and distal (enhancers, silencers) to the protein-coding sequence. While many classes focus on general transcriptional factors, students are often left with questions about the role that promoters and enhancers perform in transcriptional regulation. Hartman et al. (2015) nicely demonstrate how transcriptional regulatory elements play a critical role in distinguishing one cell from its neighbor, and how these elements can be used to generate valuable tools to be used in a research setting. It is recommended that this Primer article and Hartman et al. (2015) be read and discussed when covering regulation of gene expression in a genetics or advanced genetics course. Expression of different genes is one feature that often distinguishes a cell from its neighbor, and this is particularly important when it comes to stem cell populations, given the need to isolate stem cells to explore their therapeutic potential. Hartman et al. (2015) focused on a critical population of somatic stem cells in the Drosophila ovary, the FSCs. To allow students to more easily follow the experiments described in Hartman et al. (2015), this Primer article describes the Drosophila ovary as an experimental system, discussing the types and functions of cells found in the ovary that Hartman et al. (2015) are aiming to distinguish. The heart of this Primer article focuses on the tools developed by Hartman et al. (2015) that utilize different promoter and enhancer elements to direct gene expression. Thus, these articles are useful for introducing the concept of transcriptional regulatory elements to students in a classroom and a research setting. This Primer explains the experimental tools utilized by Hartman et al. (2015) to explore when and where these elements promote transcription. In addition, this Primer introduces students to commonly used techniques for altering gene expression in specific cell types, including RNAi and the induction of mosaic clones. It is recommended that students read the introduction to Hartman et al. (2015) and the introduction and techniques portion of this Primer article and discuss these portions of the articles in small groups. Each group can take a section of the techniques and present them in a classroom setting. The figures could then be discussed in the following class period. For discussion of the figures from Hartman et al. (2015), it is recommended that each group be assigned one to two figures for the class period to lead the discussion, wrapping up with how the genetic tools could be used for future research studies. As Hartman et al. (2015) describe how these tools are utilized for understanding the genes that function in FSCs, it is recommended that students describe how these tools could be used to explore the role of other genes in the variety of cell types found in the developing ovary. There are a multitude of articles that explore the roles of specific genes, epigenetic regulation of gene expression, and chromosomal inheritance in stem cell populations that could be incorporated for further discussion in an advanced genetics class (Jemc 2011; Sahai-Hernandez et al. 2012; Tran et al. 2012; Yadlapalli and Yamashita 2013; Luyten et al. 2014; Slaidina and Lehmann 2014).

Questions for Review and Discussion

  1. In Hartman et al. (2015), the authors are focused on identifying ways to distinguish FSCs from other somatic cell populations in the ovary. How could a failure to effectively distinguish FSCs from IGS cells or daughter follicle cells impact their results? Why is the ability to distinguish different cell types so important for studying how organs function?

  2. Why was it important for Hartman et al. to focus on region 2A/2B of the germarium in their identification of tools?

  3. How could one develop a GAL4 line in the lab that is expressed in the same pattern as a gene of interest?

  4. Why was it important to use MARCM to analyze FSC characteristics with the 109-30-GAL4 driver, as opposed to analyzing FSCs in flies containing only the GAL and UAS elements (see figure 5 in Hartman et al. 2015)? Why is the timing of clone induction important?

  5. In figure 6 in Hartman et al. (2015) the authors observe that a nutrient-poor diet induces quiescence. Why do you think lipids and proteins are important for the process of cell division to take place?

  6. Hartman et al. (2015) use UAS-GFP to label cells throughout their article. In their figure 8, they use UAS-Tau-GFP to examine cell morphology. What insights do they gain by using UAS-Tau-GFP that they would not have gained had they used a UAS-GFP containing a nuclear localization sequence (UAS-GFPnls)?

  7. In regard to the cell-autonomous requirement for integrin in FSCs, how would you have expected the results of the RNAi experiment to differ if integrin function were required cell-nonautonomously?

  8. Provide students with a gene of interest and have them design a DNA sequence that could be used for RNAi for the gene of interest.

  9. In figure 2 in Hartman et al. (2015) the authors identify weak GAL4 lines that promote low levels of expression of reporter genes, as opposed to high levels of reporter genes. Why might these weak GAL4 lines be useful for UAS-RNAi studies?

  10. The punt gene is located on the third chromosome and is required for the maintenance of the FSCs in the ovary (Kirilly et al. 2005). However, it is unknown if the FSCs mislocalize or have altered morphology. As a mutation in the punt gene is lethal to the fly, it is necessary to generate a small group of mutant cells using the MARCM technique to examine these characteristics. Using Figure 3 as a guide, what genetic elements are needed to generate FSCs that are mutant for punt and express a GFP reporter gene? What elements need to be on the third chromosome? Draw out the scheme as in Figure 3.

Acknowledgments

Many thanks to Edwin Chaharbakhshi, Matt Davis, Diana Luong, Elizabeth De Stasio, and Alana O’Reilly for reading and providing comments to improve the manuscript. J.C.J. and D.S. are supported by Loyola University Chicago.

Footnotes

Communicating editor: E. A. De Stasio

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