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Copyright © 2008 Elsevier Inc. All rights reserved.
Cell Stem Cell, Volume 3, Issue 2, 169-181, 7 August 2008
Add/View Comments (0)Six2 Defines and Regulates a Multipotent Self-Renewing Nephron Progenitor Population throughout Mammalian Kidney Development
Akio Kobayashi1, M. Todd Valerius1, Joshua W. Mugford1, Thomas J. Carroll1, 3, Michelle Self2, Guillermo Oliver2 and Andrew P. McMahon1, ,
1 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
2 Department of Genetics and Tumor Cell Biology, St. Jude Children's Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
3 Present address: Departments of Internal Medicine (Nephrology) and Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
- Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.
The metanephric kidney of the mouse initiates development at 10.5 days postcoitum (10.5 dpc). Reciprocal interactions between the Wolffian duct-derived ureteric bud and the adjacent metanephric mesenchyme population drive the process of kidney development (Costantini, 2006,Dressler, 2006,Saxen, 1987,Schedl, 2007). The mesenchymal population supports branching growth of the ureteric bud. Conversely, Wnt9b from the ureteric bud is required for a subset of cells within the adjacent mesenchyme to epithelialize, establishing the renal vesicle, the precursor for the glomerular and renal tubule compartment of the main body of the nephron (Carroll et al., 2005). The ureteric epithelium generates the collecting duct network of the mature kidney while other cell populations, notably the renal interstitium (stroma), are likely generated from the mesenchymal pool. Reciprocal interactions over weeks or months, depending upon the mammalian species, generate the full complement of nephrons.
Recently, the homeodomain transcriptional regulator Six2 has emerged as a key factor within the kidney mesenchyme. Six2 is expressed in a subset of metanephric mesenchyme, where its expression is maintained throughout kidney development (Oliver et al., 1995); no expression is detected in adult mouse kidneys (Humphreys et al., 2008). In Six2 null mice, ectopic renal vesicles form on the dorsal (cortical) side of the ureteric bud at the onset of nephrogenesis, the progenitor pool is rapidly lost, and nephrogenesis terminates after induction of only a few nephrons (Self et al., 2006). Thus, Six2 is required to maintain a nephron progenitor population.
We have addressed the tubule-forming lineage of the nephron and the cellular processes that underlie nephrogenesis. Our data suggest that Six2-expressing (Six2+) cells represent a self-renewing, multipotent nephron progenitor population throughout kidney organogenesis. Six2 acts cell-autonomously within this population to maintain a progenitor state. In this, Six2 may act, at least in part, to block Wnt9b action, thereby permitting renewal of uncommitted nephron progenitors. Thus, Six2 ensures the development of a full complement of nephrons and, consequently, a functional organ system.
Six2+ Mesenchymal Cells Are Progenitors for the Main Body of the Nephron
The mesenchymal cell populations that surround the ureteric bud during nephrogenesis are a mosaic of molecularly distinct cell types. Among these, Six2+ cells lie in close proximity to the inductive ureteric epithelium. To determine the fate map of this population of the cap mesenchyme in vivo, we generated four Six2-Cre alleles in the mouse: a BAC transgenic allele with a Tet-off-eGFPCre (Six2-TGCtg/+) cassette; and knockin alleles with TGC (Six2TGC/+), CreERT2 (Six2CE/+), and eGFPCreERT2 (Six2GCE/+) cassettes introduced into the Six2 locus at the position of the Six2 initiation codon (see Figure S1 available online). The knockin alleles remove Six2 function; however, Six2 heterozygous mice are phenotypically normal (data not shown and [Self et al., 2006]). The TGC alleles were designed to enable doxycycline-regulated control of an eGFPCre transgene within the Six2 expression domain (Bond et al., 2000,Rodda and McMahon, 2006). However, doxycycline addition did not silence Cre activity in the TGC BAC transgenic allele but disabled Cre activity in most cells in the TGC knockin allele (data not shown). The CE and GCE alleles enable tamoxifen-dependent regulation of Cre activity.
To validate expression patterns of transgenes, we examined GFP expression of the TGC and GCE alleles. In all lines, GFP expression was restricted to the cap mesenchyme from the onset of metanephric development (Figure 1 and data not shown). As expected, GFP+ cells were also Six2+, and no GFP+ cells were Six2−, though some Six2+ cells did not show detectable GFP expression in Six2-TGCtg/+ and Six2TGC/+ kidneys (Figures 1G–1I). As this slight mosaicism is observed in the TGC alleles, but not in the GCE allele, this likely reflects a component of the Tet regulatory system. In conclusion, GFP expression of all alleles was restricted to most or all Six2+ cells of the cap mesenchyme from the onset of nephrogenesis (Figures 1 and 2F–2I and data not shown).
The fate of Six2+ cells was examined during kidney development. Six2-TGCtg/+ mice were intercrossed with mice carrying a R26R-lacZ reporter allele (R26RlacZ/+) (Soriano, 1999) to permanently label descendant cells from the Six2+ population. In addition to the kidney, we observed β-galactosidase (β-gal) activity from the R26R-lacZ allele in the developing head, ear, and limb, where Six2 is also expressed (Figure 2A) (Oliver et al., 1995).
During early stages of kidney development, at the onset of nephron induction, β-gal activity was observed in the cap mesenchyme surrounding the ureteric bud epithelium (Figures 2B–2E). By 15.5 dpc, the ureteric bud has developed numerous branches and nephrogenesis is well advanced. At this time, GFP, like endogenous Six2, was restricted to the cap mesenchyme and early pretubular aggregates; no GFP activity was observed within the renal vesicle or its later derivatives (Figures 2F–2I and data not shown). All cells in the cap mesenchyme were β-gal+, indicating that all cap mesenchyme cells are derived from GFPCre-expressing cells. β-gal was also detected in early developing nephron tubules undergoing nephrogenesis and patterning (Figures 2G–2I) and in fully formed nephrons from the most proximal (renal corpuscle, Figure 2J) to the most distal (junction with the collecting duct, yellow arrow in Figure 2M) structures.
We further determined which cell types were populated by the Six2+ descendant lineage. Proteins assayed included Wt1 (cap mesenchyme, podocytes), PECAM-1 and Flk1 (endothelial cells), PDGFRb (glomerular mesangium, pericytes), uromodulin (loop of Henle), SMA (smooth muscle) and cytokeratin (collecting duct) (Figures 2J–2O and data not shown). In the maturing renal corpuscle, both parietal (Bowman's capsule) and visceral (podocyte) cells were Wt1+ and β-gal+ (Figure 2J), confirming that the Six2+ cap mesenchyme is the source of these nephron components. In contrast, the glomerular capillary system marked by PECAM-1 and Flk1 (Figure 2K and data not shown) and the glomerular mesangium marked by PDGFRb (Figure 2L) are β-gal−, indicating that these cells originate outside of Six2+ population. Interestingly, at the collecting duct-nephron junction in the cortex, no double-positive, β-gal+ cytokeratin+ cells were detected (Figure 2M), suggesting that the connecting segment derives from the main body of the nephron and not the collecting duct and that the Six2+ cap mesenchyme does not contribute to the ureteric tip. In the medullary region, adjacent epithelial tubules were identified using uromodulin (loop of Henle of the nephron) and cytokeratin (collecting duct). We detected β-gal+ uromodulin+ cell types, but no β-gal+ cytokeratin+ cells (Figure 2N and data not shown), indicating that these cell lineages are distinct. All SMA+ cells were β-gal−, suggesting that the smooth muscle is not derived from the cap mesenchyme. Overall, β-gal activity was observed specifically within the epithelial body of the nephron.
Six2+ Cap Mesenchyme Cells Continuously Contribute to Nephron Tubule Formation throughout Kidney Development
To determine whether Six2+ cells similarly contribute to nephron formation at later stages in kidney development, we used the GCE allele for tamoxifen-dependent labeling of Six2+ cells. In this allele, fusion of GFP with Cre does not alter Cre activity (Le et al., 1999). Further, analysis of the recombinase activity of the eGFPCreERT2 protein confirmed drug-dependent regulation of this fusion protein; no background recombination was observed in the absence of drug administration (Figure S2 ).
Six2+ cells were labeled in Six2GCE/+ ; R26RlacZ/+ embryos by tamoxifen induction at 16.5 dpc, and the distribution of β-gal-labeled cells was determined in the kidney at 18.5 dpc (Figures 3A–3D). β-gal activity was observed in the cap mesenchyme and developing nephron tubules of embryos from dams injected with tamoxifen (Figures 3B and 3D). No β-gal activity was observed in oil-injected controls, demonstrating that Cre recombinase activity is absolutely dependent on drug administration (Figures 3A and 3C). Thus, Six2+ cells continuously contribute to nephron formation throughout kidney development.
Self-Maintenance of the Nephron Progenitor Population from an Early Pool of Six2+ Cap Mesenchyme Cells
To obtain a more quantitative understanding of nephron formation, we used FACS of GFP+ cells in kidneys from Six2GCE/+ embryos to determine the increase in Six2+ cells from early to late stages of nephrogenesis. On average, the Six2+ compartment of a single kidney undergoes a 15.6-fold increase from 11,751 ± 3,133 cells at 11.5 dpc (n = 12) to 183,102 ± 41,382 cells at 19.5 dpc (n = 8), a substantial increase, given the continued commitment of Six2+ cells to Six2− nephron structures.
The expansion of the Six2+ nephron progenitor population may reflect two distinct mechanisms. In the first, Six2+ cells may generate more Six2+ cells; that is self-maintenance of the progenitor pool. Alternatively, a Six2− progenitor population may exist outside of the Six2+ cap mesenchyme and continuously repopulate the Six2+ cap mesenchyme component. To distinguish between these possibilities, we labeled Six2+ cells at the onset of nephrogenesis with a transient pulse of tamoxifen activity and then examined the fate of labeled cells toward the end of kidney development at 19.5 dpc (Figures 4A and 4B). If the Six2+ cell population is a self-renewing population, β-gal+ cells should be retained in the cap mesenchyme in similar proportion to that observed immediately after transient labeling (Figure 4A). In contrast, if Six2− cells give rise to additional Six2+ cells after the onset of kidney development, these β-gal− cells would be expected to dilute out the initial β-gal+ cap mesenchyme population over time (Figure 4B).
The dynamics of Cre enzymatic activity are critical in drawing a conclusion. Six2 expression commences in the mouse metanephros around 10.5 dpc prior to ingrowth of the ureteric bud (Oliver et al., 1995,Self et al., 2006). When 2 mg of tamoxifen was injected at 9.5 dpc, we rarely detected β-gal-labeled cells in the kidney (Figures 4C, 4H, and 4M and data not shown), whereas injection at 10.5 dpc led to extensive labeling (Figure 4E). These results are consistent with tamoxifen induction of CreERT2 activity being confined to a window of less than 24 hr. Labeling from 10.5 to 11.5 dpc correlates with ingrowth and branching of the ureteric bud epithelium and the first induction of Six2+ progenitors to nephron precursors (Carroll et al., 2005). Consistent with a self-maintenance mechanism, we observed extensive contribution of β-gal+ cells within both the cortical cap mesenchyme and developing nephron structures at 19.5 dpc (Figures 4D, 4E, 4I, 4J, 4N, and 4O). In contrast, when we labeled cells within the nonepithelial pretubular aggregate by transient activation of a Wnt4GCE/+ allele (Figure S3 ) by tamoxifen induction at 12.5 dpc, β-gal+ cells were predominantly in mature nephron tubule structures outside of the cortical region at 19.5 dpc (Figures 4F, 4G, 4K, 4L, 4P, and 4Q). Thus, Wnt4+ cells derived from the Six2+ cap mesenchyme form a transient population that rapidly converts to nephron fates. In contrast, self-renewal by a Six2+ population plays a major role in maintaining the nephron progenitor pool.
A Six2+ Cell Can Give Rise to Multiple Cell Types of the Nephron Tubule
The nephron tubule contains many specialized cell types along its proximodistal (glomerular-collecting duct) axis (Reggiani et al., 2007,Wingert et al., 2007), all of which derive from the Six2+ progenitor pool. To address whether this pool contains cells with extensive nephron-forming capability or a population of cells with narrowly restricted nephron fates, we performed a clonal analysis of Six2+ cells. If a single Six2+ cell is multipotent, its clonal descendants would contribute to different cell types along the axis of the nephron tubule (Figure 5A). In contrast, if a Six2+ population is a mosaic of cells already committed to generating regionally restricted cell types, the labeled clonal descendants would only contribute to restricted domains of the nephron tubule (Figure 5B). We optimized administration of tamoxifen to identify a dosage (0.1 mg) that, when injected into dams carrying Six2GCE/+ ; R26RlacZ/+ embryos at 12.5 dpc, gave rare and dispersed clusters of labeled cells at 14.5 dpc (3.58 ± 1.31 clusters per kidney, n = 12), indicative of clonal events (data not shown). Oil-injected control kidneys showed no β-gal+ cell (Figures 5C, 5E, 5G, and 5L).
Next, we repeated this 0.1 mg tamoxifen injection at 12.5 dpc but now examined nephrons at 19.5 dpc (Figures 5C–5M). We analyzed serial sections from kidneys with three clonal clusters or less; 24 clusters gave similar results. In several of these, multiple β-gal+ GFP+ cells were observed within the cap mesenchyme, confirming our previous observation that Six2+ cells undergo self-renewal (Figures 5H, 5I, 5K, and 5M). All nephron tubules that contained β-gal+ cells were mosaic, with a majority of cells β-gal−, as expected if the pretubular aggregate is derived from multiple Six2+ cap mesenchyme cells (Figures 5H, 5J, 5K, and 5K′). Within nephron derivatives, β-gal+ cells were observed in different specialized domains along the developing nephron tubule (Figure 5J). Serial section analysis demonstrated that β-gal+ cells within a single mature nephron tubule contributed to podocytes, proximal and distal tubule structures (Figures 5K and 5K′). These observations were further confirmed by confocal microscopy revealing that β-gal+ cells contribute to Wt1+ (podocytes), LTL-lectin+ (proximal tubule), and uromodulin+ (loop of Henle) cells in a clone (data not shown). These findings suggest that descendants of a single Six2+ cell can differentiate into multiple cell types within the nephron. Thus, regional commitment of progenitor cells to segment-specific cell types is likely to occur after an epithelial organization is established in the renal vesicle or its later derivatives (see the Discussion ).
Six2 Function Is Cell-Autonomously Required for Maintenance of the Cap Mesenchyme
In the absence of Six2 function, ectopic tubules form on the dorsal (cortical) side of the ureteric tip at the onset of kidney development, the cap mesenchyme is lost, and nephrogenesis arrests (Self et al., 2006). To determine the fate of the Six2-expressing cap mesenchyme cells in Six2 null kidneys, we utilized the Cre knockin alleles to remove Six2 function and label Six2-decendants. In Six2TGC/GCE ; R26RlacZ/+ embryos, activation of the Six2-TGC allele was evident from the presence of labeled, β-gal+ cells at 13.5 dpc (Figures 6A and 6B). Thus, the fate of the initial Six2 population can be assessed on a Six2 null background. All β-gal+ cells were restricted to laminin+ cytokeratin− ectopic tubules in Six2 null kidneys (Figures 6C–6J). Some cells within the tubules are not β-gal+. There are likely two possible reasons. First, as noted earlier, the Tet cassette insertion led in some unknown manner to mosaic activity of Cre; hence, we would predict that all cells would not be labeled. Second, Six2 is rapidly downregulated upon renal vesicle formation. This may not enable an adequate period of Six2 promoter activity for Cre to accumulate to sufficient levels for recombination in all cells. In summary, the data indicate that Six2 acts directly within nephron progenitors to maintain this population in a mesenchymal progenitor cell state.
To further define the requirement for Six2 activity, we performed chimera analysis, generating animals composed of wild-type and Six2 mutant cells (Figure 7A). In this analysis, all wild-type cells were β-gal+, whereas Six2+/− and Six2−/− cells were β-gal−. Approximately 40%–60% contribution was observed in 18 chimeras. As expected, when control chimeric animals (Six2+/− ↔ wild-type) were examined at 12.5 dpc, Six2 heterozygous β-gal− cells contributed to all cell types in the kidney, including the cap mesenchyme, renal vesicle, and its derivatives (Figures 7B, 7D, 7F, 7H, 7J, 7L, and 7N). Surprisingly, in chimeric animals containing Six2 null cells (Six2−/− ↔ wild-type), all kidneys examined were morphologically normal; no ectopic nephron structures were observed (Figures 7C and 7E). Interestingly, all cells within the Pax2+ cap mesenchyme and its epithelial nephron derivatives were β-gal+, indicating a wild-type genotype (Figures 7G, 7I, 7K, 7M, and 7O). In contrast, Six2 null β-gal− cells contributed normally to all other cell populations in the early kidney (Figures 7C, 7E, 7G, 7I, 7K, 7M, and 7O). Thus, whereas Six2 cell-autonomously is required to maintain nephron progenitors, in chimeras where Six2 null cells are in close association with wild-type neighbors, no ectopic renal vesicles form.
Formation of Ectopic Tubules in Six2 Null Mutants Requires Wnt9b Activity
Six2 may act by directly inhibiting the process of epithelial formation in mesenchymal progenitors or conversely by cell-autonomously inhibiting the response of these cells to Wnt9b, a signal that is required to induce renal vesicles within a subpopulation of the cap mesenchyme (Carroll et al., 2005). To understand the relationships between Six2 and Wnt9b, we examined the phenotype of Six2−/− ; Wnt9b−/− compound mutants. In Wnt9b+/− (phenotypically normal) and Six2−/− kidneys at 12.5 dpc, the histological appearance of renal vesicles provides a clear indication of nephron formation (Figures 8A and 8C). These laminin+ cytokeratin− structures were confined to the ventral (medullary) side of the laminin+ cytokeratin+ ureteric tips in Wnt9b+/− kidneys but appear also on the dorsal (cortical) side in Six2−/− kidneys (Figures 8E and 8G). In both Wnt9b−/− and Six2−/− ; Wnt9b−/− kidneys at the same stage, no renal vesicle formation was observed, suggesting that ectopic renal vesicle formation in Six2 null mutants is Wnt9b dependent (Figures 8B, 8D, 8F, and 8H).
To examine the inductive process further, we analyzed expression of the early inductive markers Wnt4 and Pax8 (Stark et al., 1994) at 11.5 dpc. At this stage, both Wnt4 and Pax8 are activated on the ventral side of the T-bud stage kidneys in initial pretubular aggregates in Wnt9b+/− kidneys (Figures 8I and 8M). Both are absent in Wnt9b−/− mutants and ectopically activated on the dorsal side of the ureteric bud in Six2−/− kidneys (Figures 8J, 8K, 8N, and 8O) (Carroll et al., 2005,Self et al., 2006). In contrast, no inductive response could be observed in Six2−/− ; Wnt9b−/− compound mutants (Figures 8L and 8P).
Whereas this may indicate that ectopic renal vesicle formation in Six2 mutants requires a direct Wnt9b signaling event, as in normal renal vesicle induction, analysis of the compound mutants suggests additional interactions that may complicate the interpretation. In Wnt9b−/− and Six2−/− mutants, the timing of ingrowth of the ureteric bud was not markedly different from wild-type (Figures 8I–8K and 8M–8O). However, ingrowth was clearly retarded in Six2−/− ; Wnt9b−/− compound mutants (Figures 8L and 8P). Further, we observed a dramatic reduction of the Pax2+ cap mesenchyme in the compound mutants, more severe than that of Wnt9b−/− mutants at 12.5 dpc (Figures 8Q–8T). Pax2 and Six2 are coexpressed within the cap mesenchyme renal progenitor (Self et al., 2006), indicating that the renal vesicle-forming compartment is depleted. Thus, a reduction in this population may influence the observed phenotype.
Cellular Dynamics, Progenitor Maintenance, and Cell Fate Specification within the Mammalian Nephron Progenitor Pool
The mammalian kidney undergoes an unusually dynamic developmental program. In this, multiple epithelial nephron precursors, renal vesicles, arise from a mesenchymal progenitor population in response to reiterative inductive signaling mediated by the tips of a branching, epithelial, ureteric network. Six2+ cells are present within the mesenchymal pools of the kidney anlagen at 10.5 dpc prior to invasion of the inductive ureteric bud, and Six2+ cells are retained within mesenchyme closely apposed to the branching bud tips until kidney development ceases in the early postnatal animal (Hartman et al., 2007,Oliver et al., 1995). We have used genetic cell fate analysis to examine the contributions of Six2+ cells to mammalian nephrogenesis. These experiments indicate that Six2+ cells are nephron progenitors throughout the extended period of nephrogenesis in the mouse. Moreover, the Six2+ subpopulation of mesenchymal cells is restricted to nephron-forming cell fates. No contribution is seen to other regions of the kidney, and no evidence is observed in this model for an ongoing recruitment of this mesenchymal population to the ureteric epithelium (Herzlinger et al., 1992,Qiao et al., 1995). Six2-mediated labeling of adult nephron structures provides new opportunity for the study of kidney regulation. A recent analysis demonstrates that nephron repair in the adult occurs through an intratubular mechanism (Humphreys et al., 2008).
Our results from this analysis are in general agreement with recent cell-fate studies of Boyle et al., 2007 with a different molecular marker, Cited1. However, unlike Six2, Cited1 is not expressed in the cap mesenchyme until some days after activation of Six2, when nephrogenesis has commenced. Once activated, Cited1 is reported to substantially overlap the Six2+ population (Boyle et al., 2007). Thus, Six2, but not Cited1, marks the nephron progenitor population throughout nephrogenesis.
At the outset of kidney development, approximately 10,000 Six2+ cells surround a single branch of the prospective ureteric epithelium of the T-bud stage kidney. Each adult mouse kidney comprises approximately 13,000 nephrons (Cullen-McEwen et al., 2003,He et al., 1996), and based on glomerular counts, 8000 are present by 19.5 dpc (Cebrian et al., 2004). As each nephron arises from a multicellular aggregate of Six2+ cells, the starting Six2+ population is clearly inadequate for the formation of the full complement of nephrons. Further, direct measurement of Six2+ cells at 19.5 dpc indicates a 16-fold expansion of the Six2+ population even though several thousand renal vesicles have formed during the intervening developmental period. At this time, 180,000 Six2+ cells surround ∼1500 branches of the ureteric tip (data herein and [Cebrian et al., 2004]). Thus, the Six2+ pool undergoes a significant expansion in conjunction with the nephrogenic process. This raises the important question of whether the Six2+ progenitor pool allocated at the onset of nephrogenesis, 11.5 dpc, maintains itself or whether Six2+ cells are replenished from another Six2− cell type.
While our studies cannot rule out a minor role for the latter mechanism, they provide strong support for the former. When a fraction of the Six2+ progenitor pool is indelibly labeled at the initiation of nephrogenesis (by 11.5 dpc), many of these maintain their progenitor status 8 days later at 19.5 dpc, a few days before nephrogenesis terminates. Six2+ cells transition to a Six2− Wnt4+ pretubular aggregate prior to renal vesicle formation. Unlike Six2+ cells, when Wnt4-expressing cells are similarly pulse labeled, Wnt4 descendants are chased into mature epithelial structures of the nephron over a shorter time course. Thus, self-renewal is restricted within the Six2+ pool and lost on induction. Importantly, these experiments, while indicating that Six2+ cells undergo self-renewal, cannot determine whether all Six2+ cells are equivalent in this ability; self-renewal may be a property restricted to a subset of the population (see below).
An argument for self-renewal of nephron precursors has recently been promoted on the basis of Cited1-based cell-labeling studies (Boyle et al., 2007). Though the conclusions reached in these studies agree with our own, they provide a less compelling case. First, the postpulse chase period was considerably shorter, as activity of the Cited1 transgene was not detected until 13.5 dpc and kidneys were examined at 19.5 dpc. Second, the pulse-labeling period was less restricted. CreERT2 was nuclear at 24 hr after induction, indicating that Cre activity was ongoing at this time. Thus, while the precise period of Cre activity was not determined, it is clear that the labeling is likely to have occurred over a longer time window than within the Six2 experiments herein.
The Six2+ Pool Contains Multipotent Nephron Progenitors
On the basis of clonal analysis in vitro and ex vivo, it was suggested that individual nephron progenitors are multipotent in their capacity to generate distinct regions of the nephron (Herzlinger et al., 1992,Osafune et al., 2006). The analysis of Six2+ cell fates following low-dose tamoxifen induction in the current study provides additional evidence in support of this conclusion in vivo and defines the population of multipotent nephron progenitor cells to the Six2+ cap mesenchyme. The appearance of a small number of labeled cell clusters per kidney is consistent with, and indicative of, a clonal level of labeling. These clusters spanned several regions of developing nephrons where distinct regional programs of cell fate specification are underway. Notably, descendants of a Six2+ cell can be found within molecularly distinct compartments of a single nephron: podocytes, proximal and distal tubule structures. Though these experiments argue that at least some Six2+ cells are multipotent, they do not address whether this is a general property of the population nor when restriction occurs in the nephron-forming lineage. For example, a clonally labeled multipotent Six2+ cell may give rise to multiple labeled cells within the mesenchymal nephron progenitor compartment, and these labeled descendants may undergo subsequent restrictions to distinct nephron compartments prior to induction. Indeed, multiple labeled cells can be observed within a Six2+ cap mesenchymal population, presumably all clonal descendants from a single cell-labeling event. Clonal labeling with Wnt4-GCE may resolve this issue. Importantly, the presence of multiple labeled progenitor cells lends additional evidence in favor of self-renewal and suggests that some Six2+ cells may have a greater self-renewal capacity than others. The simplest model for nephron patterning, multipotent precursors and regional specific patterning after renal vesicle formation (Herzlinger et al., 1992), is suggested by recent studies of Notch-based signaling in which Notch action proximalizes developing epithelial renal vesicle derivatives (Cheng et al., 2003,Cheng et al., 2007,Wang et al., 2003).
Six2 Functions in the Maintenance of Multipotent Nephron Progenitor Cells in the Cap Mesenchyme
Our studies provide new insights into the function of Six2 and more generally the regulation of the inductive process. Cell-fate analysis in Six2 mutants indicates that Six2 is cell autonomously required to prevent premature, appositional renal vesicle formation. However, while we observe this phenotype in Six2 mutants, we are unable to detect ectopic renal vesicle structures in chimeras consisting of Six2 mutant and wild-type embryos, nor of contribution of Six2 mutant cells to normal tubulogenesis. Rather, mutant cells are apparently rapidly lost from the mesenchymal progenitor pool.
There are a number of possible explanations for these different outcomes. For example, formation of a renal vesicle may depend on local induction of a critical number of cells. In the chimera model, the scattering and interspersion of mutant and wild-type cells may prevent the establishment of a critical tubule-forming cell mass. A rapid process of cell removal must follow such that Six2 mutant cells are undetectable by 12.5 dpc in the cap mesenchyme compartment. Wild-type and mutant cells may also compete for a limiting factor that may promote the propagation of wild-type cells at the expense of mutant cells, leading to rapid depletion of the latter and a failure to contribute to epithelial nephron derivatives. Recent work in Drosophila imaginal disc has demonstrated that local cell competition can trigger apoptosis in growth-defective mutants (Moreno et al., 2002).
Wnt9b secreted by the ureteric bud provides a primary inductive signal that initiates nephrogenesis in adjacent nephron progenitors through the canonical Wnt signaling pathway (Carroll et al., 2005,Park et al., 2007). Given that Six2+ cells define the nephron progenitor compartment, it follows that the Six2 population is the likely target of Wnt9b-mediated inductive signaling. Six2 mutants and Wnt9b mutants have opposite phenotypes. In one simple model, Six2 acts to inhibit a Wnt response within a subset of the Six2 progenitor pool, maintaining a nephron progenitor pool for additional rounds of nephrogenesis. Our demonstration that compound mutants lack renal vesicles and earlier molecular features of the Wnt9b-inductive response, normally observed in emerging pretubular aggregates, supports this model. However, the resulting phenotypes suggest that this view is likely too simple. In addition to observing a failure of nephrogenesis, compound mutants exhibit reduced survival of the nephron precursor compartment and, likely as a secondary consequence of a loss of signals from this population (notably the branch-growth regulator GDNF) (Costantini and Shakya, 2006), delayed and reduced ingrowth of the ureteric bud.
These observations suggest additional interactions between Six2 and Wnt9b. For example, Six2 may oppose an inductive Wnt9b signal. However, Wnt9b and Six2 may also act cooperatively to maintain the nephron progenitor pool. The distinct outcomes to Wnt9b signaling may reflect different levels of Wnt signaling—low levels promoting maintenance of Six2+ cells and high levels induction, Six2 providing a tonic level of inhibition to regulate the response. Here it is interesting to note lower levels of Wnt9b expression in ureteric epithelium underlying the Six2+ population compared with ureteric epithelium immediately beneath the branch tip (Carroll et al., 2005) where Wnt4/Pax8 inductive markers are first observed in Six2 descendant cells. Alternatively, Wnt9b may act in promoting both maintenance and induction of Six2 cells, the actions of other signals determining the specific outcome to a Wnt9b input. Here, several signaling factors have been reported to promote either survival/proliferation of metanephric mesenchyme including Bmp7 (Dudley et al., 1995,Luo et al., 1995), FGF2 (bFGF) (Barasch et al., 1997,Dudley et al., 1999), and TIMP2 (Barasch et al., 1999a) or renal vesicle induction including Wnt4 (Stark et al., 1994), FGF2 (Perantoni et al., 1995), LIF (Barasch et al., 1999b), and TGFβ2 (Plisov et al., 2001).
Experimental Procedures
Mouse Strains
The Tet-off-eGFPCre-Kan (TGC-Kan) targeting cassette for the Six2 BAC transgenic allele was generated as follows. A Tet-off cassette (tTA-2 × pA-teto-CMVmin) was generated by removing loxP-TKneo-URA-loxP from mtTAFFF (Bond et al., 2000). The Tet-off construct was combined with eGFPCre from pBS592 (Le et al., 1999) and FRT-Kan-FRT from pICGN21 (Lee et al., 2001). After adding homologous arms by PCR, the resulting TGC-Kan targeting construct was introduced by RED cloning system (Lee et al., 2001) into a mouse BAC clone RPCI23-311C1, which contains the Six2 locus in the middle of the clone. After screening for correctly recombined clones and removal of the kanamycin selection cassette, the Six2-TGC BAC clone was introduced into CD-1 zygotes (Charles River Laboratories) by pronuclear injection (Nagy et al., 2003) to generate transgenic mice. Three founders were generated, and one line exhibited the expected Six2 expression pattern, though the transgene did not respond to doxycycline. The Six2-TGC BAC transgenic was maintained on a CD-1 × Swiss Webster (Taconic) × C57BL/6J (Jackson Laboratory) mixed background.
For Six2-Cre knockin alleles, Six2 targeting vectors were generated with sequence-confirmed homologous arms subcloned by PCR from a BAC clone RPCI23-311C1. The Six2 initiation codon was replaced by Tet-off-eGFPCre (TGC), CreERT2 (CE) (Indra et al., 1999), or eGFPCreERT2 (GCE) constructs followed by an FRT-PGKneobpA-FRT selection cassette. The eGFPCreERT2 fusion construct was generated by combining eGFPCre from pBS592 (Le et al., 1999) and CreERT2 from pCre-ER(AA2) (Feil et al., 1997) constructs. After addition of MC1-TK for negative selection, the resulting targeting vectors were introduced by gene targeting (Nagy et al., 2003) into 129/Sv × C57BL/6J F1 hybrid ES cells (Eggan et al., 2001). After G418 and FIAU selection, ES cell colonies were isolated and screened by PCR using both 5′ and 3′ external primers. Chimeric mice were generated by injection of correctly targeted ES cells into C57BL/6J blastocysts. Six2TGC/+, Six2CE/+, and Six2GCE/+ mouse lines were maintained on a 129/Sv × C57BL/6J mixed background. The Wnt4-GCE allele was generated in a similar way to that of the Six2-GCE allele, except a BAC clone RPCI23-246F18 containing the Wnt4 locus was used.
Six2-Cre alleles were genotyped using the following primers: Cre-Fw1, GGACATGTTCAGGGATCGCCAGGC; Cre-Rv2, CGACGATGAAGCATGTTTAGCTG; mSix2-Fw35, CCACCTTCGGCTTCACGCAGGAGCAAGT; and mSix2-Rv36, CCGCGCAGCTTCTCCGCCTCGATGTAGT, which give a 219 bp band for the Six2-Cre alleles (Cre-Fw1 and Cre-Rv2) and a 286 bp band for the Six2 wild-type allele (mSix2-Fw35 and mSix2-Rv36). The Wnt4-GCE allele was genotyped using the following primers: Cre-Fw1, Cre-Rv2, and mWnt4-Fw56, GCCGCCGCGAGCAATTGGCTGTAAGT; and mWnt4-Rv57; GGACGCTTTCCCTCGGAGACCTGTCA, which give a 219 bp band for the Wnt4-GCE allele (Cre-Fw1 and Cre-Rv2) and a 350 bp band for the Wnt4 wild-type allele (mWnt4-Fw56 and mWnt4-Rv57).
R26R-lacZ Cre reporter (Soriano, 1999) and R26tg/tg lacZ-transgene integrated (Friedrich and Soriano, 1991) mice were purchased from Jackson Laboratory and maintained on a C57BL/6J × Swiss Webster mixed and 129/Sv inbred backgrounds, respectively. R26R-lacZ mice were genotyped using the following primers: R26-Fw11, CTCCCAAAGTCGCTCTGAGTTGTTATCAGT; R26-Rv12, CTCGGGTGAGCATGTCTTTAATCTACCT; and pBT-Rv2, GCGAAGAGTTTGTCCTCAACCGCGAGCTGT, which give a 484 bp band for the R26 wild-type allele (R26-Fw11 and R26-Rv12) and a 320 bp band for R26R-lacZ knockin allele (R26-Fw11 and pBT-Rv2).
Six2+/− ; Wnt9b+/− mice were maintained on a 129/Sv × C57BL/6J × NMRI mixed background and intercrossed to obtain Six2 ; Wnt9b compound null mutants (Carroll et al., 2005,Self et al., 2006). Littermates were used for controls. Mice were bred using timed matings, noon on the day of vaginal plug detection considered 0.5 dpc. For induction of the eGFPCreERT2 protein, tamoxifen (Sigma, T5648) was dissolved in corn oil (Sigma, C8267) and administrated by intraperitioneal (IP) injection (Danielian et al., 1998).
FACS Analysis
Kidneys from Six2GCE/+ mice were dissected and treated in 300 μl Trypsin (Invitrogen, 25200-072) at 37°C for 3–5 min. After adding 600 μl DMEM media (Invitrogen, 11965) containing 10% sheep serum (Sigma, S2263), a single cell suspension was prepared by pipetting. Cells were collected by centrifugation and resuspended in 100 μl of PBS (Mediatech, 21-031-CV) containing 2% sheep serum. FACS analysis was performed using DAKO Cytomation MoFlo.
Chimera Analysis
Six2CE/GCE morulae were aggregated with Six2+/+ ; R26tg/+ morulae. After overnight incubation, chimeric embryos were transferred to the uterus of pseudopregnant Swiss Webster females (Nagy et al., 2003). Chimeras were genotyped using the following primers: CE-Fw1, AGTCTTAAGAAGCTTGAATTCCACCA; XFP-Fw5, CCGTGCTGCTGCCCGACAACCACTA; and Cre-Rv2, CGACGATGAAGCATGTTTAGCTG, which give a 327 bp band for the CE allele (CE-Fw1 and Cre-Rv2) and a 496 bp band for the GCE allele (XFP-Fw5 and Cre-Rv2). A total of 18 chimeras were analyzed.
Dissected kidneys were fixed in 4% paraformaldehyde for 1 hr at 4°C and soaked in 30% sucrose overnight at 4°C. After embedding in OCT (Sakura, 4583), cryosections were generated at 16 μm using a Microm HM 550 cryostat.
β-Gal Staining
β-gal staining was performed as described previously (Nagy et al., 2003). Cryosections were stained with X-gal at 37°C overnight and counterstained with 0.2% Eosin-Y (Polysciences Inc.) or Nuclear Fast Red (Sigma, #N3020). Whole-mount kidneys were fixed in 4% paraformaldehyde for 1 hr at 4°C and stained at 37°C overnight for embryonic samples or at 4°C for 2–3 days for neonate samples.
To generate a Six2 antibody, rabbits were immunized with a KHL-conjugated peptide SEDEKTPSGTPDHSS corresponding to amino acids 241–255 of the mouse Six2 protein sequence, and antiserum was affinity purified against immobilized Six2 peptide (Covance Research Products). The purified antiserum was tested by immunostaining of transiently transfecting (Lipofectamine 2000, Invitrogen) COS7 cells with a mouse Six2 expression plasmid. Immunostaining on cryosections matched the expected Six2 mRNA expression pattern in metanephric kidneys at 15.5 dpc.
Sections were incubated with primary antibodies to anti-Wt1 (Santa Cruz, sc-192), anti-Flk1 (PharMingen, 555307), anti-PECAM-1 (anti-CD31, BD PharMingen, 553370), anti-PDGFRb (eBiosciences, 14-1402-82), anti-uromodulin (anti-Tamm-Horsfall glycoprotein, Biomedical Technologies Inc., BT-590), anti-SMA (Sigma, A5228), anti-Pax2 (Covance, PRB-276P), anti-GFP (Aves labs, GFP-1020), anti-β-gal (Cappel, #55976; Abcam, ab9361), anti-cytokeratin (Sigma, C2562), anti-laminin (Sigma, L9393), and LTL-lectin (Vector Laboratories, FL-1321) and detected by the secondary antibodies with Cy2, Cy3, and Cy5 (Jackson ImmunoResearch Laboratories) or Alexa Fluor 488, 568, 633, and 647 (Invitrogen). Sections were stained with Hoechst (Invitrogen, H3570) prior to mounting with Vectashield Mounting Medium (Vector labs, H-1000). Fluorescent images were photographed on a Zeiss LSM510 Axioplan inverted confocal microscope.
Whole-Mount In Situ Hybridization
Whole-mount in situ hybridization was performed as previously described (Carroll et al., 2005,Wilkinson et al., 1987).
We thank Kevin Eggan for 129/Sv × C57BL/6J F1 hybrid ES cells, Neal Copeland for reagents for BAC recombination, Brian Sauer for eGFPCre construct, and Daniel Metzger and Pierre Chambon for CreERT2 construct. A.K. was supported by a Research Fellowship from the National Kidney Foundation. M.T.V. was supported by a National Research Service Award (NRSA) award (NIH-NIDDK F32DK060319). Work in G.O.'s laboratory was supported by Cancer Center Support (CA-21765) and the American Lebanese Syrian Associated Charities (ALSAC). Work in A.P.M.'s laboratory was supported by a grant from the National Institutes of Health (NIH-NIDDK DK054364). A.P.M. is a consultant for Merck. This consultancy does not relate to the work herein.
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Publication Information
Received: January 22, 2008
Revised: April 23, 2008
Accepted: May 29, 2008
Published: August 6, 2008


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A shake-up at the La Grange Utilities Commission led to emergency legislation being passed by the city council Monday night.
The commission is currently without a director, after La Grange Mayor Bill Lammlein fired former director John M. Bennett. Lammlein said he let Bennett go because he didn't feel like Bennett was good for the commission going forward.
"I didn't feel like he was the person to move the utilities in the direction we needed to go in," Lammlein said.
As Congress slowly starts back up in 2014, Congressman Thomas Massie has a few key issues on his plate.
The two most important for Massie, who represents Oldham County and all of Northern Kentucky, is the inclusion of an industrial hemp amendment in the Farm Bill and reforming the practices of the National Security Agency and its wiretapping.
In a conference call with reporters last week, the first term congressman celebrated the industrial hemp inclusion, which will make it legal for hemp to be grown for university research and for state pilot projects.
The primary ballot for Oldham County has been officially set.
Candidates lined up for the ballot draw last week at the county clerk's office to select their positions on the 2014 primary ballot.
Sixteen county offices, including constable races, will be on the ballot for the primary.
Additionally, there will be a primary for La Grange mayor and for city council. As of the draw, the number of those running for council was 20. Keith Simpson and Linda Ballard Hall withdrew from the city council race before the ballot draw.
The Oldham County FOP Lodge 62 will be hosting a political forum.
The forum will take place Monday, Feb. 10 6:30 p.m. at the John Black Center. This forum is open to the public but there will be no public questions accepted. Candidates running for County Judge Executive, County Attorney, Sheriff, Coroner and La Grange Mayor will be asked predetermined questions pertaining to law enforcement and emergency services.
One out of a trio of familiar names hopes to become the next mayor of La Grange.
Former mayor Elsie Carter, city councilman Joe Davenport and resident Brian Oerther all have filed to run for mayor this year. Current Mayor Bill Lammlein, who's keeping a promise to serve only one term as mayor, is vacating the seat.
And while he's leaving the mayor's office, Lammlein doesn't plan to stay away from city council meetings after his term ends, because he's one of 22 who filed to run for La Grange city council.
Work on a long-awaited bridge over Interstate 71 near La Grange has finally begun.
The bridge will connect New Moody Lane and Commerce Parkway in La Grange over the interstate roughly one mile south of Exit 22 on I-71.
"This project will improve connectivity as well as reduce delay and congestion on adjacent roadways," Matt Bullock, chief district engineer for the Department of Highways Office 5, said. "The bridge will also reduce response times for emergency vehicles and improve the safety of the roadway network in Oldham County."
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It will be easier for patrons of a popular Crestwood event center to consume alcohol, thanks to the results of a special election.
The election, held Tuesday at the South Oldham Fire Station on Old La Grange road, will now allow Yew Dell Botanical Gardens to sell alcohol by the drink during events. The change was passed by an 87 percent margin.
Yew Dell is host to many business events and weddings year-round, but previously could only make alcohol available by hiring a licensed caterer to provide it.
A new map laying out magistrate districts passed by the Oldham County Fiscal Court in 2011 will finally go into effect this year.
Every 10 years, the maps that dictate district boundaries for elected officials, from Congress down to local governments, are reviewed and revised as needed. The process usually takes place six months to a year after the latest U.S. Census data is released and goes into effect during the next election cycle.


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Ken Volgamore has been appointed to serve on the Orchard City Town Board. He has stepped into a two-year term that will expire in April 2014.
Volgamore is a private contractor.
He ran for town trustee in last April's municipal election.
He lives with his wife in town, where he located to 15 years ago.
During a brief interview with town trustees prior to the Oct. 10 meeting, Volgamore said that he is the son of a Tongue Creek Valley homesteader, so he has roots in the community.
Volgamore is a retired firefighter and is proud of his service on both Delta and Cedaredge departments.
His application for the position was the only one received, said Mayor Don Suppes, and he was unanimously endorsed for the post by the trustees.
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PyF 2.0 Now Available
Version 2.0 of PyF, an open source Python framework and platform used for processing large amounts of data is now available.
In a blog post today, Jonathan Schemoul announced the release of PyF 2.0. This open source Python framework is composed of different layers: core layer--pyf.dataflow, network layer--pyf.manager, componentized layer--pyf.componentized and webservice--pyf.services. According to the website, at the core level, PyF 2.0 has reused, embedded, and extended another library called zflow and each level is based on the level directly under it.
In addition to the PyF website, users can ask questions and find out more information on the mailing list, #pfy IRC Channel on freenode.net, Google group as well as the release documentation.
Issue 161/2014
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Tag Cloud
New TLS Attack Takes the S out of HTTPS
Vulnerability affects many Linux web servers
Munich Adopts Kolab
The Bavarian capital shuns Microsoft, Google, and other alternatives to implement an open source groupware solution.
Canonical Announces Ubuntu Smartphones
Phone vendor partnerships bring Mark Shuttleworth's dream of Ubuntu on a phone a step closer to reality.
Piviti Seeks Funding
Donors will get to vote on new features for the free video editor.
Debian Tech Committee Votes for systemd
Debian project puts init out to pasture and says no to Ubuntu's Upstart.
The Mask: Scary New Face of Internet Intrusion
Ultra-sophisticated attack tool might have originated from a state-sponsored intelligence service.
Epoch 1.0 Released
New alternative for init comes with a small footprint and minimal configuration.
Wayland 1.4 Challenges X11
X marks the target for the next-generation windowing system.
Red Hat Adopts CentOS
Super-clone CentOS Linux gets beamed up to the mother ship.
W3C Promotes Media Source Extensions
HTML technology will enable new video editing and playback options.


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A collection of news and information related to U.S. Army Training and Doctrine Command published by this site and its partners.
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Newport News Public Library series to feature Jae Sinnett, TRADOC band, clogging team
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Story | Aug 25, 2011 | 8:53 AMThe Top Five Donny Osmond Donny Osmond's "Basically Yours" tour. 8 p.m. Friday at Shippensburg University's H. Ric Luhrs Performing Arts Center, 1871 Old Main Drive, Shippensburg, Pa. Tickets cost $69 to $89. Call 717-477-7469... -
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Story | Aug 29, 2011 | 7:35 AMThe Hagerstown Community College Alumni Association will host the James River Ramblers, one of the U.S. Army TRADOC Band ensembles, as part of its annual Red, White and Blue Concert Series on Wednesday at 6:30 p.m. at HCC's Alumni Amphitheater. The... -
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Story | Jun 1, 2011 | 12:05 PMThe performance by the U.S. Navy's Country Current country-bluegrass band, scheduled for Wednesday at 6:30 p.m. at the Hagerstown Community College Alumni Amphitheater, has been canceled due to weather, HCC announced in a news release. Future Red,... -
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Rangers manager Ron Washington said Wednesday that Yu Darvish will start the team's first playoff game.
Alex Wood tossed a rare spring training shutout in a rain-shortened game against the Astros on Monday.
Scott Kazmir was scratched from Monday's start due to left triceps tightness.
Jarrod Parker will undergo Tommy John surgery on his right elbow on March 25.
Orioles manager Buck Showalter on Monday named Chris Tillman the club's Opening Day starter.
Nationals manager Matt Williams said Monday that Ross Detwiler will begin the season in the bullpen.
Michael Bourn (hamstring) will miss at least the next week and is questionable for Opening Day.
Phillies manager Ryne Sandberg on Monday named Cliff Lee the club's Opening Day starter.
Anibal Sanchez was scratched from Monday's start due to inflammation in his right shoulder.
Chase Headley (calf) is hoping to make his Cactus League debut on Thursday.
Padres manager Bud Black said Monday that the conversations he's had with Joe Wieland about his injury revolved more around the triceps area rather than the elbow.
Marco Scutaro (back) is back in the Giants' lineup Monday.


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Samsung Electronics has developed what it claims is the industry's first 32GB load-reduced, dual-inline memory module (LRDIMM) for servers.
Samsung said it will begin mass producing the 32GB LRDIMM in the second half of 2010. Using 40nm-class, 4Gb DDR3 chips, the new 32GB LRDIMM accommodates next generation servers designed for virtualization, cloud computing and other high-capacity applications.
Samsung's 32GB LRDIMM prototype consists of 72 4Gb DDR3 chips and an additional memory buffer chip to help reduce the load on the memory subsystem by as much as 75%. Samsung's LRDIMMs operate at 1.35 or 1.5 volts.
- China flat panel display industry outlook, 2013-2016
This Digitimes Research Special Report outlines and analyzes the key trends and players that are influencing the continued development of China large-size and small-to medium size TFT LCD panel industry, while providing related growth forecasts for the industry through 2016.
- TD-LTE market developments and forecast, 2014-2016
TD-LTE has become the mainstream global choice for the construction of 4G asymmetric frequency spectrum networks. This Digitimes Research Special Report examines the current global status of TD-LTE, and forecasts developments in the TD-LTE supply chain in China and worldwide through 2016.
- Trends and developments in the OTT market
Companies such as Apple, Samsung Electronics and Google have become increasingly interested in over-the-top services as a means to connect ultra-mobile devices to TVs, increase cloud computing users, and boost various cloud and software services.


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Information contained on this page is provided by an independent third-party content provider. WorldNow and this Station make no warranties or representations in connection therewith. If you have any questions or comments about this page please contact firstname.lastname@example.com.
SOURCE Experian
2013 Q3 Email Benchmark Report reveals unique open rates increased 6 percent -- further evidence that personalization and cross-sell drive transactional success
NEW YORK, Dec. 16, 2013 /PRNewswire/ -- Experian Marketing Services, a global provider of integrated consumer insight and targeting, data quality and cross-channel marketing, showed that overall email volume increased 12.7 percent in Q3 2013 versus Q3 2012, according to the Experian Marketing Services 2013 Q3 Email Benchmark Report. The cross-channel marketing leader also found that unique open rates increased 6 percent year-over-year in Q3 2013.
(Logo: http://photos.prnewswire.com/prnh/20130131/LA51658LOGO)
The benchmark report's spotlight on transactional emails reveals a rise in open rates with the inclusion of a brand's name in the subject line of transactional emails, such as shipping and order confirmations, order cancellations and return confirmations. While the use of this tactic varied widely, brand-name inclusion in only shipping confirmation emails produced significant gain with a 7-percent increase in unique open rates. Other findings include:
"Our latest Benchmark Study provides even more evidence that personal connections really do matter," said Peter DeNunzio, general manager for cross-channel marketing at Experian Marketing Services. "Transactional emails showed 23-percent higher click rates when they personally addressed a customer in the body of the email. These are the kind of emails that can help marketers generate repeat sales."
Expanding cross-channel opportunities
After analyzing Twitter and Instagram campaigns, Experian Marketing Services identified a key trend to watch among social media mailings. In short, promoting social media in subject lines is an especially effective cross-channel practice.
Experian Marketing Services' research involved a year-over-year comparison of this type of subject line, revealing significant growth in the percentage of emails that mention Twitter or Instagram in the subject line. Subject-line mentions of Twitter rose from 8 percent in Q3 2012 to 41 percent in Q3 2013, while mentions of Instagram climbed from 3 percent in Q3 2012 to 30 percent in Q3 2013. While subject-line mentions of Facebook also increased, from 76 percent in Q3 2012 to 87 percent in Q3 2013, mentions of Pinterest actually fell from 9 percent in Q3 2012 to 5 percent in Q3 2013.
Holiday strategy
The benchmark report also reveals a number of significant best practices for success, which can be especially important for email marketers during the holiday sales season.
"As more customers visit their favorite online destinations for holiday shopping, more password requests will occur, providing marketers with the opportunity to optimize these emails," added DeNunzio. "Specifically, cross-sell sections in password-reset emails produced a 93-percent higher transaction rate than those without cross-sells items, leveraging subscribers' existing brand interest to maximize product interest."
Experian Marketing Services' Q3 2013 Quarterly Benchmark Report is available to download.
About Experian Marketing Services
Experian Marketing Services is a global provider of integrated consumer insights and targeting, data quality and cross-channel marketing. We help organizations around the world intelligently interact with today's dynamic, empowered and hyperconnected customers. By coordinating seamless interactions across all marketing channels, marketers are able to plan and execute superior brand experiences that deepen customer loyalty, strengthen brand advocacy and maximize profits.
For more information, please visit http://www.experian.com/marketingservices.
About Experian
Experian® is the leading global information services company, providing data and analytical tools to clients around the world. The Group helps businesses to manage credit risk, prevent fraud, target marketing offers and automate decision making. Experian also helps individuals to check their credit report and credit score, and protect against identity theft.
Experian plc is listed on the London Stock Exchange (EXPN) and is a constituent of the FTSE 100 index. Total revenue for the year ended March 31, 2013 was US$4.7 billion. Experian employs approximately 17,000 people in 40 countries and has its corporate headquarters in Dublin, Ireland, with operational headquarters in Nottingham, UK; California, US; and Sao Paulo, Brazil.
For more information, visit http://www.experianplc.com.
Experian and the Experian marks used herein are service marks or registered trademarks of Experian Information Solutions, Inc. Other product and company names mentioned herein are the property of their respective owners.
Suzanne Blackburn
Experian Marketing Services
1 212 863 4648
©2012 PR Newswire. All Rights Reserved.


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Knit this lovely valentines themed gadget cover accessory piece, a free pattern download designed by Anna Nikipirowicz and using the gorgeous yarn Felted Tweed (wool, alpaca, viscose). With a lovely heart i-cord decoration on the centre front of the cover and mostly knitted in stocking stitch, this is the perfect cover for your favourite gadget.. This knitting pattern is for the intermediate knitter.
Knit this womens slip stitch scarf, a free pattern download designed by Gemma Atkinson and using the beautiful yarn Tumble (alpaca and cotton). A long and cosy scarf for the winter months with an elegant stitch pattern, this knitting pattern is for the beginner knitter.
A message from the Rowan Team In memory of Sandra Taylor, one of our wonderful hand knitters who has knitted for Rowan for over 5 years and has recently passed away. She will be sadly missed.
Knit this cute fairisle gadget cosy, a Rowan web exclusive free download pattern, designed by Jem Weston using the beautiful yarns Felted Tweed (merino wool and alpaca) and Wool Cotton (merino wool and cotton). This cosy comes in two sizes for either your phone, camera or any other small gadget you may want to give a protective yet stylish cover for. This knitting pattern is for the intermediate knitter.
Knit this slouchy hat as a free download pattern to support woolly hat day on 26th October, a design by Gemma Atkinson using the beautiful yarn Tumble (alpaca and cotton). With oversized tuck stitch detail and a 2x2 rib band, this knitting pattern is for the beginner knitter.
Knit this lace glamour Scarf, a free download as part of the launch of our new yarn Kidsilk Haze Glamour (super kid mohair and silk). An exclusive web design by Lisa Richardson, this knitting pattern is a beautiful must knit design and is for the intermediate knitter. This free pattern PDF has also been translated into French, German and Dutch - only print the pages you need after downloading.
Knit this womens embroidered cowl, a free pattern download from the winner of our design competition. Designed by Julie Ferguson and using the gorgeous yarn Kid Classic (lambswool and kid mohair), the cowl is knitted throughout in stocking stitch, with a beautifully coloured embroidered pattern through the centre section. This knitting pattern is for the intermediate knitter. This pattern is an artist interpretation of original tribal symbols.
Adding a stripe to Rowan's much loved Kidsilk Haze in Kaffe Fassett selected colours seems a natural step and this easy to knit pattern lets you watch the colours change as you knit your own unique scarf.
This wallet is perfect if you are teaching children to knit. The perfect holder for Halloween treats! It has been designed by Jemma Weston exclusively for the web site and uses one ball of Kid Classic.
This cool mobile phone cover uses zingy shades of Handknit Cotton and has been designed exclusively for the web site.
This cute childrens beret uses Kidsilk Haze and has been designed exclusively for the web site by Sarah Hatton.


text: 10 World-Changing Innovators for 2012 Tires that maintain their own air pressure. A brain-to-computer interface. Cameras that can see around corners. This year's Breakthrough Award-winning innovators are the brains and sweat behind some of the world's biggest ideas. (And check out all our Breakthrough coverage here.) By Logan Ward Email Comments 0 Share Back 10 of 10 Next Return to Gallery Return to Gallery Return to Gallery Return to Gallery Return to Gallery Return to Gallery Return to Gallery Return to Gallery Return to Gallery Back Next NASA View Thumbnails Breaking Through the Heliopause Mechanical Lifetime Achievement Award Voyager 1 & 2 Innovators: NASA Jet Propulsion Laboratory In 1977 NASA launched twin Voyager spacecraft to take advantage of a rare alignment of the solar system's gas giants (Jupiter, Saturn, Uranus, and Neptune) that would allow both craft to swing past all four planets in a single trajectory. Engineers from NASA's Jet Propulsion Laboratory originally made plans for a 12-year mission. But by 1972 budget woes had withered their planetary grand tour to a five-year flight. Thirty-five years later, both probes are still sending back data, and within the next couple of years Voyager 1 and Voyager 2 will exit the farthest bounds of the solar system. Ed Stone, who has been JPL's project scientist for the Voyager program for four decades, is awed by the prospect of the probes entering interstellar space. "We have an object made by the human race that's traveling between the stars," he says. "It's not science fiction anymore. It's real." Return to Gallery Back 10 of 10 Next Return to Gallery Back Next Return to Slideshow Related Links


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Review: 'Veronica Mars' will delight old fans and may even win new ones
The big-screen revival of the show feels like no time has passed
www.hitfix.com/blogs/motion-captured/posts/review-veronica-mars-will-delight-old-fans-and-may-even-win-new-onesThe big-screen revival of the show feels like no time has passed
What happened on November 13? Felix Unger was asked to leave his place of res...
Kodaline also performs as another singer faces elimination
www.hitfix.com/blogs/the-fien-print/posts/recap-american-idol-season-13-top-12-results-plus-phillip-phillips-performsIt rains and everybody looks at their waterlogged hands for an hour
www.hitfix.com/blogs/the-fien-print/posts/recap-survivor-cagayan-cops-r-usThis documentary looks at why one judge worked so hard to keep kids from causing trouble.
www.hitfix.com/events/kids-for-cashBeck's 'Morning Phase' enters at No. 3
IFC Fairgrounds: March 12-14, 2014 from 2-10PM at Palm Park
Portlandia pan...
I was already rooting for Pharrell Williams' "Happy" to win the...
Adam Sandler finds out that sometimes crazy follows you
www.hitfix.com/events/grown-ups-2-two-disc-combo-blu-ray-dvd-ultraviolet-digital-copyThe all-star comedy cast from "Grown Ups" returns with some exciting new additions


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About this calendar This is a community-managed calendar. Please add any training events, related conferences or meetings, or other dates of interest to the OpenMRS community to this calendar. Times listed here are for the Indianapolis, US time zone (Eastern Time). Convert to your time zone. |
Aug 30, 2011
I've got the same question. I want to have it in my google calendar.
Aug 30, 2011
Aug 30, 2011
Thanks Michael! It's helpful, but still no way to export. Buu for Confluence calendars :( Back to proved copy & paste strategy.
Aug 30, 2011
Above method is for subscribing to the "live" calendar from another system. To export an ICS file for a 1-time import into another system, you can choose "Export to iCalendar" from the same menu.
More information from Atlassian: http://confluence.atlassian.com/display/TEAMCAL/Team+Calendars+Home
Aug 31, 2011
I heard from Atlassian today that there it's not possible to subscribe successfully with Google Calendar at this time. We now have a feature request to ensure that works in the near future - I'd encourage anyone interested to vote on it! https://studio.plugins.atlassian.com/browse/TEAMCAL-314
Nov 09, 2011
Making Sunday the first day of the week in calendars
I just discovered that Confluence Team Calendars follow user language preference. So, those of us in the U.S. who grew up with Sunday as the first day of the week on our calendars can get Team Calendars with Sunday on the left by going to Confluence → Settings and changing Language from "Automatically detect browser setting" to "English (United States)." Yay!


text:
Recent Press
The Gift of Hospice – Special to the Herald – Jacksboro Gazette-News
August 9, 2013 | By girling
August 8, 2013 "The Gift of Hospice" by Peggy Durkin, Special to the Herald "Hospice is a specialized type of end-of-life care designed to comfort and help both patients and their families as one approaches life's final journey. Many people … Read More
Helpful Videos
Interested in learning more about what hospice is and how it can benefit you or your loved one? The videos below, featuring several key members of our Girling Hospice Texas team, offer firsthand insight and information about hospice care.


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Established in 1994, Interinfo is a specialist genealogy firm. Our researchers are not amateurs. They are experienced professionals in the full meaning of this word. All of them graduated in history from Ukrainian universities. Most of them have over 20 years' experience in archival research. As professional historians with an in-depth knowledge of Ukrainian archives, they not only know how research should be conducted, but have even acquired a professional feeling where your ancestors' documents might be filed. Our services include genealogical research, location of lost relatives and probate research. We would be pleased to lend a helpful hand and look forward to assisting you in any way we can.
Here are some references on our work:
"I received the documents yesterday and I was very happy with the information. I never thought I would be able to discover anything about my Hungarian ancestors… I am excited." Loren V. New Jersey, USA "We received your envelope with all the information about my relations in Ukraine and Russia last week. Thank you very much, you did a wonderful job and I was impressed that you hand wrote the family trees out with all the information." Penny J. British Columbia, Canada "I am very pleased indeed with your excellent work and I will miss waiting for emails from you. I have spent almost the last 3 years searching and it feels strange now that I have come to the end of this particular road… I was doubtful at the start whether you would even be able to confirm where my father came from, never mind all the rest as well. Clearly, it pays to use true professionals!" Michael W. Herts, England "Thank you for the research. You discovered a lot more than I had expected!" Michael H. Ramat Gan, Israel "I and the whole family thank you so much for finding out all these interesting things about my Russian relatives!… So interesting!" Ulla S. Hamburg, Germany "Do you really mean you found everything - from Alpha to Omega? I do commend and thank you for that." Wladimir F. Chatel-Montagne, France "I would like to say I am very happy with the information I have received thus far. Hopefully I will be corresponding with my relatives soon. I am very impressed by what you have done for me in such a short amount of time… Being able to know where my heritage lies is Fantastic to say the least. Thank you once again for doing a GREAT job." Michelle S. Queensland, Australia "The results of your work are exactly what I was looking for." Andrey V. Saint-Petersburg, Russia
© 2004-2012 Interinfo All rights reserved.


text:
The Pew Charitable Trusts is driven by the power of knowledge to solve today's most challenging problems. Pew applies a rigorous, analytical approach to improve public policy, inform the public and stimulate civic life.
Stay updated with Pew News Now! We invite you to sign up to receive our weekly e-mail newsletter.
Pew offers news delivered to your desktop via RSS feed. Subscribing is easy. To learn more or get started, follow the link below.
Subscribe to News Feed
Contact: Joshua Wenderoff, email@example.com, 202.540.6542
Washington, DC - 03/09/2011 - The Pew Campaign on Human Health and Industrial Farming (HHIF) today applauded U.S. Representative Louise Slaughter (D-N.Y.) for introducing H.R. 965, the Preservation of Antibiotics for Medical Treatment Act of 2011 (PAMTA). The bill amends the Federal Food, Drug, and Cosmetic Act to withdraw seven classes of antibiotics critical for treating infections in humans from use on industrial farms unless animals or herds are sick with disease."The overuse of antibiotics in food animal production is contributing to the emergence of drug-resistant superbugs," said Laura Rogers, HHIF's project director. "Rep. Slaughter has again shown great leadership and vision by introducing PAMTA. We look forward to working with Rep. Slaughter and her colleagues on both sides of the aisle to solve this health care problem."The American Medical Association, American Academy of Pediatrics, World Health Organization and other leading medical groups all warn that the overuse and misuse of antibiotics in food animal production presents a serious and growing threat to human health because it creates new strains of dangerous, drug-resistant bacteria. As antibiotics lose their effectiveness, infections will become more difficult to treat, more costly and increasingly deadly.According to a task force led by the Centers for Disease Control and Prevention, the U.S. Food and Drug Administration and the National Institutes of Health, antibiotic-resistant bacteria could render diseases like pneumonia, meningitis and tuberculosis untreatable.


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Ryutenmon (龍天門)
In a caliber of its own, Ryutenmon's delicious cuisine has been recognized by the gourmet Michelin Tokyo Guide 2010 and 2011 with a highly esteemed Michelin star. Famous for its fresh seafood and vegetables in dishes that highlight seasonal delicacies, as well as dim sum and an à la carte menu highlighted by a steamed fish of the day, Ryutenmon serves Cantonese-style Chinese at its best.
"Ryutenmon" means "Heaven's Dragon Gate" in Cantonese, an auspicious name which Chief Chef Deng DeSheng credits with the many successes the restaurant has celebrated and the many great moments it has shared with its guests. The restaurant's dramatically lit atmosphere, featuring ornate Chinese décor in the rich hues of black, gold and blue, is luxurious yet intimate and tasteful.
One of the hotel's most popular restaurants, Ryutenmon was also awarded Best Chinese Restaurant in Tokyo by Tokyo's Best Restaurant Guide in 2007 and 2008. The restaurant is located on our second floor and accommodates 142 guests, with smoking and non-smoking areas available. Ryutenmon also offers five lavish private dining rooms with capacities of 10 to 36 persons.
Ryutenmon (龍天門)
Cucina: | Cinese |
Atmosfera: | Refined, low lighting, Chinese interior |
Codice di abbigliamento: | Casual raffinato |
Chef: | Head Chef Deng DeSheng |
Zona fumatori: | Sì |
Parcheggio: | Hotel Parking |
ore: | Monday - Friday 11:30 AM - 3:00 PM; Saturday - Sunday 11:30 AM – 4:30 PM; Daily 5:30 PM – 9:30 PM |
Telefono: | (81)(3) 5423 7787 |


text:
Unfortunately, your access has now expired. But there's good news—by subscribing today, you will receive 22 issues of Booklist magazine, 4 issues of Book Links, and single-login access to Booklist Online and over 135,000 reviews.
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March 15 2014 BOOKLIST
January 2014
Titles similar to The Dovekeepers
Hoffman makes ancient history live and breathe in this compelling story, set in 70 CE, detailing the siege of the mountain stronghold Masada, where 900 Jews held out for months against the Romans. Hoffman's novel follows four extraordinary women. Red-haired Yael has long been shunned by her father, a renowned assassin, because of her mother's death in childbirth. Forced to flee from Jerusalem, she makes a tortuous journey across the desert, during which she becomes involved with a married man, and after finally reaching Masada, is assigned to the dovecote, where she meets three charismatic women: Revka, a baker's wife who witnessed her daughter's horrific death at the hands of Roman soldiers; Shirah, a tattooed wisewoman; and Shirah's daughter Aziza, a warrior of uncommon skill. Forced to deal with the outside forces intent on eradicating them and with their people's patriarchal system, which is quick to condemn unconventional behavior, the women draw great strength from their own inner resources and from each other. This is both a feminist manifesto and a deeply felt tribute to courageous men and women of faith, told with the cadence and imagery of a biblical passage.
HIGH-DEMAND BACKSTORY: The latest novel from the best-selling and prolific Alice Hoffman will be suppported by a national ad campaign, an author tour, and a reading-group guide. Will the biblical subject matter—quite a change of pace for Hoffman—prove as popular as her domestic dramas? Stay tuned.


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Welcome to the website for the Greenleaf Singers, a singing ensemble based in St. Louis, MO. The Greenleaf Singers' repertoire consists mostly of Renaissance
music, specifically 4-part madrigals. Founded in 1976, the ensemble averages 12 to 16 singers.
The mission of the Greenleaf Singers is as follows:
The ensemble's main performances each year are at the Renaissance Madrigal Theatre and Christmas Rout for the Eliot Unitarian Chapel in Kirkwood, MO, the St. Louis Shakespeare Festival and the St. Louis Renaissance Faire in May and June.
The links above provide more information about the Greenleaf Singers. Enjoy your stay!


text: LEGO Marvel Super Heroes offers an original storyline in which Nick Fury calls upon Iron Man, the Hulk, Thor, Spider-Man, Wolverine and other heroes spanning the Marvel Universe to save Earth from such threats as the vengeance of Loki and the hunger of Galactus, Devourer of the Worlds. LEGO and Marvel fans will enjoy classic LEGO videogame adventure and humor, while playing as their favorite Marvel characters. "We're very excited to bring a myriad of classic Marvel characters to life in LEGO form for some fun, family-friendly gameplay in LEGO Marvel Super Heroes," said Tom Stone, Managing Director, TT Games Publishing. "Players will brave the ultimate mission to save the world as they explore famous landmarks in LEGO New York and take the adventure beyond Earth to iconic locations in the Marvel Universe."


text:
Current Students
Faculty & Staff
Business Partners
Contact Us
Campus Life
University Libraries
Collections and Departments
Copyright and Intellectual Property Office
About the CIPO
» University Copyright Support
The University Copyright Center: Supporting Students, Faculty, and Staff
The University Copyright Center is a valuable resource for the Ball State University community. This unit, which is a part of the University Libraries, is one of only three copyright-focused programs offered by the universities of Indiana to their academic communities. The other two are at IUPUI and Purdue University.
The Center's function is to inform faculty and educate students and staff in the lawful use of someone else's intellectual property. This is an important faculty concern for face-to-face classroom presentations and distance education classes. The Center's Manager provides training and presentations in intellectual property (IP).
In today's classrooms, faculty are both aware and involved in a new way of considering copyright." Out" are the restrictive legalities of using copyrighted materials and "in" is the practical approach of using copyrighted materials for classroom instruction. The University Copyright Center assists our faculty with this paradigm shift by being involved in a variety of approaches for making IP available for meeting instructional objectives, classroom enhancements, instructional strategies, research, and integrating technology and IP in the classroom.
The Center advances its responsibilities through nine, practical approaches.
1. The Center maintains a website, which includes a wide selection of resources in a variety of textual and audiovisual formats. Some of the instructional assets of the Center's Homepage include video spots; royalty free music, graphics, and podcast resources, PowerPoint presentations and FAQs, among others.
2. The Center offers unique services for students, faculty, and staff. For students, personalized classroom presentations are crafted on a wide variety of topics including copyright basics, fair use, peer-to-peer file sharing, and legal MP3s, and added a
Copyright for Students
Faculty have taken advantage of the Center's capacity to assist in the understanding that there is a significant role for those who teach our future and practicing teachers to incorporate a practical, useful approach to copyright in their classrooms. For faculty, special presentations have included "Fair Use or Misuse?" "Blackboard and the Copyright Law," and "The TEACH Act: What IP can you use in your Distance Education course?" Other unique services provided by the Manager involve securing musical performances for the Bracken Matinee Musicales, serving as the media consultant for the College of Fine Arts, and suggesting Digital Library Initiative collaborative projects to enhance the availability of digital learning objects.
3. The Center has several types of unique content including two nationally recognized 30-second videos. One is on the need for copyright instruction in the classroom and the second is on using the royalty-free picture databases.
4. The Center can be involved with advisement to the Ball State committees that involve IP. For example, these might include the University Senate's Publications and Intellectual Property Committee and the Presidential appointment Patent and Copyright Committee. The Center is always available to committee members for intellectual property concerns.
5. The Center's Manager promotes the Center and proper uses of intellectual property by serving as a resource in various capacities throughout the state and the country.
6. The Office organizes an annual
copyright conference
hosted at Ball State, featuring nationally-recognized IP attorneys.
7. The Manager serves as a resource person who is responsible for various specialized, intellectual property duties, such as Federal Copyright Law compliance, serving as the U.S. Copyright Office's Agent for Ball State, and the person who authorizes fair use for e-Reserves for the University Libraries.
8. The Manager is available to present at national IP conferences and in local and regional fora.
9. The University Copyright Center supports the School of Extended Education's courses, including any and all requirements for intellectual property. As copyright becomes more critical in academe, the need for an academic, intellectual property-focused copyright center grows in importance to avoid misusing someone else's intellectual property.
The University Copyright Center is a valuable resource for the Ball State University community. This unit, which is a part of the University Libraries, is one of only three copyright-focused programs offered by the universities of Indiana to their academic communities. The other two are at IUPUI and Purdue University.
The University Copyright Center is at the forefront of working with faculty by providing instructional approaches, including the tactical deployment and the amplification of the proper use of intellectual property.
For answers to your questions, please contact
Fritz Dolak
at the University Copyright Center.
About the CIPO
University Copyright Support
Fritz Dolak, D.A., M.L.S.
Request a Copyright Presentation
Fritz Dolak
Copyright and Intellectual Property Office
Bracken Library, Ball State University
Muncie, IN 47306
View E-mail Address


text:
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40 Street New York/NYC Subway/7 SignWall sign
Item #: 15180841525A
In 1998, the name "Lowery" was removed from the station and maps, but was restored in 2004 as part of a historical move. The 1999 artwork featured at the station is called "Q is for Queens" by Yumi Heo.
This sign is made to order in the USA Price
Other ways to order:
Call Us: 1-800-952-5592 |
30-DAY RETURN POLICY If for any reason you are not completely satisfied with your purchase, you can receive a replacement or refund within 30 days Learn More
Ship time indicates the typical time it takes for your item(s) to leave our facilities. This includes any framing or customizing services ordered as well as careful packaging to prevent damage in transit.
Items that ship same day normally leave our facilities on the same business day if your order is placed before 5:00pm EST. Orders that contain multiple items with different ship times will be shipped out based on the item with the longest ship time. Shipping FAQ Shipping Rates |
© Art.com Inc. All Rights Reserved. | Privacy Policy | Terms of Use | Terms of Sale
Art.com, You+Art, and Photos [to] Art are trademarks or registered trademarks of Art.com Inc.
Various aspects of this website are covered by issued US patent No. 7,973,796 and other pending patent applications.


text:
Sometimes I get burnt out. Who doesn't? I'm very sensitive to it though because I know it can be devastating to a startup, especially a single founder startup.
Over the past several years I've learned that a burnout antidote, at least for myself, is rapid prototyping of something brand new. It could be a skunk works project for the startup, but more often than not, it is something just off the wall.
Actually, it doesn't even have to be software related at all. Recently I cleaned out and organized our basement.
I didn't think too much about this process (except for that it works for me) until the other day. Upon reflection, I think why it works is the satisfaction of pushing a minimum viable product out the door.
It's fun and you're left with a great sense of accomplishment. And since it isn't your main thing (startup), you don't feel you have push past that mirage 80% completion and spend the rest of your life on the final 20%.
That's why working on a project related to your startup often doesn't work for this solution. First off, it can't be incremental because then you don't get the satisfaction. But even if it is something brand new, you could feel compelled to keep working on it, especially if it hits users. Now that may not be a bad thing at all; it just may not help with burnout.
The other thing I've learned to stave off burnout as much as possible is to get a lot of user feedback. Ideas and suggestions from real users really motivates me.
Update: also some good comments on HN.
I'm the Founder & CEO of DuckDuckGo.
More about me.
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text:
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5.1 FL OZ | Price: US$12.00
How to use
Massage Birch Body Scrub with circular motions to the body with a loofah or hands and rinse thoroughly. For best results, use in combination with Birch Cellulite Oil.
gently exfoliates and smoothes the skin
An extract of organic birch leaves, known for having purifying and detoxifying effects, promotes radiant skin that looks and feels completely refreshed. Vegetable-based cleansers free of synthetic detergents ensure even sensitive skin gets gently cleansed. Natural plant wax pearls and pure organic and natural plant oils massage your skin, supporting skin renewal without drying or disturbing your skin's natural balance. Organic sesame seed oil and apricot kernel oil, rich in essential fatty acids, promote radiant skin that feels and looks refreshed. Your skin soaks up our unique complex of skin-replenishing nutrients and feels naturally refined and silky smooth. Gently exfoliate rough surface skin to reveal the regeneration of a pure, naturally refined, beautiful you.
Key Ingredient
Birch (Betula Alba) Leaf Extract
Ingredients (INCI)Water (Aqua), Coco-Glucoside, Sesamum Indicum (Sesame) Seed Oil, Squalene (Squalene), Disodium Cocoyl Glutamate, Beeswax (Cera Alba), Copernicia Cerifera (Carnauba) Wax, Hydrogenated Castor Oil, Prunus Armeniaca (Apricot) Kernel Oil, Glycerin, Fragrance (Parfum)*, Chondrus Crispus (Carrageenan) Extract, Betula Alba (Birch) Leaf Extract, Ruscus Aculeatus (Butcher's Broom) Root Extract, Rosmarinus Officinalis (Rosemary) Leaf Extract, Xanthan Gum, Lactic Acid, Limonene*, Linalool*, Citral*.* from natural essential oils
Customer Testimonial:
Just great!! Thank you!
» Submit your product review or testimonial!


text:
Really solid. And a nice quick meal. I didn't have pimientos, so I added a touch of chili powder to the mushroom/bean mixture. Really balanced out the balsamic flavor. Used pepper jack cheese which also gave it a wee bit more kick. Served with pico de gallo, guacamole and sour cream. Will definitely add this to the regular week night rotation.
Portobello and Black Bean Quesadillas
Photo: Oxmoor House
Sautéed portobellos are delicious in ragouts; as a topping for polenta or pizza; or as a filling for fajitas, tacos, or quesadillas. The portobello mushroom stands up to the robust flavors in these quesadillas, all the while preserving its own steaklike texture.
Prep: 1 minute; Cook: 14 minutes
Yield: 4 servings (serving size: 3 wedges)
Recipe from
More From Oxmoor House
Recipe Time
Cook Time:
Prep Time:
Nutritional Information
Amount per serving
- Calories: 251
- Calories from fat: 32%
- Fat: 9g
- Saturated fat: 3.7g
- Monounsaturated fat: 0.8g
- Polyunsaturated fat: 1.8g
- Protein: 18.9g
- Carbohydrate: 25.8g
- Fiber: 7.5g
- Cholesterol: 10mg
- Iron: 2.3mg
- Sodium: 695mg
- Calcium: 318mg
- 4 (8-inch) fat-free flour tortillas
- Butter-flavored cooking spray
- 2 (4 1/2-inch) portobello caps, chopped
- 2 tablespoons light balsamic vinaigrette
- 1 cup no-salt-added black beans, rinsed and drained
- 1 (2-ounce) jar diced pimiento, drained
- 1 cup (4 ounces) preshredded reduced-fat 4-cheese Mexican blend cheese
- 1/4 cup thinly sliced green onions
- Salsa (optional)
- Chopped fresh cilantro (optional)
- 1. Stack tortillas; microwave at HIGH 1 minute. Leave in microwave to keep warm while preparing filling.
- 2. Heat a large nonstick skillet over medium-high heat. Coat pan with cooking spray. Add mushrooms; sauté 2 minutes or until tender. Add vinaigrette, beans, and pimiento; cook 1 to 2 minutes, stirring constantly, or until liquid evaporates. Mash bean mixture slightly with a potato masher.
- 3. Spoon about 1/3 cup bean mixture onto each tortilla. Sprinkle evenly with cheese and onions. Fold tortillas in half.
- 4. Wipe skillet with paper towels; heat over medium heat. Coat pan with cooking spray. Place 2 quesadillas in pan; cook 2 to 3 minutes on each side or until golden and cheese melts. Repeat with remaining 2 quesadillas. Cut each quesadilla into 3 wedges. Serve immediately with salsa and cilantro, if desired.
Portobello and Black Bean Quesadillas Recipe at a Glance
- COURSE: Main Dishes
- CONVENIENCE: Quick/Easy
- MAIN INGREDIENT: Beans, Vegetables
- DIETARY CONSIDERATION: Meatless
- PUBLICATION: Oxmoor House
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Portobello and Black Bean Quesadillas
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