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Keywords:

  • stem cells;
  • flow cytometry;
  • cell cycle;
  • BrdU;
  • proliferation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Due to their characteristic inaccessibility and low numbers, little is known about the cell-cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell-cycle dynamics is flow cytometry, which is used routinely to study the cell-cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non-cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell-cycle dynamics and follow BrdU-labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo. Developmental Dynamics 238:1111–1117, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

During the cell cycle, DNA is accurately replicated and distributed as identical chromosomal copies to the resulting daughter cells allowing for the faithful transmission of genetic material from generation to generation (Elledge et al.,1997). The eukaryotic cell cycle is generally divided into four phases: DNA synthesis (S) phase, mitotic segregation (M), and two intervening gap phases (G1 and G2) preceding the S and M phases, respectively. Strict regulation of the cell cycle ensures that the events in each phase are completed before the cell can move on to the next phase (Elledge et al.,1997). Although these phases are generally present in most cell types, important exceptions exist. For instance, embryonic stem (ES) cells in culture exhibit a very unusual cell-cycle structure, characterized by a short G1 phase and a high proportion of cells in S-phase. As ES cells differentiate, the cell cycle changes dramatically, with the emergence of a significantly longer G1 phase (Smith,2001). The unique cell-cycle structure and likely mechanisms underpinning cell-cycle control in ES cells indicates that the cell-cycle machinery plays a role in the establishment and/or maintenance of the stem cell state.

In addition to variations in phase lengths, the cell cycle of many cells (including stem cells) is regulated in response to physiological and/or traumatic changes such as the replacement of cells lost to tissue homeostasis and the regeneration of injured body parts (Orford and Scadden,2008). This is observed in the stem cells of the hematopoietic compartment, which can reversibly switch from quiescence to self-renewal during homeostasis and repair (Wilson et al.,2008). In fact, such regulatory mechanisms appear to be widespread among distantly related metazoans. For example, the Drosophila germ line and follicle somatic stem cells of the ovary have been shown to adjust their proliferative rates in response to nutritional levels (Drummond-Barbosa and Spradling,2001). Similarly, mouse embryonic stem cells have been shown to respond to environmental insults such as irradiation by regulating the G1-phase checkpoint of the cell cycle to remove damaged cells from the population (Hong and Stambrook,2004). Furthermore, classical studies have shown that in planarians, the stem cells can respond to both global and local homeostatic changes (Dubois,1948; Baguñà,1974). For instance, starvation leads to an allometric reduction in size of the entire organism (Oviedo et al.,2003) and, locally, amputation leads to a burst of stem cell proliferation near the amputation plane without significantly affecting the proliferative rates of stem cells elsewhere in the animal (Baguñà,1975). These attributes, therefore, make planarians a good model system for studying the population dynamics of adult somatic stem cells in vivo. Ultimately, in vitro studies can only provide us with an approximation of the nature and functions of stem cells. Therefore, the opportunity to systematically study in vivo a cohort of stem cells such as those found in planarians is likely to provide novel insights on the mechanism controlling their proliferative activities.

In order to better understand the in vivo regulation of stem cells using the planarian Schmidtea mediterranea as a model system, we aimed to develop whole animal flow cytometric methods to measure cell-cycle dynamics under normal and experimental conditions. In the past, fraction of labeled mitosis (FLM) has been successfully used to study the planarian cell cycle (Newmark and Sánchez Alvarado,2000). However, this method is labor intensive as it involves whole-mount preparations, confocal microscopy, and cell-by-cell quantitation. An alternative method that is routinely used to monitor the cell cycle of yeast and mammalian cells is flow cytometry (Pozarowski and Darzynkiewicz,2004). This method takes advantage of the different DNA concentrations found in cycling cells, which allows measuring the distribution of cycling cells along the various phases of the cell cycle (G0/G1, S, G2, and M). Using flow cytometry, cell-cycle distribution can be analyzed rapidly based on the DNA content of individual cells. In addition, subpopulations of each cell-cycle phase can be quantified. Therefore, adaptation and optimization of flow cytometry to monitor the status of the cell cycle and regulation of planarian stem cells could emerge as a powerful, larger throughput alternative to FLM methods. Here, we report on our efforts to establish such methods and demonstrate their utility in the analysis of cell-cycle parameters both in wild type and experimentally manipulated organisms.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Cell-Cycle Analyses Using Flow Cytometry

To analyze the planarian cell cycle using flow cytometry, we tested and optimized several methods to dissociate and fix cells, stain DNA, and analyze the resulting data. We found that maceration solution (Newmark and Sánchez Alvarado,2000) and absolute methanol were the most efficient reagents for fixation (see Experimental Procedures section). In flow cytometric cell-cycle analyses, the staining method is critical because of cell-to-cell differences, cellular/nuclear concentrations, and stoichiometry of nuclear material. After several titration series of the concentration and incubation time courses of propidium iodide (PI), we determined that optimal staining was obtained reproducibly by using 5 μg/ml of PI for 2 hr at RT. Since PI is known to bind stoichiometrically to DNA, and cells at different stages of their cell cycle will have different amounts of DNA, it is possible to discriminate these subpopulations of cells based on fluorescence intensity (Robinson,1998). In order to analyze the resulting flow profiles, we tested various off-the-shelf software programs (e.g., Cyflogic, FlowJo, Flowexplorer). Because of ease of use and reliability, we selected Modfit LT to deconvolute flow curves (Fig. 1B, see Experimental Procedures section). We also adjusted the gate used for cell sorting during forward scatter measurements in order to avoid overestimating the number of cells in the G2/M phase, thus avoiding counting doublets made of groups of cells that are not fully dissociated from each other.

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Figure 1. Flow cytometric cell-cycle analyses. A: Diagrammatic representation of key steps involved in the preparation of samples for flow cytometry. B: Representative frequency curve of the cell-cycle profile obtained from analyzing the heterogeneous mixture of cells obtained after dissociating complete animals into individual cells. Cell cycle was determined by counting all the cells in the sample and plotting their respective DNA content. The two prominent peaks (in red) represent G0/G1 and G2/M phase cells, respectively. The intermediate region between peaks (blue hashes) represents S phase cells. C: Anti-phosphorylated histone H3 (H3P) immunostaining time course in whole-mount animals after feeding. Asterisks represent planarian photoreceptors. D: Quantitation of H3P-positive nuclei in animals over time after feeding. The graph represents at least five animals counted per time point. Error bars represent standard deviation. E: Cell-cycle profile of planarians 24 hr after feeding. Note the prominent increase in the area under the curve representing cells in S (blue lines) when compared to B. F: Graphic representations of time course following the percentage of cells in G0/G1, S, or G2/M phase of the cell cycle after feeding. The graphs represent three independent biological replicates. Scale bar in C = 0.2 mm.

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Once the dissociation, fixation, and labeling methods were optimized, we analyzed the cell-cycle profile of planarian cells obtained from wild type animals starved for one week (Fig. 1B). DNA content distribution of planarian cells consists of two predominant peaks corresponding to G0/G1 and G2/M phase cells, respectively. The intermediate region between both peaks includes cells in the S phase of the cell cycle. G2/M phase cells have twice the amount of DNA of G0/G1 phase cells, and S phase cells possess variable amounts of DNA as they progress through S between G1 and G2. Analyses of the PI profiles using Modfit LT provided us with an estimate of the percentage of cells in G0/G1, S phase, and G2/M (Robinson,1998). Average percentage of planarian cell populations in G0/G1, S, and G2/M phases are 81.99% (SD=2.4), 5.78% (SD=1.4), and 12.23% (SD=1.4), respectively. The representative cell distribution obtained in Figure 1D is comparable to that observed in mammalian tissue culture preparations, indicating that the developed method is sensitive enough to detect proliferating cells in a pool of postmitotic, differentiated, and heterogeneous cells derived from whole planarians. Using the methods described, canonical flow cytometric profiles such as those shown in Figure 1B can be obtained from as little as two average-sized planarians (4–6 mm in length).

Effects of Environmental Changes on the Cell Cycle Can Be Detected by Flow Cytometry

A mitotic burst of planarian stem cells has been observed after feeding (Baguñà,1974). Thus, we wished to determine if we could detect and reproducibly measure such changes by flow cytometry. To confirm whether the mitotic activity of stem cells changes in response to feeding, we first carried out whole-mount fluorescent immunostaining using the phosphorylated histone H3 (H3P) antibody at different time points after feeding (Fig. 1C). The inherent flatness of the animal and the finite number of mitotic figures in any given animal allowed us to easily quantify mitoses, and thus to measure changes in the number of mitotic cells at various time points after experimental manipulation. Our data show that the number of mitotic cells was increased significantly by 12 hr after feeding and eventually decreased to pre-feeding levels 72 hr later (Fig. 1D). To determine whether the cell cycle of planarian stem cells reflects the changes in the mitotic activity induced by feeding, animals were dissociated at 24 hr after feeding followed by flow cytometry (Fig. 1E and F). Cells found in G0/G1 phase are decreased from 84.98 to 80.41% (P = 0.02), while cells in S and G2/M phases are increased from 4.33 and 10.68% to 6.52% (P = 0.0106) and 13.07% (P = 0.012), respectively. This result indicates that environmental changes can modulate the cell-cycle dynamics of the planarian stem cell population. Since the length of S-phase and mitosis are relatively fixed, changes in cell-cycle parameters induced by feeding are likely to be manifested by shortening the length of G1. Thus, an accelerated transition from G1 to S phase may result in an increase in the number of mitotic cells. We tested this hypothesis by measuring changes in cell proliferation in fed animals by flow cytometry, and observed that the percentage of the cell population in each cell-cycle phase changed with time in a general trend toward a decrease in the number of cells in G0/G1-phase and an increase in the numbers of S-phase cells through 72 hr after feeding (Fig. 1F). These results demonstrate the ability of flow cytometry to detect the response of stem cells to mitogenic stimuli provided by changes in nutritional status (feeding), data that are consistent with the results obtained in whole-mount immunostainings with the anti-H3P antibody (Fig. 1C and D). From this study, we concluded that the regulation of planarian cell cycle by environmental changes could be reproducibly detected and measured using the flow cytometric assays developed.

Cell-Cycle Analyses of Animals Subjected to RNAi Treatments

To test if RNAi-induced phenotypes affecting stem cell functions can be reliably measured by the flow cytometric cell-cycle analysis developed above, two previously characterized cell-cycle-associated genes were tested: the planarian cell division cycle 23 (Smed-cdc23) and Smed-sec61 genes (Reddien et al.,2005). Prior studies have shown that planarians fed double-stranded RNA (dsRNA) of Smed-cdc23 have high mitotic activity, while those fed Smed-sec61 dsRNA have low mitotic activity (Fig. 2A and B) (Reddien et al.,2005). Flow cytometry was performed on dissociated cells from RNAi animals (Fig. 2C). C. elegans unc22 dsRNA was used as a control for RNAi feeding. If the effects of RNAi on the planarian cell cycle can be detected by the DNA analysis using flow cytometry, Smed-cdc23(RNAi) animals would be expected to have a higher percentage of cells in the G2/M phase relative to controls. Accordingly, we expect that planarians fed Smed-sec61 dsRNA will have a lower percentage of cells in the G2/M phase than controls. As expected, the percentage of cells in S and G2/M phases in Smed-cdc23(RNAi) animals increased from 16.46 and 8.81% to 21.42% (P = 0.0221) and 12.93% (P = 0.0017). This result probably reflects the fact that cdc23, a component of the anaphase-promoting complex (APC), is known to promote the progression from M to G1 phase (Irniger and Nasmyth,1997). Hence, the number of cells entering the G1 phase of the cell cycle is decreased in Smed-cdc23(RNAi) animals. In contrast, in Smed-sec61(RNAi) animals, the percentage of cells in G2/M phase is drastically reduced to 4.52% (P = 0.004). This results in a marked accumulation of cells in S phase (37.2% compared to 16.5% in controls, P = 0.009) accompanied by a notable decrease in the cells populating G0/G1 (58.3% compared to 75%, P = 0.0132). Such a decrease in G0/G1 may reflect the fraction of cells that re-enter the cell cycle during G1 progression and that are found in this region of the flow cytometry curve. These results clearly indicate that experimentally induced changes in the cell cycle can be detected using the flow cytometry methods developed, and that these types of analyses are likely to provide insight on various regulatory aspects of the planarian cell cycle at the molecular level.

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Figure 2. Changes in mitotic activity and the cell cycle in RNAi-treated animals. A: Immunostaining using H3P antibody was carried out in trunk fragments regenerating both heads and tails 14 days after RNAi feeding. Anterior is to the right. Scale bars = 0.2 mm. B: Quantitation of H3P-positive nuclei in RNAi animals. The data represent three independent biological replicates and demonstrate the range of hyper- and hypoproliferation achieved by silencing Smed-cdc23 and Smed-sec61. Error bars are standard deviation. C: Cell-cycle profiles for RNAi animals treated as in A, but analyzed by flow cytometry. Significant changes are seen in the S (area under the curve marked by blue lines) and G2/M phases of the RNAi-treated animals when compared to unc-22(RNAi) control (second peak shaded in red in both histograms). The first peak in red represents cells in G0/G1.

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BrdU Analyses Using FACS

Although we could detect changes in the cell cycle caused by environmental and experimental changes by flow cytometry using PI, the changes are relatively subtle because most cells of the animal are differentiated (G0) and the cell cycle of the stem cells is not synchronized (Newmark and Sánchez Alvarado,2000). To increase the resolution of cell-cycle measurements in planarians, various methods to differentiate stem cells from their postmitotic progeny were tested using Hoechst, TO-PRO-3, and BrdU. Among these, BrdU was the most effective. BrdU is a thymidine analog that becomes incorporated into newly synthesized DNA during S-phase, and so in planarians a short pulse of BrdU labels the cycling stem cells (Eidinoff et al.,1959). Therefore, it should be possible to cytometrically follow the fate of cells labeled with a single pulse of BrdU through multiple rounds of cell division. For instance, labeling cells with BrdU would allow us to distinguish the fraction of cells that become post-mitotic (G0) from the fraction of cells that will re-enter the cell cycle (G1).

In order to label cycling stem cells, a single pulse of BrdU is provided to the animals via feeding or injection. The animals are then dissociated into single cells at various time points, and the cells analyzed for BrdU incorporation along with DNA content using PI. Cells labeled with both of these markers were then subjected to flow cytometry. At 48 hr after BrdU, we observed that the G0/G1-phase of the cell cycle contains a mixture of both BrdU-unlabeled and labeled cells, while BrdU-positive cells were found occupying the majority of the regions representing the S and G2/M phases in the cytometry curve (Fig. 3A). In addition, we were able to follow the fate of the BrdU-labeled stem cells by dual flow cytometry time courses after introduction of a single pulse of BrdU (Fig. 3B). BrdU-positive cells with a DNA content corresponding to diploid cells (2n) were first detected between 1 and 2 hr after the administration of the thymidine analog. This observation supports prior observations that at any given time, planarian stem cells are rapidly entering the cell cycle (Newmark and Sánchez Alvarado,2000). We also detected BrdU-positive cells with a DNA content higher than 2n at this time point, indicating that proliferation in vivo is not synchronized and that cycling cells are found at multiple places along their progression through S phase at the time of BrdU delivery. This distribution profile of diploid (early S phase) and higher than 2n (late S, G2 and M phases) BrdU-positive cells was maintained for the next 16 hr. By 22 hr, however, we observed a marked accumulation of diploid BrdU-positive cells, most likely due to the generation of postmitotic, nonproliferating division progeny (Eisenhoffer et al.,2008). These results allowed us to deduce an approximate total length of the cell cycle of planarian stem cells to correspond to about 21 hr, which accounts for the time required for BrdU to be first detected in stem cells after its introduction (∼1 hr).

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Figure 3. BrdU analyses of planarian cells using flow cytometry. A: Forty-eight hours after labeling cycling stem cells with a single pulse of BrdU, animals were dissociated into single cells and processed for dual flow cytometry (PI plus BrdU). First, the PI profile was obtained (red plot), followed by selection of the BrdU-negative (blue plot) and BrdU-positive (green plot) cells. By superposing the BrdU-negative and -positive profiles, their location in the flow cytometry distribution can be defined (overlap). Note that most BrdU-negative cells are in G0/G1 phase, while BrdU-positive cells are found through G1, S, and G2/M. B: After a single injection of BrdU, animals were dissociated into single cells every 2 hr for 24 hr. Each time point was processed for BrdU fluorescent immunostaining and PI labeling. The resulting cells were analyzed for PI and BrdU fluorescence. The green rectangle indicates the region of the plot in which robust BrdU signal can be detected. The line perpendicular to the X-axis separates diploid (2n) from non-diploid cells (2n≤4n) as DNA is synthesized and cells traverse G2 and M as tetraploid (4n) cells.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

It is clear that cell-cycle regulation is critical for maintaining stem cells functions (Orford and Scadden,2008). Most studies on cell cycle in stem cells have been done in vitro using ES cells. The cell cycle of these cultured cells is constitutively primed for DNA replication. Hence, ES cells have a very short G1 phase with newly formed division progeny rapidly entering a new phase of DNA replication. In contrast, little is known about the cell cycle of both ES cells not undergoing clonal expansion (Rossant,2008), and adult somatic stem cells (D'Urso and Datta,2001). Given the abundance of stem cells and the number of biological functions that are driven by these cells in the planarian S. mediterranea (Newmark and Sánchez Alvarado,2002; Sánchez Alvarado,2006), these organisms provide a unique opportunity to explore cell-cycle regulation and dynamics of an adult stem cell population in vivo. Thus, we endeavored to develop optimal methods of flow cytometry to study stem cell proliferation in this animal.

First, cell-cycle distribution measurements allowed us to rapidly and reproducibly measure changes in cell-cycle dynamics of stem cells in planarians in response to environmental stimuli. Making such measurement with established methods such as FLM (Newmark and Sánchez Alvarado,2000) would require capturing via confocal microscopy large sets of optical stacks for multiple animals at multiple time points, followed by individual analyses of single- and double-labeled cells in each of the resulting optical sections. Using flow cytometry, for example, we were able to quickly detect that feeding results in a rapid G1 to S phase progression (Fig. 1F). These results suggest that specific mechanisms must be in place to regulate the rapid transit through the G1 phase in this adult somatic stem cell. Whether the shortening of G1 is due to repression of the early phase of G1 and/or the restriction point (Zetterberg et al.,1995; Hullemann et al.,2004), or whether similar regulatory events occur in the somatic stem cells of other organisms are important considerations for future studies. Likewise, the characterization of the molecular events effecting nutritional status into cell-cycle phase changes remains to be determined. The connection between nutrition and cell-cycle regulation is widespread among metazoans. Germline stem cells and ES cells are affected differently by nutritional changes. Drosophila germline stem cells promote their proliferation rate in response nutrients (Drummond-Barbosa and Spradling,2001), but ES cells are independent of serum stimulation (Schratt et al.,2001) or mitogenic signals (Burdon et al.,2002). ES cells can efficiently progress through the G1 to S phase constantly. Generally, in other systems, the rate-limiting transition from G1 to S is regulated by cyclin D, which itself is under the regulation of the retinoblastoma protein, a target of signaling pathways responding to environmental changes (Shen,2001). Moreover, changes in proliferative activity can also be detected by the flow cytometric method described here (data not shown), allowing us to monitor local changes in cell-cycle dynamics during regeneration. By combining the relative ease with which genes can be silenced in planarians (Reddien et al.,2005), the sensitive response to both nutritional status and amputation, and the flow cytometric methods reported here, a systematic dissection of G1 phase progression, the restriction point, and cell-cycle re-entry of an adult stem cell population is now within experimental grasp.

Second, the methods reported here allowed us to reproducibly detect differences in the cell-cycle distribution profiles of normal animals and RNAi-treated planarians in which different cell-cycle deficiencies are known to occur. Our data clearly indicate that the methodologies described here can be deployed to both characterize cell-cycle phenotypes caused by loss-of-function of cell-cycle-related genes, and as tools to screen for cell-cycle defects in RNAi screens. The number of mitotic cells and the percentage of cells in each phase of the cell cycle can serve as readouts for the functional characterization of the screen-identified molecules. For example, if a molecule promotes the progression of cell cycle after environmental change, animals fed dsRNA against this gene will show a phenotype of low mitotic activity and/or cell-cycle arrest. If, as expected, the planarian stem cells possess autonomous cell-cycle regulators, these factors are likely to modulate cellular proliferation in response to environmental changes. Alternatively, the cell cycle of the planarian stem cells could be regulated by cell non-autonomous signals responding to environmental changes, which could also be characterized in detail using the flow cytometric protocols described here.

Finally, BrdU labeling of cycling stem cells provided us with a remarkable improvement in the resolution of cell-cycle analyses based on flow cytometric measurements (Fig. 3). BrdU labeling in combination with flow cytometry will be instrumental in characterizing the cell-cycle characteristics of the planarian stem cells. Recently, we utilized this method to characterize the cell-cycle features of three planarian cell subpopulations defined by their sensitivity to irradiation in Fluorescent Activated Cell Sorting (FACS) methods (Hayashi et al.,2006; Eisenhoffer et al.,2008). These subpopulations are known as X1, X2, and Xins cells (Hayashi et al.,2006). X1 cells are extremely sensitive to irradiation and disappear in 1 to 2 days after irradiation, followed by the cells in X2, which persist for a total of 3 to 5 days post-irradiation. Xins cells, on the other hand, are not noticeably affected. By analyzing each of these subpopulations at different time points after BrdU incorporation, we were able to follow the movement of labeled cells from one subpopulation to another, and demonstrated that the cycling X1 cells can give rise to a portion of the cells found in the X2 population, allowing us to establish for the first time a lineage relationship between these cell types (Eisenhoffer et al.,2008). Future experiments will aim to incorporate mitotic measurements to combine BrdU and flow cytometry methods using, for instance, the anti-H3P antibody. Since histone H3 is phosphorylated at the G2 to M transition, mitotic cells could be resolved from G2 cells found in the G2/M peaks of flow cytometry profiles (Juan and Darzynkeiwicz,2004). Nevertheless, in its present state, the flow cytometric methods reported here provide us with a unique opportunity to utilize S. mediterranea as an experimental paradigm to investigate the cell-cycle dynamics of animal stem cells, an understudied aspect of in vivo stem cell biology.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Organism

The asexual clonal line CIW4 of S. mediterranea (Sánchez Alvarado et al.,2002) was maintained and used as described (Reddien et al.,2005; Gurley et al.,2008). All animals used in the experiments reported in this report were of similar sizes (4–6 mm in length). Control animals were starved for 1 week and used to obtain baseline information on proliferation status.

Flow Cytometry

In order to study planarian cells by flow cytometry, reproducible tissue dissociation methods were identified. We took advantage of the observation that animals placed in maceration solution (planarian water, glycerol, and acetic acid in a 13:1:1 ratio) result in both fixation and full tissue dissociation (Newmark and Sánchez Alvarado,2000). Briefly, animals were placed in maceration solution at 4°C for 16–18 hr. Fixed cells were then washed with calcium-magnesium-free media (CMF: 15 mM Hepes, 400 mg/L NaH2PO4, 800 mg/L NaCl, 1,200 mg/L KCl, 800 mg/L NaHCO3, 240 mg/L glucose, 1% BSA, pH 7.3) as previously described (Reddien et al.,2005) and treated with 5 μg/ml of RNaseA for 10 min at RT, followed by staining with 5 μg/ml of propidium iodide (PI), a DNA-binding dye for 2 hr at RT. The ribonuclease treatment is essential as PI can bind to RNA, thus introducing variability between samples during flow cytometric measurements. The cells were then subjected to flow cytometry using a Becton Dickinson FACScan. The resulting data were analyzed using the Modfit LT software (Verity Software House, Inc.), which is capable of deconvoluting DNA content into frequency histograms.

Immunostaining

One-week-starved or egg-yolk-fed animals were killed in 2% HCl for 5 min at room temperature (RT) and fixed in Carnoy's for 2 hr on ice (Sánchez Alvarado and Newmark,1999). The animals were then dehydrated in 100% methanol for 1 hr at −20°C and bleached in 6% H2O2 in methanol overnight at RT. Following rehydration through a methanol dilution series in PBST (PBS + 0.3% Triton X-100), animals were labeled with the phosphorylated histone H3 antibody (1:300, Upstate) and the goat anti-rabbit conjugated to HRP (1:150, Molecular Probes) as previously described (Newmark and Sánchez Alvarado,2000). Signals were amplified by tyramide conjugated to Alexa 568 (1:100, Molecular Probes), followed by washing with PBST including 0.25% BSA (Bovine Serum Albumin). Animals were mounted in Vectashield and imaged in a Zeiss Lumar microscope equipped with a Zeiss Axiocam.

RNAi

Wild-type animals starved for 10–14 days received three RNAi feedings (days 0, 4, and 13) as previously described (Reddien et al.,2005). To examine regeneration phenotypes, RNAi-treated animals were amputated 4 hr after RNAi feeding on days 4 and 13 and assayed for phenotypes on day 14.

BrdU Analysis Using FACS

Animals were injected with BrdU (about 100 nl of 5 mg/ml of BrdU), washed, and dounced in CMF at appropriate times after introduction of the thymidine analog. Dissociated cells were incubated in CMF on a nutator for 15 min at RT and filtered through a 50-μm Nitex filter. Cells were then fixed in 100% chilled methanol for 30 min on ice, collected by centrifugation at 1,200 rpm for 10 min, resuspended in 2N HCl in PBST (PBS + 0.5% Triton X-100), and incubated for 30 min at RT to denature their DNA. After neutralization by adding 0.1M Borax in PBST, cells were collected by centrifugation at 1,200 rpm for 10 min and washed in PBST. This was followed by blocking in PBST including 1% BSA for 1 hr and incubating the cells with a BrdU antibody (1:10, Oxford Ltd) overnight at 4°C. Once the primary antibody incubation concluded, the cells were washed twice with PBST for 15 min each, and then incubated for 1 hr at RT with an HRP-conjugated anti-rat secondary antibody (1:200, Upstate) preabsorbed wild-type animals fixed in Carnoy's. After several washes with PBST for 2 hr, cells were incubated with tyramide conjugated to Alexa 488 (1:200, Molecular Probes) for 30 min at RT. Cells were subjected to a final washing step with PBST and incubated in RNaseA (20 μl of 100 μg/ml) in PBS for 10 min at RT. Finally, cells were stained with PI (5 μg/ml) for 2 hr at RT. Analyses were performed using a BD Biosciences FACS Aria equipped with 630- and 488-nm lasers to resolve the PI fluorescence from the BrdU signal. The BD FACSDiva software was used to analyze the resulting profiles.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank James C. Jenkin, Nasum-Hawk Oh, and Wayne Green for technical flow cytometry support, particularly for discussions on the BrdU labeling experiments. A.S.A. is an investigator of the Howard Hughes Medical Institute.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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