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Cell Reports, Volume 2, Issue 4, 1048-1060, 18 October 2012
An In Vivo Functional Screen Uncovers miR-150-Mediated Regulation of Hematopoietic Injury Response
Brian D. Adams1, Shangqin Guo1, Haitao Bai1, 5, Yanwen Guo1, Cynthia M. Megyola1, Jijun Cheng1, Kartoosh Heydari2, Changchun Xiao3, E. Premkumar Reddy4 and Jun Lu1, ,
1 Department of Genetics, Yale Stem Cell Center, Yale Cancer Center and Yale Center for RNA Science and Medicine, Yale University, New Haven, CT 06520, USA
2 Department of Immunobiology, Yale Flow Cytometry Core Facility, Yale University, New Haven, CT 06520, USA
3 Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
4 Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA
5 Department of Hematology, Shanghai Jiaotong University Affiliated Shanghai First People's Hospital, Shanghai 200080, China
Graphical Abstract
► In vivo miRNA screen identified regulators of hematopoietic injury response ► miR-150 expression inhibits hematopoietic recovery across multiple lineages ► Upon injury, miR-150 null cells contribute faster to peripheral blood recovery ► Partial loss of the miR-150 target c-myb delays hematopoietic recovery
- Hematopoietic stem and progenitor cells are often undesired targets of chemotherapies, leading to hematopoietic suppression requiring careful clinical management. Whether microRNAs control hematopoietic injury response is largely unknown. We report an in vivo gain-of-function screen and the identification of miR-150 as an inhibitor of hematopoietic recovery upon 5-fluorouracil-induced injury. Utilizing a bone marrow transplant model with a barcoded microRNA library, we screened for barcode abundance in peripheral blood of recipient mice before and after 5-fluorouracil treatment. Overexpression of screen-candidate miR-150 resulted in significantly slowed recovery rates across major blood lineages, with associated impairment of bone marrow clonogenic potential. Conversely, platelets and myeloid cells from miR-150 null marrow recovered faster after 5-fluorouracil treatment. Heterozygous knockout of c-myb, a conserved target of miR-150, partially phenocopied miR-150-forced expression. Our data highlight the role of microRNAs in controlling hematopoietic injury response and demonstrate the power of in vivo functional screens for studying microRNAs in normal tissue physiology.
Tissue stem and progenitor cells, such as hematopoietic stem and progenitor cells (HSPCs), are responsible for sustaining tissue homeostasis and replenishing the loss of mature lineages after tissue injury. A major cause of hematopoietic injury stems from side effects associated with chemotherapy for solid tumors, which often indiscriminately kill or impair the function of HSPCs, leading to bone marrow toxicity with an acute reduction in peripheral blood cell counts (Wang et al., 2006). The affected cell types include platelets and myeloid cells, which are responsible for blood coagulation and controlling infection, respectively. Such undesired side effects can lead to life-threatening conditions in the weeks during and following administration of chemotherapy and often limit therapeutic doses (Wang et al., 2006; Lyman et al., 2003). Thus, understanding the molecular mechanisms governing the recovery rate of circulating mature blood cells remains an important question.
MicroRNA (miRNA)-mediated posttranscriptional control has emerged as a critical regulatory mechanism in diverse biological processes. These small noncoding RNAs target endogenous messenger RNAs, largely through cognitive sites in the 3′ untranslated regions (UTRs), resulting in target degradation or translational inhibition (Bartel, 2009). Multiple laboratories, including ours, have reported that specific miRNAs can control hematopoiesis (Baltimore et al., 2008; Guo et al., 2010; Lu et al., 2008; Garzon and Croce, 2008), yet it remains largely unknown whether miRNAs can regulate hematopoietic recovery rate after injury.
Functional screens can provide a direct and powerful approach to identify gene functions in mammalian biology. Indeed, in vitro screens based on miRNA expression libraries have proven highly useful (Voorhoeve et al., 2006; Izumiya et al., 2010; Huang et al., 2008; Poell et al., 2011). For instance, miR-372/miR-373 were found to be potent oncogenes that have a stronger effect than p53 knockdown in relieving oncogene-induced senescence (Voorhoeve et al., 2006). Despite the successes of in vitro miRNA screens, in vivo miRNA screens have not been reported in mammals. Specifically, questions regarding physiological processes, such as studying HSPCs, are often difficult to model in vitro because hematopoietic stem cells easily lose stemness outside the host environment. Thus, genetic screens to study HSPCs were rarely attempted in vitro or in vivo, often requiring large cohorts of mice (Ali et al., 2009; Deneault et al., 2009; Hope et al., 2010; Wang et al., 2012). Recent studies with barcoded shRNA libraries (Zender et al., 2008; Bric et al., 2009; Meacham et al., 2009; Wang et al., 2012) suggest the prospect of an in vivo barcoded miRNA screen to study HSPC physiology, with multiple miRNAs screened in each individual mouse, and analysis of barcode abundance to deconvolute the contribution of each miRNA. There are, however, two limiting factors for a barcoded HSPC screen. One is the limited number of HSPCs from in vivo sources, making it challenging to obtain enough cell coverage for large numbers of genetic constructs. Second, only a limited number of normal HSPCs may engraft each lethally irradiated transplantation recipient (Lu et al., 2011), creating a bottleneck for analyzing a large number of genes per recipient mouse. In this regard, miRNAs are especially favorable candidates for medium-sized library screens because only a few hundred miRNAs are predicted to target >30% of the protein-coding transcriptome (Bartel, 2009), thus allowing for a small number of miRNAs to represent substantial gene regulatory perturbations.
In this study, we established an unbiased, in vivo, gain-of-function screen to identify miRNAs involved in modulating hematopoietic recovery in mice upon chemotherapeutic challenge. The term "hematopoietic recovery" in this paper is used to describe the recovery of peripheral blood cells after chemotherapy-induced suppression of peripheral blood parameters, thus reflecting underlying mechanisms of both regeneration and apoptosis of bone marrow progenitors. We modeled chemotherapy response using 5-fluorouracil (5-FU), a clinically used drug for solid cancers (Longley et al., 2003). With a library of 135 hematopoietic-expressed miRNAs, we assayed the function of 35–71 miRNAs in each recipient mouse. Three out of four screen candidates were validated to inhibit hematopoietic recovery. Among them, gain- and loss-of-function experiments established the importance of miR-150, partially through targeting c-myb, a conserved miR-150 target. Our data highlight the role of miRNAs in controlling response of HSPCs toward tissue injury and demonstrate the feasibility and power of functional in vivo barcode screens for studying tissue physiology.
Injury from 5-FU Induces Vast miRNA Expression Changes in the Bone Marrow
To identify miRNAs that regulate hematopoietic response after 5-FU-induced injury, we initially analyzed miRNA expression changes in bone marrow cells from mice with or without 5-FU treatment. Global miRNA expression profiling of the 5-FU and untreated cohorts showed that 92 miRNAs (among 168 that passed the detection threshold) were significantly upregulated or downregulated, with false discovery rates <5% (Figure S1A; Table S1. Global miRNA Expression Profiling of Bone Marrow Samples from 5-FU-Treated or Untreated Cohorts, Related to Figure S1). This large number of altered miRNAs made it difficult to identify which miRNAs serve as functional regulators of hematopoietic injury response. We thus went on to perform an in vivo functional screen.
Developing a miRNA Expression Library and Bead-Based Barcode Detection System
As a first step toward establishing a physiologically relevant in vivo gain-of-function screen, we constructed an expression library comprised of individual miRNAs that are expressed in the hematopoietic system (Figure S1B). These miRNAs (Table S2. Primer Sets for Cloning and Barcode Detection Probe Sequences, Related to Figures 1 and S1) were chosen from publicly available deep-sequencing data of hematopoietic tissues (Landgraf et al., 2007), largely based on their abundance. In anticipation of limited numbers of HSPCs that may engraft in each mouse (Lu et al., 2011), we restricted the size of the library to 135 miRNAs. For those miRNAs that can be produced from multiple loci in the genome, only a single representative locus was picked. With these design principles, we PCR amplified genomic regions harboring the miRNA hairpins and flanking sequences and individually cloned them into a pMSCV-based retroviral vector, which is widely used to support overexpression in the hematopoietic system (Lu et al., 2008; Guo et al., 2010).
With a miRNA expression library in hand, we then developed an inexpensive bead-based system that can readout the contribution of each library construct among a pool of library-transduced cells (Figure S1C). We took advantage of unique sequences (referred to as barcodes below) present in the hairpin-flanking regions of miRNA-coding genes. Complementary oligonucleotides to barcode sequences were coupled to polystyrene beads embedded with unique combinations of fluorophores. Genomic DNA samples from library-transduced cells could be PCR amplified with biotinylated primers specific to the expression vector, hybridized to the barcode-detector-coupled beads, and labeled with streptavidin-phycoerythrin. A flow cytometer was then used to report the identity of the beads and quantify the barcode abundance detected on each bead.
This bead-based detection system has the specificity and reproducibility required for robust barcode quantification of the miRNA library. To test detection specificity, genomic DNA from miR-29a-transduced HL-60 cells was assayed, and signal was only observed from the respective miR-29a detector (Figure S1D). We next pooled the 135 library constructs, produced virus, and transduced HL-60 cells. The majority of barcodes (130 out of 135, or 96%) could be robustly detected above background (Figure S1D). Independent PCR amplifications from the same genomic DNA sample produced low variance in signals, indicating that the detection system is reproducible (Figure S1E). Thus, with the availability of the expression library and a reliable barcode detection system, we proceeded to the in vivo screen.
An In Vivo Gain-of-Function Screen Reveals Candidate miRNAs Involved in Regulating Hematopoietic Recovery
To uncover miRNA-mediated regulation of hematopoietic injury response upon 5-FU treatment, we performed an in vivo functional screen as illustrated in Figure 1. Adult murine donor bone marrow cells were transduced with the miRNA expression library as a pool and transplanted into lethally irradiated recipient mice. To ensure that donor cells contained largely single copy integrations of the viral constructs, we titered the miRNA library to a transduction efficiency of ∼10%–25%. After transplantation, we harvested peripheral blood of recipients at defined intervals, both to monitor the level of engraftment and to prepare genomic DNA for barcode detection. Once engraftment reached relatively stable levels at 11 weeks posttransplantation (Figures S2A and S2B), the recipient animals were treated with 5-FU. Day 10 post-5-FU reflected the time point with strong hematopoietic suppression, whereas day 22 represented normalization of the blood parameters (Figure S2C). We reasoned that miRNAs with barcode signals decreased after 5-FU treatment would suggest an inhibitory role upon 5-FU-induced hematopoietic recovery, whereas those that increased would be potential enhancers of this process.
A key consideration for the screen is the functional heterogeneity of HSPCs. Some dominant HSPC clones may have a stronger contribution to peripheral blood than other clones. Thus, miRNAs landing in such clones could be misclassified as enhancers of recovery, even though they do not function in this capacity. Conversely, miRNAs integrating into weak HSPC clones could be misclassified as inhibitors. To avoid HSPC heterogeneity confounding interpretation of barcode results, we used a short duration of viral transduction to minimize proliferation of HSPCs during ex vivo culture, thus reducing the likelihood that descendants of the same HSPC clone could be transplanted into different animals. We also performed three independent viral transductions and transplanted into a total of 15 mice, further reducing the possibility of dominant HSPC clones rendering false-positive results in multiple recipients.
Quantification of barcodes revealed several interesting features from this screen:
(2) On average, the effect of 52 different miRNAs could be assayed in each recipient mouse. At 3 weeks posttransplantation, each recipient contained 62–108 detectable barcodes. This number decreased with time and was largely stabilized by week 11 posttransplantation to 35–71 detectable barcodes in each animal (Figure 2A). This drop in barcode number is likely due to preferential retroviral transduction of transiently amplifying progenitors that only support short-term contribution to peripheral blood, whereas contribution from the less-transduced but more primitive HSPCs supports longer-term engraftment. Constitutive expression of miRNAs that antagonize engraftment may also contribute to this reduction in barcode number.
(3) Barcode patterns differ between mice. Unsupervised clustering of barcode abundance showed that after 3 weeks posttransplantation, there were more differences in barcode patterns between mice than across different time points within the same mouse (Figure 2C). This result suggests that different sets of miRNAs were transduced in different mice and indicates that barcode abundance changes should only be compared within the same recipient across time points.
(4) A large number of barcodes could be collectively detected within the entire screen cohort. At week 3 posttransplantation, 124 barcodes out of 135 (92%) could be detected in at least one recipient, and 115 barcodes (85%) could be detected in at least five different recipients (Figure S3D). At the time points specifically associated with the 5-FU assay (week 11 and 10 days post-5FU), 124 barcodes (92%) could be detected in at least in one mouse, whereas 88 barcodes (65%) were present in at least five mice (Figure 2B). Taken together, the above-mentioned data indicate that it is possible to use a small number of animals to quantify the effects of a moderate number of genes.
To assess the effect of miRNAs during hematopoietic injury response to 5-FU administration, we considered the effect of HSPC heterogeneity and reasoned that miRNA barcodes that showed consistent changes in multiple animals were more likely true candidates rather than false positives. To reflect this concept, we used a consistency score to quantify the barcode abundance changes in the screen cohort (see Experimental Procedures ). After comparing day 10 and day 22 post-5-FU time points with the closest pre-5-FU time point (week 11), we assigned downregulation of barcode signals in each mouse as −1 and upregulation as +1. Thus, miRNAs that antagonized hematopoietic recovery would have a negative consistency score, whereas miRNAs that enhanced recovery would have a positive score. Ranking miRNAs based on these scores, we found that a small number of miRNAs showed consistent trends (Figure 3A), and the rankings were insensitive to variations in analysis parameters (Experimental Procedures ; Table S3. Consistency Scores Are Largely Insensitive to the Variation of Analysis Parameters for Most Barcodes, Related to Figures 3 and S3), indicating minimal analysis artifact.
Upon close inspection, upregulated barcodes showed less consistency, with changes sometimes driven by a single screen cohort. We thus focused on the top four miRNAs whose barcode abundance was decreased in the screen and was distributed across multiple cohorts and across both post-5-FU time points (Table S3. Consistency Scores Are Largely Insensitive to the Variation of Analysis Parameters for Most Barcodes, Related to Figures 3 and S3; Figures 3A, 3B, and S3E). Thus, miR-153-2, miR-150, miR-301, and miR-652 (ranked from top down) were chosen for further experimental validation.
Forced Expression of miR-150 and Other Candidates Inhibits Hematopoietic Recovery upon 5-FU Treatment
To validate screen candidates, we transduced miR-153-2, miR-150, miR-301, miR-652, or a vector control into donor bone marrow cells and assayed the recovery of these cells in transplant recipients after 5-FU treatment. A constitutive GFP on the vector labeled transduced cells, so competitive recovery of transduced cells versus nontransduced cells within the same recipient mouse could be reflected by GFP+/GFP− ratio changes in the peripheral blood. In addition, by using lineage-specific antibodies, we could determine the effects of the miRNA candidates on Mac1+ (CD11b) myeloid cells, B220+ (CD45R) B cells, CD3+ T cells, as well as anuclear CD41+ platelets. For transplant recipients, we followed the GFP+/GFP− ratio after transplantation and waited until the ratio largely stabilized before treating mice with 5-FU (Figures S4A and S4B; data not shown). In control recipients, the GFP+/GFP− ratio in all examined lineages showed relatively minor perturbations following 5-FU treatment, likely due to mild toxicity of GFP expression (Figure 4). In contrast, the expression of miR-153-2 or miR-150 strongly inhibited both myeloid and platelet recovery (Figures 4A and 4B). Interestingly, expression of the weaker candidate miR-652 led to a weaker yet significant suppression of myeloid and platelet recovery (Figure 4C). Candidate miR-301 did not cause significant changes and is thus a false positive (data not shown). In total, we successfully validated three out of four candidate miRNAs from the screen. Because miR-150 caused a strong inhibitory effect in the validation experiments and is the only miRNA among the three validated candidates that has an available knockout model (Xiao et al., 2007), we thus focused on this miRNA for follow-up experiments.
The miR-150 recipient cohort displayed a significant and sharp decrease in the GFP+/GFP− ratio in both myeloid and platelet lineages as early as 7 days post-5-FU (Figure 4A). This strong suppression of the GFP ratio persisted for ∼2–3 months and then gradually recovered to levels similar to that of the control cohort. The strongest ratio differential between miR-150 and the control cohort was at 16 days, with platelets suppressed by 4.8-fold and myeloid cells by 2.6-fold. B and T lymphoid lineages were overall constitutively inhibited by the expression of miR-150 (Xiao et al., 2007; Lu et al., 2008), reflected by a lower initial GFP+/GFP− ratio (Figure S4C). Nevertheless, a ratio drop was also observed post-5-FU (Figure 4A), albeit to a lesser extent than myeloid and platelet lineages. Similar to myeloid cells, the GFP+/GFP− ratio of lymphoid cells recovered close to control levels after long duration. The time course of GFP+/GFP− ratio normalization suggests that forced expression of miR-150 possibly negatively regulates hematopoietic output from multiple bone marrow progenitor populations, including multipotent progenitors, common myeloid progenitors, and lineage-committed progenitors, but with a lesser effect on long-term stem cells.
miR-150 Knockout Bone Marrow Confers an Accelerated Recovery Response to Hematopoietic Injury
We asked whether loss of miR-150 induces an opposite phenotype as compared to miR-150 gain of function, i.e., accelerating hematopoietic recovery. The germline miR-150 knockout mice were viable and normal in fertility, development, and behavior (Xiao et al., 2007). Further examination of hematopoietic tissues revealed largely normal steady-state hematopoiesis. Peripheral myeloid, lymphoid, and red cell counts of miR-150−/− mice were indistinguishable from wild-type littermates (Figures 5A and S5B). Similarly, proportions of bone marrow and spleen Ter119+, Mac1+, CD3+, and B220+ cell lineages were similar to controls (Figure S5C). Although peripheral platelet counts and spleen megakaryocyte frequency trended lower in the knockouts, the differences were not statistically significant (Figures 5A and S5D). Note that although changes in specific B cell subsets (Figure S5A), NK, and iNKT cells have been reported in miR-150 knockout mice (Xiao et al., 2007; Bezman et al., 2011; Zheng et al., 2012), changes in these populations were not expected to change the overall peripheral blood cell counts.
To investigate hematopoietic injury response, we conducted competitive recovery experiments, as illustrated in Figure 5B. We genetically labeled the majority of platelets and nucleated blood cells by crossing miR-150−/− mice with β-actin-GFP mice (Okabe et al., 1997). Bone marrow cells from GFP+ miR-150−/− or GFP+ miR-150+/+ mice were mixed 1:1 with GFP− wild-type bone marrow and transplanted into wild-type recipients. After reestablishment of hematopoietic homeostasis and GFP+/GFP− ratio stabilization (Figure S5E), recipient mice were challenged with 5-FU. Examination of peripheral blood GFP+/GFP− ratios indicated that miR-150−/− myeloid cells and platelets recovered faster than wild-type controls (Figure 5C). Significant differences of the competitive ratio for platelets between knockout and control cohorts were observed from day 12, with the maximum increase of 34.5% at 22 days post-5-FU. For myeloid cells, a maximal ratio increase of 50.1% was also observed at day 22. B and T lymphocytes showed only minor ratio alterations, similar to the aforementioned forced expression model.
Unexpectedly, the GFP+/GFP− ratios of myeloid and lymphoid cells (but not platelets) showed a time-dependent fluctuation, even in the wild-type control cohort, which may be partially due to incomplete GFP labeling of hematopoietic cells in β-actin-GFP mice (∼69% GFP+ in peripheral Mac1+ cells; ∼72% in B220+ cells; ∼75% in CD3+ cells; ∼81% in platelets). To eliminate the possibility that the differences we observed were due to artifacts of GFP+/GFP− ratio fluctuation, we performed competitive recovery experiments using different isoforms of CD45. Similar to the GFP model, bone marrow from CD45.1+ littermates of miR-150−/− or miR-150+/+ mice was mixed 1:1 with CD45.2+ wild-type bone marrow cells and transplanted into lethally irradiated CD45.2+ recipients (Figure 5B). 5-FU was administered following recovery from transplantation and CD45.1+/CD45.2+ ratio stabilization (Figure S5F). Consistent with the GFP model, the myeloid lineage showed faster recovery in the knockout cohort, whereas B and T lymphocytes were largely similar to controls (Figure S5G). Because CD45 is not expressed on platelets, platelet ratios could not be assessed. Taken together, the aforementioned experiments established that loss of miR-150 accelerated the recovery of myeloid and platelet lineages after injury.
Expression of miR-150 Impairs Bone Marrow Clonogenic Potential upon 5-FU Treatment
To further elucidate how miR-150 inhibits hematopoietic recovery, we reasoned that forced expression of miR-150 could impair the function of multiple bone marrow progenitors upon 5-FU administration. This hypothesis is consistent with our observation in Figure 4A, in which the ratio of miR-150-transduced to -nontransduced cells in the peripheral blood was sharply decreased for several weeks and then gradually recovered after an extended period of time. To test this, we generated bone marrow transplant recipients expressing miR-150 or the vector control. After peripheral GFP+/GFP− ratios stabilized, we isolated GFP+ (transduced) and GFP− (nontransduced) bone marrow populations from the same recipient mouse and tested their clonogenic potential in vitro. The GFP+ and GFP− cells from both miR-150 and control vector recipients gave rise to similar frequencies of myeloid colonies (Figure 6A), indicating that expression of miR-150 did not adversely affect colony formation under homeostatic conditions. In contrast, for bone marrow cells harvested 6 days after 5-FU administration, GFP+ cells from miR-150 recipients showed an ∼2.9-fold decrease in myeloid clonogenic potential, whereas GFP− cells from the same recipient produced similar frequencies of colonies compared to control vector recipients (Figure 6A). Pre-B cell colonies were less severely affected by miR-150 (Table S4. Results from Myeloid and Pre-B Colony Formation Assays in miR-150 Overexpression, miR-150 Knockout, and c-myb Heterozygous Knockout Models, Related to Figures 6 and S7). Upon closer examination, this decrease in myeloid colony formation of miR-150-expressing cells impacted all types of colonies, including erythroid, granulocytic/monocytic, and the more primitive GEMM colonies (Table S4. Results from Myeloid and Pre-B Colony Formation Assays in miR-150 Overexpression, miR-150 Knockout, and c-myb Heterozygous Knockout Models, Related to Figures 6 and S7), supporting the notion that multiple types of hematopoietic progenitors were compromised.
We then examined the miR-150 knockout model. Under steady-state hematopoiesis, no difference in myeloid colony number was observed between knockout and wild-type mice. In contrast, when we examined bone marrow after 5-FU treatment, miR-150 knockout cells generated a significant increase in the number of myeloid colonies compared to controls (Figure 6B; Table S4. Results from Myeloid and Pre-B Colony Formation Assays in miR-150 Overexpression, miR-150 Knockout, and c-myb Heterozygous Knockout Models, Related to Figures 6 and S7). These data indicate that miR-150 inhibits bone marrow colony formation upon 5-FU-induced injury.
This colony inhibition effect could be due to miR-150 inhibiting hematopoietic progenitor function during stressed hematopoiesis or sensitizing progenitors to 5-FU-induced cell death, or both. We tested the first possibility using a postirradiation hematopoietic recovery model. Wild-type bone marrow cells were transduced with miR-150 or vector control, sorted for GFP+-transduced cells, and transplanted into irradiated recipients. Ten days after transplantation, miR-150 expression resulted in significantly reduced spleen colony formation (Figure 6C), which is an in vivo measure of hematopoietic progenitor activity (Till and McCulloch, 1961). Importantly, because only the transplant hosts were irradiated but not the donor cells, this experiment measured postinjury responses rather than injury itself. Thus, we conclude that miR-150 can inhibit hematopoietic progenitor function during states of stress or injury.
To investigate whether miR-150 sensitizes bone marrow cells to 5-FU-mediated apoptosis, we first transduced wild-type HSPCs (Lin−Sca+ Kit+) with mir-150 or control vector. Cultured HSPCs were then treated with 5-FU in vitro and monitored for apoptosis by flow analysis of the AnnexinV+7AAD− population in transduced (GFP+) and untransduced (GFP−) fractions. Compared to controls, miR-150-transduced cells showed higher basal apoptosis, which was further elevated upon 5-FU treatment (Figures 6D and S6A), indicating that miR-150 can sensitize HSPCs to apoptosis in vitro. However, we did not observe changes in apoptosis in vivo using the miR-150 overexpression model described above (Figures 6E and S6B), at 6 days post-5-FU when apoptosis was prevalent (data not shown). These data suggest that the inhibitory effect of miR-150 on bone marrow progenitors may also be due to enhanced apoptotic sensitivity toward injury. Taken together, the data above show that forced expression of miR-150 suppressed the bone marrow clonogenic potential after 5-FU administration.
Heterozygous Loss of the miR-150 Target c-myb Partially Phenocopies miR-150 Overexpression
To understand the molecular mechanism by which miR-150 inhibits hematopoietic recovery, we focused on the conserved miR-150 target c-myb (Xiao et al., 2007; Lu et al., 2008; Barroga et al., 2008). We asked whether heterozygous loss of c-myb could phenocopy miR-150 overexpression, i.e., having a hematopoietic-autonomous effect to suppress hematopoietic output in response to 5-FU-induced injury. We examined a conditional heterozygous c-myb model because germline knockout of c-myb is embryonic lethal (Mucenski et al., 1991), and conditional homozygous knockout of c-myb in the hematopoietic system quickly depletes hematopoietic stem cells (Lieu and Reddy, 2009). In addition, because miRNAs do not completely suppress the expression of their target gene(s), heterozygous loss of the target better mimics miRNA action.
Competitive recovery experiments were performed to assay the role of c-myb (Figure 7A). CD45.1+/CD45.2+ bone marrow cells from Mx1-Cre+;Mybfl/+ mice or Mx1-Cre−;Mybfl/+ mice were mixed 1:1 with CD45.2+ wild-type bone marrow cells and transplanted into CD45.2+ recipients. All recipient mice, regardless of the status of the Mx1-Cre allele, were injected with pIpC, a standard treatment to induce Mx1-Cre expression in the hematopoietic system. After the excision of c-myb was confirmed in the Cre+;Mybfl/+ recipients (Figure S7A) and blood parameters stabilized (Figures S7B and S7C), recipient mice were treated with 5-FU, and the ratio between CD45.1+ cells and competitor CD45.2+ cells was quantified. Note that we waited 35 days between the pIpC and 5-FU treatments to avoid an effect on stem cell activation (Essers et al., 2009). As expected, nonexcisable Mx1-Cre−;Mybfl/+ cells behaved similarly to wild-type competitor cells. In contrast, Mx1-Cre+;Mybfl/+ cells showed slower recovery in the myeloid lineage, but not in lymphoid lineages (Figure 7B). We also crossed the Mx1-Cre+;Mybfl/+ mice with β-actin-GFP mice to examine platelets. Indeed, heterozygous loss of c-myb also decreased the platelet recovery rate (Figure S7E), although the level of inhibition on myeloid and platelet lineages was less compared to miR-150 overexpression (Figure 4A). Also similar to the miR-150 overexpression model, myeloid clonogenesis trended lower after 5-FU treatment in heterozygous c-myb knockout cells (Figure S7D; Table S4. Results from Myeloid and Pre-B Colony Formation Assays in miR-150 Overexpression, miR-150 Knockout, and c-myb Heterozygous Knockout Models, Related to Figures 6 and S7). Overall, these data demonstrate that heterozygous loss of c-myb partially recapitulated the phenotypes associated with the miR-150 gain-of-function model.
This study demonstrates the feasibility to perform in vivo miRNA screens in live mice with a barcoded library and the power to uncover regulators of complex physiological processes. The finding through the screen that miR-150 impaired hematopoietic injury response contrasts the miRNA expression-profiling results comparing bone marrow samples with or without 5-FU treatment, which found 92 out of 168 (55%) miRNAs with significant expression changes (Figure S1A). Interestingly, miR-150 was among the 45% of miRNAs that were not significantly changed after 5-FU treatment (Table S1. Global miRNA Expression Profiling of Bone Marrow Samples from 5-FU-Treated or Untreated Cohorts, Related to Figure S1). These results reiterate usual problems associated with gene-profiling studies: that the resulting miRNA changes are often too numerous to study. On the other hand, functionally important genes may be missed with profiling. Even after we identified miR-150, measuring miR-150 expression in Kit+ or other potential bone marrow progenitor populations did not yield intuitive expression patterns associated with its functional importance: miR-150 expression was higher and c-myb lower in post-5-FU samples than those without treatment (Figure S6C). Similarly, the complex nature of our measured hematopoietic injury response, which reflects the clinically relevant rebound of circulating mature cells within the peripheral blood after acute injury, can be challenging to model in vitro. We performed two experiments to measure the effect of forced miR-150 expression in cultured Ficoll-purified bone marrow cells but did not observe any differential competitive expansion/survival disadvantage in the presence of varying amounts of 5-FU (data not shown). We only detected changes in apoptosis when starting with purified HSPCs, suggesting that it would have been difficult to discover the role of miR-150 in hematopoietic injury response in a culture dish without the initial in vivo screen.
The bead-based detection system for genetic barcodes is specific and reproducible for functional screens. Compared to next-generation sequencing, it is inexpensive, flexible to design, and more readily accessible. The inexpensiveness makes it more attractive to run small sample batches, such as those during assay set up, or screens with a medium-sized library. It is likely less sensitive compared to next-generation sequencing and less powerful for large genetic libraries, but our detection rate in mouse (92% of the library at 3 weeks) suggests that sufficient sensitivity of detection can be achieved for libraries of ∼100–200 genes in complexity.
The data presented in this study highlight the role of miR-150 in controlling hematopoietic recovery after injury. Although the functional screen was designed to uncover regulatory miRNAs that control short-term peripheral blood recovery after acute injury, which is highly relevant to clinical management of patients receiving chemotherapy, the effects with miR-150 overexpression and knockout studies could be seen within the short-term window of 1–3 weeks and also beyond. The miR-150 overexpression model showed weak but significant lymphoid recovery changes, which was not seen in the miR-150 knockout model. This difference may be due to forcing miR-150 expression in cell types that normally do not express miR-150. Our finding, that miR-150 suppresses multiple types of hematopoietic progenitor activities, including both immature and more mature myeloid progenitors upon 5-FU treatment, provides one explanation for the observed short-term and longer-term effects of miR-150. It is important to note that this progenitor cell effect of miR-150 does not contradict previous knowledge of miR-150 in specific lineage differentiation (Lu et al., 2008; Barroga et al., 2008; Xiao et al., 2007; Bezman et al., 2011; Zheng et al., 2012). For example, even though the forced expression of miR-150 elevates platelet production in comparison to myeloid cells (Figure S4C) under homeostatic conditions as expected, upon hematopoietic recovery from acute injury, miR-150 affected more upstream progenitors that largely masked this platelet production bias.
Mechanistically, we show that hematopoietic-intrinsic heterozygous loss of c-myb produced similarly delayed recovery after 5-FU. It is known that c-myb regulates proliferation and apoptosis-related genes, such as c-myc, cyclins, KIT, Bcl-2, and Bcl-XL (Ramsay et al., 2003; Ramsay and Gonda, 2008), some of which may mediate c-myb's function in hematopoietic recovery. The delayed recovery in the c-myb model was both shorter and weaker than the gain of function of miR-150 and did not affect the lymphoid lineages. We speculate that additional targets may also be involved, or the difference in phenotype may be due to quantitative differences in c-myb suppression in the two models.
It is intriguing that loss of miR-150 had mild effects on steady-state hematopoiesis (other than B cell subsets, NK and iNKT cells), yet knockout cells displayed the beneficial effect of accelerated rate of hematopoietic recovery in the peripheral blood. This is somewhat puzzling given that miR-150 is evolutionarily conserved from human to frog, although it is possible that the roles of miR-150 in specialized immune cell production, or anticancer effect in lymphoma (Chang et al., 2008), contribute to the pressure for genetic conservation. Nevertheless, our data suggest that an interesting avenue to explore in the future will be to temporarily suppress miR-150 activity for the clinically beneficial outcomes of faster platelet and myeloid cell recovery. Because mice null of miR-150 show largely normal behaviors, it suggests that inhibition of miR-150 may have limited side effects. The recent progress in in vivo delivery of miRNA inhibitors (Stenvang et al., 2012) will, in the future, help to test this concept in a more translational setting.
Experimental Procedures
Mouse Strains and Maintenance
All mouse studies were conducted in accordance with federal guidelines and were approved by the Institutional Animal Care and Use Committee of Yale University. See Extended Experimental Procedures for more details.
Constructing a miRNA Expression Library
Design of the retroviral expression library was carried out based on sequence information present in miRBASE v9.0 (Kozomara and Griffiths-Jones, 2011). Desired miRNA hairpins for the library were identified in the human genome, and their ∼200 bp flanking sequences were scanned for the presence of other miRNAs or polyA signals. Specific primers (see Table S1. Global miRNA Expression Profiling of Bone Marrow Samples from 5-FU-Treated or Untreated Cohorts, Related to Figure S1) were designed so that they can uniquely amplify the corresponding miRNA, and through a two-step approach, cloned into the pMIRWAY-GFP vector (Guo et al., 2010; Lu et al., 2008). Retrovirus was produced in 293T cells from a pooled plasmid preparation of the pMIRWAY-miRNA library. See Extended Experimental Procedures for more details.
In Vivo miRNA Screen and Barcode Detection
For the in vivo screen, donor adult bone marrow cells were transduced with pooled miRNA viral library, and murine bone marrow transplantation was performed similar to published procedures (Guo et al., 2010). Starting from 3 weeks posttransplantation, genomic DNA isolates from the peripheral blood samples were prepared for barcode detection by first performing PCR to amplify the miRNA cassette. PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN), and miRNA barcodes were detected using a Luminex bead-based detection system. Acquired median fluorescence intensity values were then used in subsequent data analysis. See Extended Experimental Procedures for more details on transplantation, sample preparation, barcode detection, and signal analysis.
Murine Bone Marrow Transplantation
For single miRNA overexpression studies, murine bone marrow transplantation was performed similar to published procedures (Guo et al., 2010). For the miR-150 and c-myb competitive transplant assays, bone marrow was collected from donor animals, mixed at a 1:1 mononuclear cell ratio with CD45.2 wild-type competitor cells, and injected into 9.0 Gy irradiated recipient mice.
For determining the GFP ratios of different peripheral blood lineages, peripheral blood was lysed and stained with the following antibody mixture: Mac-1-PE, B220-APC, and CD3-PE-Cy5 in PBS +2% FBS. For experiments using the 45.1 congenic system, CD45.1-FITC was added to the antibody mixture. To assess GFP ratios in platelets, peripheral blood was stained with CD41-PE in sodium citrate buffer, fixed in 1% PFA, and analyzed on FACSCalibur. See Extended Experimental Procedures for details.
Statistical Analysis
Student's t test was used for p value calculation unless specified otherwise.
Mouse Strains and Maintenance
All mouse studies were conducted in accordance with federal guidelines and were approved by the Institutional Animal Care and Use Committee of Yale University. All CD45.1 and CD45.2 wild-type mice (C57Bl6/J) were purchased from the Jackson Laboratories. The generation of miR-150 knockout mice and Mx1-Cre Mybfl/fl mice was described previously (Lieu and Reddy, 2009; Xiao et al., 2007). β-actin-GFP mice were obtained from Jackson laboratory. Genotyping of the miR-150 knockout allele, floxed c-myb allele, or Mx1-cre allele followed previously described protocols (Lieu and Reddy, 2009; Xiao et al., 2007). To generate miR-150−/− β-actin-GFP mice and littermate controls, miR-150−/− mice maintained on a C57bl6/J background were crossed with heterozygous β-actin-GFP mice, also maintained on a C57bl6/J background. To generate CD45.1 miR-150−/− mice and littermate controls, CD45.2 miR-150−/− mice were crossed with CD45.1 wild-type mice. CD45.1/.2 Mx1-Cre Mybfl/+ mice were produced by crossing the CD45.1 wild-type line with CD45.2 Mx1-Cre+/−Mybfl/fl mice.
miRNA Profiling
Female 6wk-old C57Bl6/J mice were left untreated or given a single dose of 5-FU (150mg/kg). Four days post treatment whole bone marrow was isolated from each animal and lysed for total RNA using TRIzol (Invitrogen). Samples were profiled for miRNA expression using a bead-based profiling methodology (Lu et al., 2008). Profiling data were normalized assuming equal total miRNA expression across samples. Differential miRNA expression was analyzed by student's t test and then calculated for false discovery rate using the Benjamini and Hochberg method. The data is provided in Table S1. Global miRNA Expression Profiling of Bone Marrow Samples from 5-FU-Treated or Untreated Cohorts, Related to Figure S1 and has been submitted to GEO (GSE39211).
Constructing a miRNA Expression Library
Design of the retroviral expression library was carried out based on sequence information present in miRBASE v9.0 (Kozomara and Griffiths-Jones, 2011). Desired miRNA hairpins for the library were identified in the human genome, and their ∼200bp flanking sequences were scanned for the presence of other miRNAs or polyA signals. Specific primers (see Table S2. Primer Sets for Cloning and Barcode Detection Probe Sequences, Related to Figures 1 and S1) were designed so that they can uniquely amplify the corresponding miRNA, but avoid amplification of full hairpin sequences of neighboring miRNAs. The number of potential polyA core sequence (AAUAAA) was minimized whenever possible. PCR-primers contained recombination recognition sites for Gateway cloning (Invitrogen). The forward primers contain the following at the 5′ end: GGGGACAAGTTTGTACAAAAAAGCAGGCT, whereas reverse primers contain the following at 5′ end: GGGGACCACTTTGTACAAGAAAGCTGGGT.
The retroviral miRNA expression library was cloned through a two-step approach into the pMIRWAY-GFP vector (Guo et al., 2010; Lu et al., 2008), which was a modified pMSCV vector containing a separate constitutive PGK promoter-driven GFP. PCR-amplicons from genomic DNA were first recombined into pDONR221 entry vector (Invitrogen). Each entry construct was then recombined into the pMIRWAY-GFP destination vector with the miRNA expression cassette under the control of viral LTR promoter. Each construct was sequence verified and maintained as a glycerol stock.
Other Plasmids
The control GFP-containing retroviral vector, pMIRWAY-GFP-control, was described previously (Guo et al., 2010).
Retrovirus Production
Retrovirus was produced following published protocol (Guo et al., 2010; Lu et al., 2008). Briefly, 293T cells were cotransfected with the miRNA-library, or individual miRNAs in the pMIRWAY-GFP construct, along with the viral packaging vectors. The miRNA-library DNA was prepared by inoculating individual miRNA expression constructs separately in 96-well deep-well plates, and then pooling the bacteria mixture for plasmid preparation. 24 hr post transfection, the supernatant was replenished and multiple virus collections were taken over a 48 hr period. Viral supernatant was filtered and used to infect either primary bone marrow cells or HL60 cells in the presence of appropriate cytokines and 8 μg/ml polybrene (American Bioanalytical).
Murine Bone Marrow Transplantation
For the in vivo screen, and single miRNA overexpression studies, murine bone marrow transplantation was performed similar to published procedures, with modifications (Guo et al., 2010). Briefly, donor C57Bl6/J mice were primed with 150 mg/kg 5-FU for 4 days. Bone marrow HSPCs were enriched by Ficoll (GE Healthcare) density gradient centrifugation, and suspended in X-VIVO 15 medium (LONZA) supplemented with 10% BSA, 1% Pen/Strep, 100μM β-ME, 100ng/ml mSCF, 50ng/ml mTPO, 50ng/ml mFlt3L, and 20ng/ml mIL-3. Cells were transduced by spin-infection onto retronectin-coated plates (Takara). Following overnight infection, cells were washed in PBS and injected into the tail veins of lethally irradiated (9.0 Gy) recipient mice. For overexpression of miR-150 and vector controls, 7-8x106 cells were injected into each recipient. For the screen, 4-7x106 cells were injected into each recipient mouse. Three independent cohorts of mice were generated where each cohort of 5 recipient mice were generated from independent transductions.
Normalization of hematopoietic parameters occurred approximately 6-7 weeks post transplantation as determined by complete blood count (CBC). During this period, percent GFP+ cells in Mac-1, B220, CD3, and CD41 peripheral blood compartments were also assessed, as to determine engraftment efficacy as well as baseline GFP ratios prior to 5-FU challenge.
Competitive transplant assays were performed for miR-150 and c-myb knockout studies. In this paradigm, whole bone marrow was collected from donor animals by crushing and washing in PBS + 2% FBS. These cells were mixed at a 1:1 mononuclear cell ratio with GFP− wild-type competitor cells, prepared in a similar manor, and approximately 5x106 cells were injected into 9.0 Gy irradiated recipient mice. Recipients were monitored for engraftment three weeks after transplantation by assessing 45.1/.2 or GFP+/− peripheral blood ratios.
For c-myb studies, marrow was collected from CD45.1+/.2+Mybfl/+Mx1Cre+ or Mybfl/+Mx1Cre− donor animals, mixed with CD45.2 wild-type competitors, and injected into irradiated recipient mice. Recipients were allowed to recover for 5 weeks posttransplantation before excision of c-myb floxed allele was initiated. Recipient mice were injected with 25mg/kg pIpC (P-0913; Sigma) by i.p. injection every other day for 1 week, at which time peripheral blood was taken for assessing the presence of the deleted c-myb allele. After pIpC treatment, mice recovered for 5 weeks (35 days) prior to implementation of the 5-FU-induced injury model.
In Vivo miRNA Screen Sample Preparation
Starting from 3 weeks post transplantation, mice for the screen were bled for barcode analysis every two weeks. Specifically, 30μl of peripheral blood was lysed in FACS Lysis buffer, quenched and washed twice in PBS+2% FBS, and lysed in Tail Digestion Buffer (Viagen) supplemented with Proteinase K at 50°C overnight. Samples were then treated with RNase (Invitrogen) for 20min at 50°C, and heat inactivated at 85°C for 30min. Genomic DNA was purified by one round of phenol extraction, followed by one round of extraction with 25:24:1 phenol:chloroform:isoamyl alcohol, and two rounds of purification with a chloroform:isoamyl alcohol mixture. Genomic DNA and carrier glycogen were then precipitated by isopropanol in the presence of 300mM sodium acetate (pH 5.2). Following centrifugation, the precipitated genomic DNA samples were washed in 70% EtOH, air-dried, and re-suspended in 30μl DNA suspension buffer (5mM Tris-Cl, pH8.0 and 0.5mM EDTA).
Genomic DNA isolates were prepared for Barcode-Detection by first performing PCR for amplification of the miRNA-Cassette. Briefly, 250ng of gDNA was added to a standard PCR mix containing Taq in ThermoPol buffer (NEB). Primers used were /5Biosg/C∗C∗TCCCTTTATCCA GCCCTCA and CTAAAGCGCATGCTCCAGAC. The PCR reaction was performed with the following program [95°C – 5min, (95°C – 45 s, 58.2°C – 45 s, 72°C – 1.45 min) x30 cycles, 72°C – 10 min, 4°C – hold)]. For each batch of PCR, multiple water controls were performed, by using water as empty template, to allow for estimation of detection background (see below). PCR products were then purified using a QIAquick® PCR purification kit (QIAGEN) to remove salt, free dNTP and primers.
MicroRNA Barcode Detection
miRNA barcodes were detected using a Luminex-bead-based detection system. To create Luminex beads specific for miRNA barcodes, 5′ amino-labeled bar-code detection oligos (IDT) were coupled to carboxylated MagPlex microspheres (Luminex), with each oligo coupled to beads with a unique color combination (referred to as region by the manufacturer). Refer to Table S2. Primer Sets for Cloning and Barcode Detection Probe Sequences, Related to Figures 1 and S1 for barcode-sequences. Coupling reactions followed standard protocols from the manufacturer. After coupling, beads of different colors were mixed together, washed with Tris-EDTA buffer (10mM Tris, 1mM EDTA),and stored at 4°C before being used in subsequent assays.
Hybridization to the MagPlex miRNA beadset was performed similar to previous studies (Lu et al., 2008). Briefly, beads were re-suspended in 1.5X TMAC buffer. A known amount of post hybridization control oligos (/5Biosg/CAACGGAATTCCTCACTAAACCCTGGACAGGCG AGGAATACAGTTTAACCGCGAATTCCAGTA, /5Biosg/CAACGGAATTCCTCACTAAATGA CTCTCAGCGAGCCTCAATGCTTTA ACCGCGAATTCCAGTA, /5Biosg/CAACGGAATTCCT CACTAAACTGCGGGAGCCGATTTCATCTTTAACCGCGAATTCCAGTA) were spiked into the biotin-labeled barcode samples. Samples were then hybridized by denaturing at 95°C for 10 min and incubating at 53°C overnight. Following hybridization, beads were washed in 1X TMAC, incubated with 10 μg/ml SA-PE (Invitrogen) at 50°C for 10min, washed for four times with 1X TMAC to remove free SA-PE, and re-suspended in a 45ul final volume of 1X TMAC. Raw barcode signals from each miRNA construct were acquired on a Luminex-FlexMap3D instrument. The acquired median fluorescence intensity values were then used in subsequent data analysis. Examples of raw data are provided in Table S5. Examples of Raw Data from Bead-Based Detection Platform for the Screen, Related to Figures 1, 2, and 3.
Barcode Signal Analysis
Barcode signal analyses were performed using custom MATLAB programs, through multiple steps as detailed below.
Data Preprocessing and Normalization
First, samples with extremely low signals, representing unsuccessful PCR amplification, were eliminated from further analyses. Particularly, samples of the 22 day post 5FU time point from the 2nd mouse cohort were poorly detected and thus not used. Second, cutoffs were established to call a particular signal as detected or not detected. This detection cutoff step was performed for barcode samples from each cohort of mice separately because samples for each cohort were detected separately. To this end, we compared the overall signal histograms (as shown in Figures S3A and S3B) between samples from the in vivo screen and the corresponding water controls, detected with the barcode bead set. In addition, signals from a set of unrelated detection probes were also used to determine the cutoffs (see Figure S3C as an example). By doing this, we set the detection cutoff to be 6.3, 7 and 7 (on log2 scale) for each of the three cohorts, respectively.
We then performed data normalization across different time points for each mouse from each cohort separately. This is due to our finding that different mice harbor different sets of barcodes, precluding us to normalize between animals. We used the invariant probe method of normalization. The basis of this method is based on the assumption that some miRNAs within the library do not functionally alter the biology under examination, and therefore their barcode signal intensity should be similar at the different time points of analysis (thus are "invariant" miRNAs or barcode probes). To identify invariant probes, we ranked all barcode probes within any sample according to their intensities, and calculated the standard deviation (std) of the ranks of probes across multiple time points from the same animal. Invariant probes were defined as having a low std of ranks for samples that were harvested on time points of interest (week 11 post transplantation, 10 days post 5-FU and 22 days post 5-FU), as well as having signals well above detection background and within linear range of detection on the Luminex platform. The latter criterion was defined as having median signal (across the above time points) between detection cutoff+2 and 12 on log2 scale. We used 4 different conditions to define probes with low variation ranks. The first three conditions used cutoffs of 3, 4 or 5 on the std of ranks, each generating 8 to 22 invariant probes across animals. In addition, we applied a forth condition to use the 5 probes with the lowest std of ranks. All 4 conditions of invariant probe definitions generated largely similar results in follow up analyses (see Table S3. Consistency Scores Are Largely Insensitive to the Variation of Analysis Parameters for Most Barcodes, Related to Figures 3 and S3). After obtaining invariant probes, we linearly normalized all detected signals so that the sums of log2 signals of invariant probes of different samples were the same across all time points, whereas non-detected signals were set as 32 (background level). If more than one PCR replicate existed, the data were independently normalized and then averaged.
Quantification of the Number of Detectable Barcodes
Raw barcode detection data were pre-processed (as described above) and the number of barcode probes that surpass the detection cutoff was used to calculate the number of detectable barcodes. If more than one PCR replicates existed, the data were averaged between replicates.
Data Clustering
Data clustering was performed using the hierarchical clustering method, with correlation similarity and average linkage on the normalized data.
Consistency Score Calculation
We used consistency scores to describe the consistent trend of barcode abundance changes across animals. Comparisons were made between the day 10 post 5-FU time-point and the 11 week post transplantation time-point, as well as between the day 22 post 5-FU time-point and the 11 week post transplantation time-point, using the normalized data. To calculate the consistency score, the log2 of fold changes were first calculated between the corresponding time points for the same recipient mice. Then a threshold (called change threshold) was applied to define increases, decreases, or no changes. An increase in any mouse will be scored as +1, a decrease as −1 and no change as 0. The sum of all these scores across all recipient mice under examination was defined as the consistency score for a given barcode probe. We have applied 5 different change thresholds: 0.3, 0.4, 0.5, 0.6 and 0.7 (applied on data of log2 scale). For example, for a threshold of 0.7, the log2 of fold change of less than −0.7 will be scored as −1, that of over 0.7 scored as +1 and those between −0.7 and +0.7 scored as 0. If a probe was not detected in both time points of comparison in a particular recipient mouse, that mouse was labeled as not detected for that probe, and was not considered for calculating the consistency score for that probe. All 5 cutoffs produced largely similar results (see Table S3. Consistency Scores Are Largely Insensitive to the Variation of Analysis Parameters for Most Barcodes, Related to Figures 3 and S3).
Peripheral Blood Assays
After transplantation and/or 5-FU treatment (IP injection, 150mg/kg), mice underwent several standard peripheral blood assessments. CBC analysis was performed on 10μl of peripheral blood collected in an EDTA coated microtainer tube using the Hemavet 950FS system (Drew Scientific).
For determining the GFP ratios of different peripheral blood lineages, 30μl of peripheral blood was lysed in 300μl of FACS Lysis Buffer, quenched with PBS + 2% FBS, and stained with 100ul of the following antibody mixture (Mac-1-PE, B220-APC, and CD3-PE-Cy5 in PBS +2% FBS). For those experiments using the 45.1 congenic system, CD45.1-FITC was added to the antibody mixture. Cells were then washed, and analyzed on a FACSCalibur.
To assess GFP ratios in platelets, 1.5μl of peripheral blood was stained in 40μl of antibody mixture containing CD41-PE in 0.38% sodium citrate. Cells were fixed in 800μl of 1% PFA, and analyzed on FACSCalibur. Total number of platelets could also be determined in this assay by spiking in 4ul of diluted APC conjugated beads (AlignFlow, Invitrogen) and normalizing number of CD41+ cells to total bead counts.
Colony Formation Assays
Bone marrow was collected day six post 5-FU-treatment from miR-150 and control-vector mice. After treatment with PharmLyse (BD Biosciences), marrow was sorted (FACS Aria) for GFP+ or GFP− populations and re-suspended in M3434 Methocult® media (Stem Cell Technologies) at 5x104 cells/ml. 1ml of this cell mixture was seeded into 35mm dishes placed in a humidified chamber, and incubated at 37°C for ten days, where resulting GEMM, G/M, and BFU-E colony number and types were determined. For vehicle-treated control mice the number of cells plated per dish was reduced to 2.5x104 cells. Additionally, colonies were picked, washed, cytospun at 500rpm, and stained with Wright's-Giemsa to confirm morphological assessment of colony-type. Pre-B assays were performed in a similar fashion using MethoCult® M3630 media (Stem Cell Technologies) and seeding sorted marrow at 5x104cells/dish.
For colony studies of the c-myb heterozygous knockout model, mice were injected with 25mg/kg pIpC (P-0913; Sigma) by i.p. injection every other day for 1 week, at which time peripheral blood was taken for assessing the presence of the deleted c-myb allele. After a 3 week recovery period, implementation of the 5-FU-induced injury model occurred, and bone marrow from Mybfl/+Mx1Cre mice were collected for myeloid colony assays as described above.
Spleen colony assays were performed by transducing whole bone marrow with miR-150 or control-vector, and after three days of ex vivo culture, marrow was sorted for GFP+ or GFP− populations and injected into 9GY irradiated mice (∼1.5x104cells per recipient). After 10 days, spleen weights from each mouse was assessed, fixed in Bouin's for 2hr, and number of yellow/off-white hematopoietic colonies were counted. Data was normalized to the initial number of cells injected into each recipient.
Quantitative RT-PCR
Bone marrow samples from day 6 post-5-FU treated wild-type mice or untreated cohorts were sorted for c-Kit+, c-Kit− Lin− Sca-1−, and c-Kit− Lin− Sca-1+ progenitor populations. Cells were then lysed in TRIzol (Invitrogen), and total RNA (80ng) was used as input for RT-PCR. cDNA was generated for all samples using the miScript system (QIAGEN), which allows for detection of both miRNA and mRNA within the same RT product. For miR-150 assays (QIAGEN), we used 2ng RNA equivalent as total input and data from U6 RNA were used for normalization of the qPCR results. For c-myb assays, a TaqMan assay (ABI) specific for mouse c-myb was used for qPCR, along with 18S for normalization.
Apoptosis Assays
Apoptosis assays were performed on both bone marrow samples collected from miR-150 and miR-control transplant recipient mice six days after treatment with 5-FU, as well as on in vitro cultured HSPCs (Lin− Kit+ Sca+), which were transduced with either miR-150 or miR-Control and then treated with 0nM or 250nM 5-FU for varying time-points. In all assays GFP+ cells marks the miRNA-containing cells, while GFP− cells marks the untransduced population. For bone marrow samples, cells were stained with 100ul of the following antibody mixture (Lineage cocktail-biotin + SA-PE, 7-AAD, and AnnexinV-APC in 1x Annexin Binding Buffer; lineage cocktail contains: CD11b, GR-1, Ter119, CD3e, CD4, CD8, and B220). AnnexinV staining follows manufacturer suggested protocol (BD PharMingen). For cells of HSPC culture, cells were similarly analyzed, but without lineage cocktail.
We thank Geoffrey Lyon (Yale Cell Sorter Core Facility) for assistance with FACS and analysis. We thank Stacey Baker and Crew Smith for technical assistance. This work was supported in part by the National Institutes of Health Grants 1R01CA149109 (to J.L.), 5K01DK082982 (to S.G.), and 5T32HL007262-34 (to B.D.A.), and the American Cancer Society Institutional Research Grant #58-012-51 (to J.L.).
Accession Numbers
The GEO accession number for the miRNA profiling data is GSE39211.
Supplemental Information
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Publication Information
Received: April 24, 2012
Revised: August 4, 2012
Accepted: September 12, 2012
Published online: October 18, 2012


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School districts, supermarkets, and even whole countries have banned genetically engineered foods, fearing that they are not safe. Discover the evidence that confirm that these dangerous foods should never have been approved, and find out how to protect yourself, your family and the next generation.
Three videos in one: includes an interview with Jeffrey M. Smith, footage of scientists, and a look at the miraculous improvement in student behavior that accompanied a change in diet at a Wisconsin school. Also included is a lecture by Smith on "The Health Dangers of Genetically Engineered Foods and Their Cover-up."
International bestselling author of Seeds of Deception and Genetic Roulette, Jeffrey M. Smith is a widely popular spokesperson on the documented health risks of genetically modified organisms (GMOs) and how biotech companies rig research, gag critics, hijack regulators, and spin fantastic unfulfilled promises. His Institute for Responsible Technology's Campaign for Healthier Eating in America is designed to achieve the tipping point of consumer rejection of GMOs to force them out of the food supply. The Campaign encourages public and private showings of the Monsanto film and offers support material at www.ResponsibleTechnology.org. He lives in Fairfield, Iowa.
Visit Jeffrey's ...
View Jeffrey M.'s full profile page >
Sign up for our e-newsletter today and get 25% off your next purchase in our bookstore. Please note that discount codes do not combine with other offers or books already on sale.


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Singer Patti Page dies at age 85
... (WASHINGTON POST) Patti Page, the Singing Rage who became one of the most successful female singers ... but irresistibly likable (How Much Is) That Doggie in the Window, died Jan. 1 in Encinitas, Calif. She was 85 ... . With a husky, often alluring voice, Ms. Page was a superstar of the post-World War II era. It was a time
Gore: Dust Bowl coming unless we act
... Valid until Mar 01 See More Circulars JobStart Top Jobs Enter Keyword(s): Select a Category ... on the world's food supply. "Think about that," he said. "The Dust Bowl ... strategies, Gore mentioned the Vatican's increasing use of solar technology. Vatican City, Gore
Bono is a hypocrite
... control of the Internet is what it takes to prevent piracy of his music, U2's front man is happy ... Technology fuels popular entertainment. It has visited on the music industry a sea change in sales ... music has supplanted the now antiquated "go to the music store and buy it" consumer model
Limbaugh jokes: I, 'the pope,' am not quitting
... [caption id="attachment_366987" align="alignright" width="300" caption="On Feb. 7, 2013, Time ... magazine likened radio giant Rush Limbaugh to a pope, saying the conservative icon was "like a pope ... announcement by 85-year-old Pope Benedict XVI that he was stepping down from his position for health
The king of … the Internet? … is dead
... The headline "Jackson dies, almost takes Internet with him" pretty much summed it up. Without ... in just over an hour, receiving 20 million page views in the hour the story of Jackson's death broke ... , with nearly 2.3 million page views in one hour, more traffic than during any single hour last Nov
Public pays respect to Ronald Reagan
... Nancy Reagan approached the casket, accompanied by daughter Patti Davis and son Ron Reagan. She ... will include a four-plane flyover, a 21-gun salute and music from military bands and the U.S. Army chorus ... anything to anyone." Previous stories: Reagan remembered from coast to coast Reagan dies after
Ray Nagin indicted
... http://www.nola.com/crime/index.ssf/2013/01/ray_nagin_becomes_first_new_or.html (Times Picayune
Family name dogs George P. Bush
... 130 as a toll road despite an October 2013 decision by the rating agency Moody's Investors ... . By October 2013, Moody's had lost confidence SH 130 was capable of drawing the traffic required ... Herald Tribune had featured a front-page story headlined "Hitler's Angel Has $3 Million in U.S
Beltway gunrunners
... [poll id="85"]
10 things you didn't know about Ahmadinejad
... Ahmadinejad"s presidency (2005-2013). According to the latest statistics released by the United Nations ... Conference on Trade and Development (UNCTAD; 2013 World Investment Report), Iran managed to attract $2 ... , $3.64 billion in 2010, $4.15 billion in 2011, and $4.87 billion in 2012 (see table on page 214


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Grief and Bereavement (cont.) Medical Author:
Lynn Barkley Burnett, EdD, MS, LLB
Medical Editor:
Melissa Conrad Stöppler, MD, Chief Medical Editor
Melissa Conrad Stöppler, MD, Chief Medical EditorMelissa Conrad Stöppler, MD, is a U.S. board-certified Anatomic Pathologist with subspecialty training in the fields of Experimental and Molecular Pathology. Dr. Stöppler's educational background includes a BA with Highest Distinction from the University of Virginia and an MD from the University of North Carolina. She completed residency training in Anatomic Pathology at Georgetown University followed by subspecialty fellowship training in molecular diagnostics and experimental pathology. IN THIS ARTICLE
DecidingIt is absolutely essential to discuss the medical steps that should and should not be taken as the illness progresses. In the U.S., the most common approach to shared decision-making is discussions between the doctor and the person who is ill -- and, under most circumstances, his or her family. The patient, exercising autonomy, makes the choices about medical care, based on the options that are medically appropriate. Patients from some cultures, however, prefer "family-centered" decision-making, rather than " patient-centered." In the family-centered style of decision-making, medical choices are primarily the province of the family, whose choices "serve the good of the whole family, which includes but is not limited to the patient." A key factor in end-of-life decision-making is the goal of treatment. The goal of treatment is either curative, supportive, or comfort care. In a terminal illness, cure is obviously not possible. Early in the illness, it may be appropriate to actively support vital functions to "weather a passing storm." Such support may include use of antibiotics for infection, temporary use of breathing machines, and other interventions. In the last stages of illness, such steps only prolong dying, and it may be more appropriate to forgo them and to institute aggressive care only to provide comfort and to relieve pain and suffering. These decisions, which most often should be mutually agreed upon by the ill person and the doctor, represent the values and desires of the ill person in light of the medical facts.
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Lead Electrical Engineer |
STR Global
Ref: MP484-103
The RoleThe RoleSTR Global in partnership with a multinational EPC company is recruiting for a Lead Electrical Engineer to be based in Qatar. Competencies/Skills: � Should be capable of handling multiple tasks and functions well under pressure � Should be familiar with latest international standards � Experience in dealing with clients and preparing tenders � Should have knowledge in Cost Control and Project Planning � Very good planning, leadership and management skills |
RequirementsRequirementsWe are looking for candidates who have: � Bachelor�s Degree in Electrical Engineering � Ten (10) years of experience working in Oil and Gas Industry Communication and Reporting skills are mandatory Salary: QAR 20 -25,000/- (All inclusive) |
About the CompanyAbout the CompanySTR Global is a niche resourcing solutions business focused on the energy sector. STR Global have a network of 7 offices worldwide - London, Dubai, Doha, Rio De Janeiro, Hong Kong, Perth and Houston. From these locations STR Global is capable of delivering recruitment solutions for any size and location of project. Our professional recruiters have an understanding of the global energy market and the issues that are relevant to individual locations gained through their hands on experience working directly for clients and contractors in the sector. We are members of the Recruitment and Employment Confederation (REC) and follow a strict code of ethics combined with an in-depth awareness and understanding of employment and recruitment law and health, safety and environmental matters. |
This Position is closed or expired


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a place for zinesters - writers and readers
April 16, 2009 at 6pm to April 30, 2009 at 7pm – Usa The IPRC (Independent Publishing Resource Center) is requesting submissions for the Zine-of-the-Month. Getting a zine in the mail each month is a benefit of membership at the $100+ level. If your zi… Organized by Marilyn Zornado | Type: zine, submissions, requested
267 members
68 members
21 members
384 members
181 members
Ist preference given to distros and zines. Rates and details are here. Limited space. Very Low Cost!
© 2014 Created by Krissy PonyBoy Press. Powered by


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Definition of acellular in English:
Line breaks: acel¦lu|larPronunciation: /eɪˈsɛljʊlə
Biology- 1Not consisting of, divided into, or containing cells.More example sentences
- Briefly, a cell volume distribution was obtained and gates were set to select for counting only intact cells excluding acellular debris.
- This isolate contains an internal acellular compartment containing filaments linking the cells.
- Besides being segmented, the body wall of annelids is characterized by being made up of both circular and longitudinal muscle fibers surrounded by a moist, acellular cuticle that is secreted by an epidermal epithelium.


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Pricing and Economic Regulation of Medicines |
One of the principle determinants of access to essential medicines is price. Countries in the Region of the Americas face important challenges in medicines pricing including: lack of information and transparency in pricing; inappropriate price referencing in public procurement; weak regulatory frameworks (procurement, financing, intellectual property) that impact medicine price; fragmented strategies promoting generic medicine penetration in markets; and differential pricing policies that are not necessarily equity based. The issue of price is especially important when we consider that in some countries up to 50% of household expenditure in health is spent on medicines.
PAHO is approaching the issue of medicines price from the Economic Regulatory perspective, recognizing that a medicine price may be directly impacted by policy and regulatory frameworks within countries, developed and implemented by national governments.
Components of the Technical Cooperation Program
Assessment of regulatory and policy frameworks impacting price: (download presentation)Assessment of prices (particularly HIV/AIDS, TB, and Malaria):
-Work of the GAN, Price Negotiations Outcomes
-PAHO Publications:
- "A multidisciplinary approach to scaling up HIV/AIDS treatment and care: the experience of the Bahamas," (Fitzgerald et al., 2005)
- "An Open Competition Model for Regional Price Negotiations Yields Lowest ARV Prices in the Americas" (Fitzgerald J, Gomez B, 2003)
Development and implementation of Generic Medicines Policy (download article on the situation in the Americas)
Development and implementation of strategies to promote and rationalize Access to High Cost Medicines (download document)
Working Group to promote the establishment of Pricing Databases for transparency and referencing in public procurement (download terms of reference)
Listserve ECONMED
Implementation of medicines pricing studies using the WHO/HAI methodology
-Medicine Prices, Availability, Affordability and Price Components in Peru (2007)
-Precio, Disponibilidad y Asequibilidad de Medicamentos y Componentes del Precio en Colombia (2009)
-Estudio de Precios, Disponibilidad y Acceso de Medicamentos y Componentes del Precio en Bolivia (2009)
-Precio, Disponibilidad y Asequibilidad de Medicamentos y Componentes del Precio en Ecuador (2008)
Regional Office for the Americas of the World Health Organization |


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Mounting brackets for Gas Lift Springs. The perfect solution for attractively and efficiently opening hinged panels. Sold in pairs.
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worked well for the Boat Rod Locker, to prevent us breaking any more fishing rods.
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In the second installment of the acclaimed MANDIE series, Mandie (Lexi Johnson, Sugar Creek Gang, Mandie and the Secret Tunnel) is forbidden by her Uncle John (Terrell Anthony, Guiding Light, Mandie and the Secret Tunnel) to join him on a dangerous quest to keep an old promise. Mandie and her long-lost mother take matters into their own hands. Joined by friends, they embark on a harrowing journey by train to protect Mandie's stubborn uncle. Grand adventure ensues as they race to outwit evil conspirators and dare to face the secrets of Lost Antler Cave. Mandie's most difficult challenge arises when she realizes that success will land her face to face with the one person she never wanted to meet. Starring Lexi Johnson and Hayley Mills. Dove approved!
Please add a name and description in order to save your playlist to your user profile.


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Have a question about Men's Rugby at SBU? Contact us.
Clarence Picard Head Coach (716) 375-2253 email@example.com
Over the past several years, experienced players from high school teams across New York, the Northeast, Midwest, and Mid-Atlantic regions have helped St. Bonaventure rugby become a premier Division 1AA team. Our current roster includes players with extensive regional, national and international experiences.
While more and more players are entering the collegiate ranks with rugby experience, several top players throughout the team's history have stepped on campus without any previous experience. Football, basketball, hockey and soccer players, wrestlers and track athletes can all find skills that easily transition to the rugby pitch. Our full-time, dedicated coaching staff is committed to ensuring your transition to rugby is as smooth as possible. With the proper amount of dedication and hard work, you can quickly develop into a qualified player.
SBU Rugby Recruitment Form Rugby Scholarships Support SBU Rugby


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Sophia Grace & Rosie Lands at Claire's
Claire's assortment spans 10 styles of apparel, accessories and beauty products ranging in price from $6 for a hair bow to $16 for a sequined tutu.
— Mar 6, 2014
Natalie Bader Named President of Clarins Brand
The executive, who started in the role on Monday, had most recently served as chief executive officer of Prada France.
— Mar 6, 2014
Sally Hansen Signs Madeline Poole as Global Ambassador
As part of the collaboration, she will create looks for the nail sponsorship with Stella McCartney.
— Mar 2, 2014
Masahiko Uotani Prepares to Take Helm at Shiseido
The incoming president and chief executive officer plans to accelerate the company's international growth and strengthen its portfolio of brands.
— Feb 28, 2014
Alessio Rossi Discusses Lancôme's Digital Strategy
The beauty firm's vice president of interactive marketing and e-business challenged attendees to modernize their view of the digital universe.
— Feb 28, 2014
Mally Roncal's Social Media Truths
Taking to the stage with her trademark pep and personality, the makeup artist and founder of Mally Beauty illustrated the parallels between makeup and digital.
— Feb 28, 2014
A Cocktail for Marc Jacobs Beauty's Europe Launch
The line is to come out on Thursday in Sephora in France and the Marc by Marc shop in Paris.
— Feb 28, 2014
Charlotte Rampling Named New Face of Nars
The beauty brand is gearing up for its 20th anniversary in September.
— Feb 26, 2014
Revlon Reshuffles Executives
Chris Elshaw, Revlon's executive vice president and chief operating officer, has decided to leave the company, effectively immediately.
— Feb 25, 2014
Rodolphe d'Ornano Dies at 93
The former L'Oréal executive was credited with helping establish Lancôme in the United States.
— Feb 23, 2014
Briogeo Enters Natural Hair Care Space
Nancy Twine left a high-paying job at Goldman Sachs in 2012 to launch Briogeo, a range of natural hair-care products.
— Feb 21, 2014
Ryan Reynolds Named L'Oréal Paris Face
The actor will front advertising for the Men Expert brand.
— Feb 19, 2014
Horst Rechelbacher Dies at 72
The sustainability-minded founder of Aveda and Intelligent Nutrients died Saturday at his home after battling pancreatic cancer.
— Feb 16, 2014
Soap & Glory Names Vincenzo Minore as CEO
Executive joins London-based brand from Aveda.
— Feb 14, 2014
Milani Taps Five New Executives
The appointments come as demand for Milani's multicultural positioning is hitting on all cylinders.
— Feb 13, 2014


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Bupa Great Run
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28 September 2014
Sheffield City Centre
Enter Now
Open for Entry
28 September 2014
Sheffield City Centre
Weekend Activities
Throughout the weekend runners and spectators can join in the family fun at the Bupa Great Yorkshire Run.
Thousands of runners will be entertained along the 10k course by the Sheffield Bands on the Run. Local bands and artists playing anything from rock to pop to funky jazz will keep you motivated every step of the way.
The entertainment doesn't stop once you cross the finish line, as the live music and dance continues in the Sheffield Peace Gardens until 16:00. We'll post up the confirmed timetable of bands and dance acts at greatrun.org/yorkshire the week of the event.
A host of charity and sponsor stalls will be in the area, along with the Capital FM Street Stars giving you the red carpet treatment with fun, games and instant wins!
This year the Italian Market will be on Fargate in the city centre across the entire Bupa Great Yorkshire Run weekend. Just a stone's throw away from the Peace Gardens, it's perfect for market lovers looking for a range of products, gifts and food.


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Please Don't Pass The Nuts™
Just because you have a restricted diet, doesn't mean you have a restricted life.
Allergen-friendly "chipwich" cookies
Tuesday, August 05, 2008
BBQ at Blue Smoke
Yum oh yum,
Worry-Free Dinner last night at Blue Smoke
I'm still stuffed.
Have a look at the pix on the
WFD site
and enjoy!
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Published on | Description | |
MondayOctober 10, 2005 |
Novelist Richard Ford to read for Annual Writers HarvestPulitzer Prize-winning novelist Richard Ford reads from his work at 8 p.m., Thursday, Oct. 13, in Chase Hall Lounge, Campus Avenue, Bates College. |
TuesdayNovember 9, 2004 |
Poet Mathis featured in Writers Harvest readingThe annual Writers Harvest reading at Bates College will be given by Cleopatra Mathis, professor of English at Dartmouth College and author of five collections of poetry, at 8 p.m. Wednesday, Nov. 10, in Chase Hall Lounge, Campus Ave. |
TuesdayNovember 5, 2002 |
Richard Russo to read at Bates for annual Writer's HarvestRichard Russo, the Camden author who won the 2002 Pulitzer Prize in fiction for his novel "Empire Falls," reads from his work at 8 p.m. Thursday, Nov. 14, in Chase Hall Lounge, 56 Campus Avenue. The public is invited to attend the event, part of the Writers Harvest, the annual literary benefit to fight hunger and poverty sponsored by the national hunger organization Share Our Strength (SOS). Donations will be accepted and proceeds will benefit the Maine Coalition for Food Security and the Good Shepherd Food Bank. |
TuesdayOctober 20, 1998 |
Bates to host Seventh Annual Writer's HarvestPoet Sonia Sanchez, author of "Does Your House Have Lions?" (1997), will read from her work at Bates College during the Seventh Annual Writers Harvest Author Reading Oct. 29 at 8 p.m. in Chase Hall Lounge. The public is invited to attend and donations will be accepted. Proceeds will benefit the hungry as part of the Writer's Harvest program sponsored by Share Our Strength (SOS). |


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Halloween Costumes
Search Results
Rule with grace, even if youre evil, in this officially licensed Disney Alice in Wonderland Sassy Red Queen adult womens costume. The gold, black and red dress comes complete with petticoat, stand-up collar, gold crown and matching knee socks.
Average Rating
Cons Best Uses
Comments: I attended a costume party and receive lots of compliments, everyone like it and congratulate me for the originality and great quality of the costume. I was very glad that I bought it. |
Cons Best Uses
Comments: The best Red Queen costume in my opinion. Shortened it, added a white tutu and a bunch of rhinestones and I was good to go. Lots of compliments. |


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Christmas Fun – All In One Holiday Spirit HD !
By Kids Games Club by TabTale
Open iTunes to buy and download apps.
>Enjoy lovable Christmas songs and exciting educational Christmas games
>The largest collection of Christmas activities for kids of all ages
>Top#1 educational Christmas app in USA , UK and over 50 countries
>Over 3,500,000 happy users | Selected New & Noteworthy by Apple
>Adorable HD graphics and delightful soundtracks
This year Christmas Fun app is better than ever! With 14 fun filled Christmas songs, games and exciting activities. Sing along with classic songs such as Jingle Bells and Santa Claus is Coming to Town and have hours of laughs with wonderful games such as Simon Santa or Gingerbread Peekaboo. Christmas Fun is an all in one, special edition Christmas app which is fun for the whole family.
~3 Interactive Sing along Songs:~
>Jingle Bells
>We wish you a Merry Christmas
>Santa Claus is Coming to Town
Animated, Interactive Sing-Along story book for children - The classic children's Christmas song comes to life in an interactive and fun experience.
~Santa and Snowman Dress Up Games~
Help Santa and Snowman dress up for Christmas in a fun and intuitive game. Select hats, faces, shirts, pants and more to combine the perfect Christmas outfit. Then take a picture. Get the outfit just right to win.
~Christmas Paint - Coloring pages~
Enjoy wonderful Christmas spirited coloring pages in a unique and fun coloring game. Select from the collection of Christmas pages, create your own drawing and save your drawing in the photo album.
~Gingerbread PEEKABOO Game~
Where is the gingerbread man hiding? Touch the moving objects on screen and find him. The joyful interaction of animations and sounds create a fun educational activity for babies, toddlers and children.
~Christmas Adventure~
Join Little Paul and Emma on their wonderful Christmas Adventure in this beautiful interactive story. Enjoy this magical animated treat with interactions in every feature and a lovely christmas story.
~Christmas Train~
All aboard the Christmas Train! See all the the beautiful train's carts with toys. Learn spelling and vocabulary as you interact with the train and toys.
~Simon Game~
Come play with the most beautiful Simon Game in the app store! Listen to the holiday tunes and then repeat with three fun festive modes!
~Mix and Match~
Santa and the Snowman have gotten all mixed up! Can you help put them back together again? Create hilarious combinations with Christmas Fun Mix and Match game.
~Memory Match~
Can you find all the Christmas toy's identical pairs? This memory game is a great way to develop memory skills and have fun all at the same time.
~Puzzle Fun~
High quality, super fun and simple to master puzzle game, with 16 unique Christmas puzzles filled with holiday spirit.
~Maze Challenge~
Help the gingerbread man find his way to the gingerbread house! In 20 colorful delicious gingerbread mazes.
Note: First two pages of each song and the story are completely free with full features, Santa dress up game the Christmas Train are completely free and so are the first few stages of each game. Many more games, pages, activities and stages are available with an easy to use in-app purchase inside the app.
Sound doesn't work?
Increase the volume and check that your device is not set to "Mute". The Mute button is the switch next to the volume buttons on the side of the device.
Still no sound?
On the iPad it's possible that your side switch is set to lock screen rotation. In this case you can control sound volume by:
Double tap the home button
Swipe from left to right
Tap the speaker icon to the far left
It should show "Mute Off" below the play button
You can also set the side switch functionality by going to "Settings > General > Use side switch to" and set it to to control mute.
By downloading this application you agree to the Terms of Use of TabTale Ltd. which are available to your review on http://tabtale.com/terms-of-use.
What's New in Version 4.0
> Optimized for iOS 7
> Performance & Stability Improvements
> Bug Fixes
> Like to stay updated? facebook.com\tabtale
Customers Also Bought
- Free
- Category: Education
- Updated: Dec 30, 2013
- Version: 4.0
- Size: 35.5 MB
- Language: English
- Seller: Kids Games Club by TabTale
- © Kids Games Club 2014
Compatibility: Requires iOS 5.0 or later. Compatible with iPhone, iPad, and iPod touch. This app is optimized for iPhone 5.
Top In-App Purchases
- Full Version$3.99
- Full Peekaboo Game$1.99
- Full Jingle Bells Song$1.99
- Full Puzzle Game$1.99
- Full Snowman Dress Up Game$1.99
- Full Merry Christmas Song$0.99
- Coloring Pages pack$1.99
- Full Santa Claus is Coming to Town Song$1.99
- Full Christmas Adventure Story$1.99
- Full Simon Game$1.99


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The Deal
$34.99 for one pair of Bucco braided wedges ($89.99 list price). Choose from the following options:
- Alamea style; available in black, blue, and natural
- Ariel style, available in coral, mint green, and tan
- All options are available in half sizes 5.5–9 and 10
Click here to view an image of each option.
Bucco Braided Wedge Sandals
A versatile summer shoe, wedge sandals traditionally pair well with jeans, capris, and shorts, as well as maxi skirts and sundresses. The details make Bucco's Alamea and Ariel styles eye-catching as well as practical. The braided straps have a breezy, laid-back sense, and the two-tone wedge heel adds a pop of color to outfits. Though the heels stand a towering 4.5 inches—the height needed to sneak over the fence into Six Flags—wedges are more stable and comfortable than traditional high heels.
Alamea Braided Wedges
- Braided straps
- 4.5-inch heel
- Back zipper
- Polyurethane upper
- Rubber outsole
Ariel Braided Wedges
- Woven straps
- Cork-look wedge platform
- 4.5-inch heel
- Lightly padded footbed
- Side buckle
- Polyurethane upper
- Rubber outsole
How to get your goods: by purchasing this Groupon and providing your name and shipping address, your order is complete!
For questions pertaining to this deal, please visit the deal Q&A on this page. For post-purchase inquiries, please contact Groupon customer service.
View the Groupon Goods FAQ for additional information, including how list price is determined.
In a Nutshell
Versatile summer sandals with braided straps and 4.5 in., two-tone wedge heels
The Fine Print
Limit 3 per person, may buy 2 more as gifts. Free returns. Free shipping. Does not ship to AK/HI/Canada/Puerto Rico. Most orders are delivered within 2 1/2 weeks from the purchase date. Does not ship to PO boxes. See return policy. Must pay applicable tax and provide name and shipping address at checkout, which will be shared to facilitate shipping. Goods sold by Groupon Goods. See the rules that apply to all deals.