The 256-slice CT enables the entire brain to be scanned in a single examination. We evaluated the application of 256-slice whole-brain CT perfusion (CTP) in determining graft patency as well as investigating cerebral hemodynamic changes in Moyamoya disease before and after surgical revascularization.t test or matched-pairs signed-ranks test.Thirty-nine cases of Moyamoya disease were evaluated before and after surgical revascularization with 256-slice CT. Whole-brain perfusion images and dynamic 3D CT angiographic images generated from perfusion source data were obtained in all patients. Cerebral blood flow (CBF), cerebral blood volume (CBV), time to peak (TTP) and mean transit time (MTT) of one hemisphere in the region of middle cerebral artery (MCA) distribution and contralateral mirroring areas were measured. Relative CTP values  were also obtained. Differences in pre- and post- operation perfusion CT values were assessed with paired P<0.01 for all). All graft patencies were displayed using the 3D-CTA images. Postoperative CBF, rCBF and rCBV values of surgical side in the region of MCA were significantly higher than those before operation . Postoperative MTT, TTP, rMTT and rTTP values of the surgical side in the region of MCA were significantly lower than those before operation .Preoperative CBF, MTT and TTP of potential surgical side were significantly different from those of contralateral side (The 256-slice whole-brain CTP can be used to evaluate cerebral hemodynamic changes in Moyamoya disease before and after surgery and the 3D-CTA is useful for assessing the abnormalities of intracranial arteries and graft patencies. Moyamoya disease is characterized by a chronic progressive steno-occlusive vasculopathy affecting the terminal internal carotid arteries This study was approved by the local ethical committee , and written informed consent forms were obtained directly from patients or the next of kin. From August 2009 to October 2010, 39 subjects  with Moyamoya disease confirmed by DSA according to Suzuki's (1969) criterion were included. 39 patients with Moyamoya disease including 30 cases of ischemic-type and 9 cases of hemorrhagic-type were studied before surgery and after surgery. The surgical treatment procedures of direct superficial temporal artery-middle cerebral artery (STA-MCA) bypass combined with encephaloduromyosynangiosis were performed in the side of ischemic hemisphere. The patency of the anastomosis was verified using intraoperative ultrasonic Doppler.The imaging protocol consisted of whole brain CTP, dynamic 3D CT angiography (CTA) before and after surgery. All subjects were evaluated with 256-slice CT. CTP studies were performed in the transverse plane by using a 256-slice CT scanner  before and after surgical revascularization. A 50-ml bolus of nonionic contrast media  was administered into an antecubital vein by using a power injector  with an injection rate of 5 ml/s. CT scanning was initiated 5 seconds after the start of the injection with the following acquisition parameters: JOG mode(back and forth toggle move of the table),120 kV tube voltage,125 mAs,5 mm slice thickness,128×0.625 mm collimation,0.4 s rotation time,1.9 s cycle time, 22-cm field-of-view (FOV),512×512 image matrix size, 24 slices. Helical scanning was not performed. A total of 312 slices were obtained with a scan time of about 50 seconds and 120 mm scan length. Brain standard reconstruction was performed with the CT system. The gantry angle was parallel to and above the orbital roof to avoid radiation exposure to the lens.surgical side/absolute CTP values contralateral side.The whole-brain plain images, dynamic 3D-CTA and whole-brain perfusion images were reconstructed from source data acquired with dynamic scans. The dynamic 3D-CT angiographic images were generated from volumetric data acquired in the arterial, capillary, and venous phases of CTP source data. The 3D volume-rendered images of cerebral arteries performed at the peak of the arterial time-attenuation curve were the same as conventional CTA images using advanced vessel analysis software installed in the CT workstation. CTP data were analyzed using brain perfusion software . The software relies on the central volume principle to calculate perfusion parameters from the time-concentration curve. The software applies curve fitting by a least-mean squares method to obtain mathematical descriptions of the time-attenuation curves, and the mean transit time (MTT) map was calculated by a closed-form (noniterative) deconvolution operation from the time-concentration curve of a particular voxel and the arterial input function (AIF). The first artery to reach peak enhancement on the time-attenuation curve was selected as the AIF. The vein with the largest area under the time-attenuation curve was selected as the venous outflow function. For each voxel, the cerebral blood volume (CBV) map was calculated from the areas under the time-concentration curves. The cerebral blood flow (CBF) map for each voxel was finally calculated according to the following equation combining the CBV and MTT value: CBF = CBV/MTT. Large cortical vessels were automatically excluded via brain perfusion software. Two experienced neuroradiologists independently drew 2 standardized elliptical mirrored regions of interest (ROIs) manually on the basal ganglia (BG) section level of the reference CT image  over the25–P75). Differences in pre- and post- operation perfusion CT values were assessed using paired t test and t values were obtained, while skew distribution of measurement data were assessed with matched-pairs signed-ranks test and z values were derived. Values with P<0.05 were considered statistically significant. All raw data were analyzed using statistical software .For CTP parameters, the mean of ROI values on each ipsilateral and mirrored contralateral hemisphere was calculated before and after surgery. The preoperative mean ROI values between ipsilateral and mirrored contralateral and mean ROI values in surgical side between pre-surgery and post-surgery were compared in all 39 cases. Normal distribution data were expressed as mean±SD while skew distribution of measurement data were expressed as median  . Postoperative MTT, TTP, rMTT and rTTP values of surgical side were significantly lower than those before operation  (P>0.05).Preoperative CBF, MTT and TTP of potential surgical side were significantly different from those of contralateral side  . The CBFfor all) . Postopectively) . HoweverMoyamoya disease is a cerebral arterial angiopathy characterized by chronically progressive stenosis and occlusion of the intracranial internal carotid arteries and their proximal branches together with the formation of collateral vessel Studies have reported early diagnosis of Moyamoya coupled with the expeditious institution of therapy is of paramount importance. Revascularization surgery appears to be effective in preventing stroke in patients with Moyamoya disease Many techniques such as conventional angiography, transcranial Doppler, MRI, CTA, CTP, Xe-CT, PET, and SPECT with acetazolamide challenge have all been used in the evaluation of patients with Moyamoya. However, each technique has its own advantages and drawbacks. In terms of evaluating hemodynamic status in Moyamoya disease, SPECT has been considered a reference standard The 256-slice CT, which enables the entire brain to be scanned in a single examination with administration of one contrast medium bolus, does not have the spatial coverage limitations that other multi-detector row CT systems possess and can be used to obtain the whole-brain perfusion data. Meanwhile, in our study, the whole-brain dynamic 3D-CTA images were reconstructed from source data acquired with dynamic scans. Both quantitative method of measuring cerebral hemodynamic and CTA images were performed in one examination. Whole brain perfusion imaging can be performed to ascertain the occurrence and the extent of ischemia, and CTA can be performed to investigate the morphologic status of cerebral arteries. Although cerebral angiography is considered the 'gold standard' imaging modality for diagnosis and postoperative follow-up of Moyamoya disease, its invasive nature has a number of limitations including procedural risks, costs, and limited availability that makes it less desirable than CTA for screening for intracranial stenosis and occlusion or following patients with these conditions. CTA has relatively fewer risks, costs less, is more readily available and appears to be highly accurate, suggesting that this approach may be preferred for identifying patients with intracranial stenosis and occlusion Patients with Moyamoya disease have reduced CBF and increased CBV, MTT and TTP. These hemodynamic changes in Moyamoya disease are explained by decreases in the cerebral perfusion pressure and associated vasodilation There were limitations in the present study. First, the radiation exposure is unavoidable in perfusion CT, the radiation dose is increased because of repeat scans. Further research is needed to determine whether the radiation dose can be reduced. Recently, an attempt was made to reduce the radiation dose using iDose technology with relatively low-dose conditions The second limitation concerns AIF. In the CBF calculations with CT perfusion imaging, the choice of a reference artery is critical. An artery of the anterior circulation such as anterior cerebral artery (ACA) or MCA is usually selected as reference artery automatically. However, in a patient with Moyamoya disease, ACAs and/or MCAs might be occluded. Therefore, we propose that it is optimal to select basilar artery or posterior cerebral artery (PCA) as reference artery manually, because PCA is hardly affected by revascularization surgery The third limitation concerns the accurate drawing of MCA territories before and after revascularization surgery. Drawing the same level and size of ROI was challenging The 256-slice whole-brain CTP and 4D-CTA have the potential for the non-invasive assessment of the abnormalities of intracranial arteries, the graft patency and cerebral perfusion changes in Moyamoya disease before and after surgery. The postoperative volumetric CTA acquired in the arterial phase of whole-brain CTP could display the graft patencies instead of conventional CTA; meanwhile reducing the radiation exposure in a single examination.


Hemoglobin variability (Hb-var) in patients with chronic kidney disease has been stipulated to be a result of exogenous treatment with erythropoiesis stimulating agents (ESA) and has been related to mortality in dialysis patients. We hypothesized the existence of Hb-var independent of ESA administration and compared it to that in healthy adults using data from the Scripps-Kaiser and NHANES III databases. We studied the Hb-var in 1571 peritoneal dialysis patients which included 116 patients not requiring treatment with erythropoietin. We systematically studied the differences between the groups that needed ESA therapy and those who did not. White race and male sex were significant predictors of need for erythropoietin therapy. We found peritoneal dialysis patients to exhibit significantly increased Hb-var independent of treatment with exogenous erythropoietin . We found age to be a significant determinant of Hb-var in the ESA treated group. Hb-var in younger patients (<30 years) was increased by 50% compared to young healthy adults. The Hb-var in elderly (>60 years) peritoneal dialysis patients was similar to that seen in healthy elders, suggesting similarity with anemia of aging. We conclude that exogenous ESA administration does not explain Hb-var entirely but may enhance it. Intrinsic factors affecting erythropoiesis including age may be the major determinants of Hb-var. The basal rate of Red Blood Cell (RBC) production is diminished in patients with chronic kidney disease (CKD). Usually, the anemia is proportional to the severity of renal dysfunction, caused due to a deficiency of erythropoietin (EPO) Data on Hb-var in peritoneal dialysis (PD) patients are limited to small reports from the Netherlands This was a retrospective observational study. The study protocol was approved by the University of Florida Institutional Review Board and adhered to the declaration of Helsinki. A limited data set consisting of de-identified data was provided by Dialysis Clinics Inc. through a data use agreement. It was not possible to identify individual patients directly or through identifiers linked to the patients. Therefore, it was exempted from requiring informed consent by the IRB. Prevalent peritoneal dialysis patients receiving services for 6 months or longer at any of the centers operated by Dialysis Clinic Inc., a large nonprofit dialysis provider in the United States between January 2007 and December 2009 were included in this study. Children less than 18 years of age were excluded from the study population. Demographic data including - age, sex, race, dialysis vintage were included in the data provided. Date of clinic visit, Hb measured per clinic protocol (monthly); ESA dose administered, types of ESA used were also recorded. Laboratory values obtained during routine clinical care including serum iron, ferritin, TIBC, percent saturation , serum Albumin, PTH, and Kt/V measured per clinic protocol were recorded.All data analysis was carried out using STATA v.9.0. Patients were stratified into those requiring any ESA therapy (n = 1455) and those not requiring ESA therapy (n = 116). Patients who were administered even a single dose of an ESA during the study period were stratified in the ESA group. ESA naïve patients did not receive any doses of an ESA during the entire follow-up period. Hb-var was calculated using a 1 month standard deviation (SD-Hb) and the coefficient of hemoglobin variation. The coefficient of hemoglobin variation is defined as the ratio of the SD-Hb to the mean Hb. Results are reported as mean ± standard deviation. Comparisons between groups were based on a two sided student's t-test of means. The patients were divided into deciles of age to compare Hb-var with the population means reported in the Scripps-Kaiser and NHANESIII databases Data was available for a total of 1571 patients followed over a mean 19 months (range 7–156 weeks). The data revealed that 7.4% (n = 116) of our sample did not require any ESA therapy over the entire follow-up period. The two groups did not differ with regards to mean patient age, dialysis vintage and follow up period in the study. The proportion of diabetics in either group was also similar. The group not requiring ESA had a greater proportion of males (69%). The proportion of sexes was equally distributed in the ESA group . Since females may have menstrual blood losses which can partly account for variability in their hemoglobin, males were analyzed separately as well. By limiting the analysis to males only, the results did not change. The male no-ESA group still had a standard deviation of 1.0 gm/dL compared to 1.15 gm/dL in males who received ESA.Patients in the no-ESA group had significantly more residual renal function, perhaps explaining the lack of EPO requirement in this group. The no-ESA group had slightly higher serum albumin and lower ferritins and PTH. Other iron indices  were lower in the ESA treated group. A racial difference was noted in EPO requirement. A greater proportion of whites than blacks (9.1% vs. 4.2%) did not need ESA therapy to maintain similar levels of Hb. Separate subgroup analysis of the no-ESA group by race did not find any significant differences in Hb levels, iron indices, ratio of sexes, dialysis vintage or Kt/V. The black patients had significantly higher PTH overall- an effect that was consistent across the ESA and no-ESA groups.0) or a proliferative state. In dialysis patients the effects of peripheral factors such as varying iron stores and circulating inflammatory mediators such as hepcidin In the absence of a disease condition RBC production is controlled by a negative feedback system. The rate of change of RBC concentration is the difference between their rates of production and destruction. Within the bone marrow there exists a population of committed lineage specific pluripotent stem cells, also called colony forming units (CFU). CFUs are capable of differentiating into mature RBC under the influence of hormone erythropoietin produced by the kidneys. Low density of circulating RBC signals EPO production, whereas an increase in the numbers of circulating RBCs provides negative feedback to EPO. The pluripotent cells in the bone marrow exist in either a resting state (GThe existence of hemoglobin variability in the patients on dialysis not requiring ESA therapy provides useful insights into understanding RBC dynamics in CKD patients. Previous investigations have focused on the relationship between ESAs and Hb-var Peritoneal dialysis patients lack large volume shifts and do not have ongoing blood losses into dialysis tubings and dialyzers, which characterize hemodialysis which can affect Hb-var. Access related blood loss and use of hemodialysis catheters are also known to effect Hb-var The inverse relationship between age and Hb-var in ESA treated hemodialysis patients has been observed previously Higher EPO requirements have been observed in African American patients on dialysis in previous observational studies We conclude that Hb-var cannot be attributed to exogenous ESA administration alone. The physiologic bases of Hb-var are not completely understood. Deregulated homeostatic mechanisms in chronic kidney disease may enhance Hb-var. Hb-var in peritoneal dialysis patients younger than 50 years of age may be 50% greater than in healthy adults. The normal biology of aging may determine Hb-var. Effects of aging such as increased levels of inflammatory mediators and diminished capacity to secrete erythropoietin may mimic chronic kidney disease. The interaction and effects of exogenous ESA administration on these factors remains poorly understood and should be investigated. Identification of these mechanisms may help us identify unique targets for novel therapies. By choosing appropriate physiologic targets for therapy we may be able to improve patient outcomes and enhance our understanding of disease processes.


Mirounga leonina). Using teeth recovered from dead males on îles Kerguelen, Southern Ocean, we first aged specimens. Then we used stable isotopic measurements of carbon . Here we investigated the relationship between foraging strategy and life-span in an elusive and large marine predator: the Southern Elephant Seal ( Diomedea exulans) may live up to Identifying individual factors affecting life-span has long been of interest for biologists and demographers In the case of mammal species, most studies investigating the relationship between longevity and fitness have focused on females Mirounga leonina) is the most dimorphic and polygyneous mammal among extant species. The biology of this elusive carnivore, which can spend up more than The Southern Elephant Seal  model of Studying of the life-span of wild animals is a difficult endeavour: ecological correlates can be uncovered but a large amount of variation usually remains unaccounted for in the analysis Institut Paul Emile Victor - IPEV) approved this study. All animals in this study were cared for in accordance with its guidelines. This study is part of a national research program  supported by the French Polar Institute (IPEV). The approval ID for this study is IPEV research program no. 109, which is evaluated every year by the ethics committee of the French Polar Institute.The ethics committee of the French Polar Institute , Southern Indian Ocean. Canines grow continuously throughout the whole life of males without closing of the pulp cavity, allowing for age determination et al.et al.As a recent study raised concerns about non-linear offsets of organic %C, %N and For measurement of carbon stable isotopes and et al.Data were first analyzed using growth mixture models shape of individuals with the same foraging strategy is identical, which is not reasonable given the variety of profiles observed in For model identification purposes, we constrained Change-point models aim at finding an abrupt rupture in a time-series. The time-series is assumed to be the juxtaposition of piece-wise linear homogeneous segments, each segment separated from the next by a change-point. These models are very flexible as they allow specifying different probability distributions to describe different parts of a time series. Different curve shapes can thus be generated. Change-point models thus seem appropriate to describe ontogenetic shifts Because teeth were sampled from dead animals that were subsequently aged, all survival times are observed: there is no censoring in the data. Denoting The shape parameter xpected  . The parTo assess the appropriatedness of our joint model, we compared it with a null model with with no individual-level covariate;a random-effect model wherein an individual-specific deviation from the mean a mixture model wherein each male was first assigned to a group depending on its mean Strictly speaking, both the random and mixture models are also joint models, as time-series of versus at-sea with a Kolmogorov-Smirnov test. Except for one individual, there was no statistically significant differences . This time-span is commensurate with, if not longer than, the typical haul-out duration of a Southern Elephant Seal. Assuming similar equilibration time for cattle and elephant seals, this may explain why no statistically significant differences were found  to illustrate the different strategies: males mainly forage in the Antarctic Zone, on the Kerguelen Plateau or in Subantarctic waters (waters lying between the Sub-Antarctic Front and the Southern Antarctic Circum-Polar Front). Îles Kerguelen (Ker), and the Antarctic coastline's contour are depicted in black, and while the 2000 metres isobath is depicted in light grey. Dotted lines symbolized fronts (EPS)Click here for additional data file.Figure S2Checking the appropriatedness of the Accelerated Failure Time model. With the BUGS parametrization:versus(EPS)Click here for additional data file.Figure S3Goodness-of-fit of the joint change-point/survival model. The selected model, although being the best in term of predictive performance among the set of competiting model, could be still improved. A Kolmogorov-Smirnoff goodness-of-fit test indicated that the considered covariates were not a sufficient set for these data ((EPS)Click here for additional data file.Figure S4Correlations between the predictors of the Accelerated Failure Time survival model. The large correlations means that these parameters are not independent. Further model checking revealed that only (EPS)Click here for additional data file.Figure S5Histogramm of the observed longevity of males included in the sample.(EPS)Click here for additional data file.Table S1Kolmogorov-Smirnoff tests for comparing dentin layers grown onshore or at sea. For only one individual was there a significant difference. Removing this individual did not change the analysis.(XLS)Click here for additional data file.Text S1WinBUGS code to fit the Growth Mixture Model.(TXT)Click here for additional data file.Text S2WinBUGS code to fit the joint Accelerated Failure Time\Change-point Model.(TXT)Click here for additional data file.


Various spatial pattern analyses applied to the data extracted from the 2-dimensional coordinate positioning of individual bacterial cells at single-cell resolution indicate that microbial colonization within natural biofilms is not a spatially random process, but rather involves strong positive interactions between communicating cells that influence their neighbors' aggregated colonization behavior. Geostatistical analysis of the data provide statistically defendable estimates of the micrometer scale and interpolation maps of the spatial heterogeneity and local intensity at which these microbial interactions autocorrelate with their spatial patterns of distribution. Including in situ image analysis in cell communication studies fills an important gap in understanding the spatially dependent microbial ecophysiology that governs the intensity of biofilm colonization and its unique architecture.This paper describes how the quantitative analytical tools of CMEIAS image analysis software can be used to investigate  The long-range goal is to build a computing toolkit that strengthens microscopy-based approaches for understanding microbial ecology at spatial scales directly relevant to their ecological niches, without the need for cultivation . The sof website . The CME website  includesin situ spatial ecology are designed to scrutinize patterns of distribution at a given spatial scale and produce predictive models of microbial colonization behavior that can help to reveal ecophysiological processes on surface habitats. Of central importance in that assessment are tests for patterns of complete spatial randomness. The essence of the statistical pattern analysis is to distinguish between spatial distributions of the organisms that can be explained by chance versus those that cannot. Complete spatial randomness implies that no microbial interactions affect the events resulting in their patterns of distribution. In contrast, significant deviations from complete randomness in spatial patterns indicate that regionalized microbial interactions between neighboring cells have affected their colonization behavior. Aggregated (clustered) spatial patterns of microbial colonization imply positive interactions that promote growth physiology among neighboring cells, whereas uniform patterns of distribution imply negative (inhibitory) microbial interactions resulting in their maximal, over dispersed, self-avoiding colonization behavior. This information is of significant ecological importance because spatial heterogeneity resulting from both types of nonrandom interactions between individuals tends to stabilize ecological systems [A central goal in spatial ecology is to define what a measured characteristic at one location can reveal about that same variable at neighboring locations. Analyses of  systems ,4. Also,i.e., structured to operate at spatial scales that extend sufficiently to affect neighboring attached cells. Other papers in this special issue of Sensors describe the biochemical and genetic details of bacterial cell-cell communication. This paper describes 18 different experimental tests on data derived from CMEIAS computer-aided microscopy to analyze the spatial ecology of bacterial colonization within biofilms, providing (in the broadest sense) both indirect and direct evidence of bacterial cell-to-cell communications mediated by environmental sensing phenomena and the geospatial scale at which they occur in situ at individual, single-cell resolution.At the core of these positive (cooperative) and negative (conflict) interactions that reduce spatial randomness are various types of microbial cell-to-cell communication events that regulate genes affecting their colonization behavior. The key connection between biofilm spatial ecology and cell-to-cell communication occurs when positive or negative interactions are found to be spatially autocorrelated, 2.2.1.This first experimental system involved many types of spatial pattern analyses to test for microbial interactions indicative of cell-to-cell communication among individual microbes in natural biofilms. Microbial assemblages were allowed to develop on clean glass microscope slides submerged for four summer days in the Red Cedar River on the campus of Michigan State University . Slides were retrieved and their underside wiped clean. The top surfaces of the slides were mounted in water with a cover slip and examined by phase-contrast light microscopy using a 100X Planapochromat Phase 3 objective lens to resolve individual bacterial cells. Digital 8-bit grayscale images of the biofilms were acquired using a monochrome digital camera, then segmented to binary, spatially calibrated and analyzed using CMEIAS image analysis software ,5,6 to pst and 2nd nearest neighbor distances was evaluated using a high-resolution 3-frequency grid distortion target  as ground truth. The accuracy of CMEIAS color segmentation software used to process the color images for analysis was previously measured as 99+% [The accuracy of CMEIAS image analysis software to measure and compute the object centroid-to-centroid 1 as 99+% .2.2.Pseudomonas putida reporter strains during their colonization of tomato roots. Details on construction of fluorescent reporter derivatives of P. putida IsoF serving as the source and sensor strains for these bacterial cell communication studies using N-acylhomoserine lactone (AHL) signal molecules are previously described [P. putida F117 (pKR-C12) has a mutation in its single copy of the ppuI-gene that abolishes its ability to synthesize AHLs, but contains an AHL-inducible reporter plasmid pKR-C12 with a green fluorescent protein (GFP)-encoding sensor cassette. This sensor cassette contains an AHL-regulated promoter for ppuA (encoding acetyl-CoA ligase) coupled to the Gfp gene that responds with high specificity and differential sensitivity to defined AHLs (C10- and C12-HSLs and 3-oxo-C10 to 3-oxo-C14-HSLs) at threshold concentrations as little as 10 nmol/L [The second experimental system was designed to further advance our understanding of bacterial cell-to-cell communication during their colonization of plant roots. CMEIAS image analysis was used to reevaluate the spatial scale of calling distances and the variations in intensity of gene expression activated by extracellular signal communication molecules produced by neighboring cells of escribed –16. BrieP. putida at 109 cells per plant, grown gnotobiotically, harvested and examined by laser scanning microscopy in the epifluorescence confocal mode [in situ spatial relationships and luminosity of segmented red-fluorescent AHL-source and green-fluorescent AHL-sensor cells colonized on the root surface. To avoid errors in measurement of in situ calling distances and gene expression, communicating cells attached to root hairs and cells located above or below the optisections used to prepare the montage of X | Y projected images were excluded from analysis, as was done in our previous study [Axenically grown seedling roots were inoculated with both the AHL-producer and AHL-sensor strains of cal mode . Georefecal mode , and anacal mode ,5,6 to eus study .3.3.1.st and 2nd nearest neighboring cell, the cumulative Empirical Distribution Function of the 1st nearest neighbor distance, and the CMEIAS Cluster Index that measures the clustering intensity of each cell in relation to its local environment [st and 2nd nearest neighbor distances used to compute these spatial attributes were 3.2% and 2.3%, respectively, with an overall combined accuracy of 97.2% (n = 38).ironment ,17. The st and 2nd nearest neighbor distances and cluster indices of cells in these biofilms were significantly skewed and lacked normality  for complete randomness in spatial distribution of cells. These 4 tests indicate spatially aggregated patterns when the corresponding upper class limits of their indices computed from the 1st and 2nd nearest neighbor distances are >0.5, <1.0, <1.0 and >1.0, respectively. The results of all of these point pattern tests rejected the null hypothesis of spatial randomness in favor of the alternative hypothesis of aggregated distributions, and the intensity of this colonization behavior was significantly higher in the CS25 biofilm sample  bacterial cell-to-cell interactions affecting their colonization behavior over the entire spatial domain. High values of this metric extracted from surface-attached cells indicate that they are arranged in spatially aggregated patterns that facilitate cell communications resulting in positive metabolic cooperations promoting their localized growth into populations of microcolony biofilms [The cluster index, computed as the inverse of 1st nearest neighbor distances, is a sensitive, local biofilms ,17,20.in situ in the CS4 and CS25 biofilms. This pseudocolored classification scheme indicates a preponderance of well-dispersed cells and a few small clusters (cluster indices < 0.4) in the CS4 biofilm  maps of the local intensity of the measured Z-variate parameter within that spatial domain, based on analysis of its weighted average from sampled locations. From the microbial ecology perspective, cellular interactions that are autocorrelated with spatial patterns of microbial distribution within biofilms statistically indicate that communication events (in the broadest sense) significantly influence the in situ colonization behavior (cooperation vs. conflict) of neighboring cells over a defined scale within the spatial domain. At the core of geostatistical analysis is the variogram plot, which defines the extent to which the measured Z-variate exhibits spatial dependence between sample locations. It does so by examining how the values of the Z-variate become different as the spatial separation between the sample points increases. In this example, geostatistics using a simulation analysis of 10,000 subsamples were performed to test for connectivity of each georeferenced cell's cluster index values to their spatial patterns within the 2 biofilm image domains. Semivariance analyses of the clustered indices of the bacteria in the CS4 and CS25 biofilms indicated that this regionalized variable positively autocorrelated with the spatial pattern of distribution  is spatial autocorrelated, tructure . The anaribution , and Morribution .in situ spatial scale of the separation distance at which bacterial cells exhibit positive, autocorrelated clustering behavior was more than 10-fold longer for cells in the CS25 biofilm sample  and a maximal in situ separation ‘calling distance’ of up to 78 μm . In addition, individual bacteria in small clusters (2–3 cells) communicated with each other even when separated from dense populations by even longer distances. Thus, during colonization of plant roots, one individual bacterium is able to produce sufficient AHL signal molecules to communicate with another single bacterial cell neighbor even when those two cells are separated apart by a wide range of distances. Geostatistical modeling of local spatial densities predict that AHL-mediated cell-to-cell communication is governed more by the in situ spatial proximity of cells within AHL gradients than by a quorum requirement of high population density, and therefore this type of cell-to-cell interaction is likely to be more commonplace in biofilms than originally thought [It is generally thought that high bacterial population densities are needed to exceed the threshold level of AHL signal concentrations required to activate genes and their physiological functions. Hence, this specific type of microbial communication has become known as ‘quorum sensing’ that functions primarily as sensors of high population density, thus optimizing the expression of functions that are most beneficial when simultaneously performed by dense populations. Despite its wide appeal, this quorum sensing paradigm has been challenged since the methods commonly used to detect it nstrated . Recentlnt roots . These s thought . Follow- thought ,24 have in situ evidence of AHL-mediated cell-to-cell communication during bacterial colonization of a tomato root is provided in st nearest neighboring (red-fluorescent) AHL-source cell  differentiated the intensity of autocorrelated spatial structure in aggregated colonization behavior between two natural freshwater biofilms. Two other tests using the second type of sensor  indicated the range of calling distances between individual bacterial cells in situ, and predictably, that autocorrelated colonization behavior facilitates positive metabolic cooperations that promote their growth into microcolonies [Spatial ecology studies reported here confirm and expand upon previously acquired data ,23,24 incolonies ,20. The colonies .Future work should continue to further test and validate these ecophysiological models of cell communication and colonization behavior by microbes at single-cell resolution during dynamic stages of biofilm development, including their spatiotemporal redistribution within various natural habitats.


However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency  Tumor-initiating cells (TICs), often termed cancer stem cells, are functionally defined by their capacity to re-grow a new tumor after transplant into experimental animals that recapitulates the phenotype of the primary tumor from which the cells were isolated, and which can be serially transplanted thus demonstrating their capacity to differentiate and self-renew Studies of human breast TICs are challenging for a number of reasons. Breast tumors are generally small at the time of resection thus providing relatively few bulk tumor cells for TIC purification and analyses Mouse mammary TICs have been identified in p53-null mice and in transgenic mice genetically engineered to overexpress Wnt-1 or Neu (Erbb2 or Her-2) in their mammary epithelium neu proto-oncogene We employed several mouse models of human breast cancer, where the mouse mammary tumor virus (MMTV) promoter directs transcription of the rat in vitro to derive TIC-rich or -poor cell populations for comparative studies.We determined the TIC fraction in multiple mammary tumors arising in each of the models and found that, independent of the model, all the tumors comprised between 15% and 50% TICs. Culture of primary tumor cells from tumors of the MMTV-Neu model in serum-free medium, conducive for stem cell self-renewal ad libitum. All animal experiments were approved by the McMaster University Animal Research Ethics Board (AUP: 10-01-04).All mice used in these experiments were housed in an Canadian Council on Animal Care (CCAC)-approved facility at McMaster University. Mice were provided with food and water http://wehi.edu.au/software/elda/index.htmlWe have reported the methods used to isolate, process and transplant mammary epithelial cells and tumor cells Mammary fat pads from recipient mice were spread onto glass slides, allowed to air-dry and subsequently stained in Harris Haematoxylin . Excess stain was removed from the whole mounts by washes in 70% ethanol and 1% hydrochloric acid, and were dehydrated in 100% ethanol, cleared in xylenes and mounted on glass slides with Permount (Fisher Scientific). Stained whole mounts were examined on a Leica Diaplan  dissecting microscope and photographed.Tumors were fixed in 4% paraformaldehyde  for 24 hours at room temperature, embedded in paraffin, cut into 4 µm sections and stained with Harris Haematoxylin and Eosin (H&E) solution. All images were captured using a Leica inverted microscope (Leica) and Open Lab Improvision software .The expression of mammary epithelial cell lineage markers in tumor sections was determined as described previously Freshly isolated primary tumor cells were placed in serum-free medium comprising Dulbecco Modified Eagle Medium (DMEM): Ham's F-12 (3∶1), 4 μg/ml of B-27, 20 ng/ml Epidermal Growth Factor (EGF), 40 ng/ml Fibroblast Growth Factor-2 (FGF-2) and 4 ng/ml Heparin, and plated into ordinary T25 tissue culture flasks  to generate tumorspheres 4 or 105 tumor cells. Small tumor-like masses, which we termed tumor nodules, were found near the cell injection site, corresponding in size to the number of transplanted tumor cells  strain that express the wild type rat Neu cDNA in mammary epithelial cells bona fide tumors, we repeated the transplants with cells from a different mammary tumor and sacrificed the recipient hosts 4 or 16 weeks thereafter. Single tumor cells, visualized microscopically prior to transplant, were injected into syngeneic female mice. Tumor nodules were found in many of the recipients 4 weeks after cell transplantation as observed previously  and those to each of two myoepithelial-lineage markers (CK14 and α-SMA) were used to characterize the cellular composition of the tumors. The morphology and cellular composition of the mammary tumors in the Neu (3) comprising ∼5,000 tumor cells from the single cell-seeded tumors from each of the three models formed secondary tumor grafts in over 80% of transplanted hosts, which could be similarly serially transplanted for up to 10 successive passages before this experiment was discontinued (data not shown). Hence the tumors of all three breast-cancer prone mouse models comprised TIC capable of recapitulating the phenotype of the primary tumors from which they were isolated and were capable of self-renewal, even when the tumors originated from transplant of single tumor cells.To determine whether the tumors resulting from single cell transplants comprised self-renewing TIC, we performed serial transplants of the engrafted tumors High TIC frequency, lack of an apparent cellular hierarchy and the inability to prospectively enrich TIC from bulk tumor cell populations using antibodies to a diversity of cell-surface proteins are features that have been ascribed to tumor types that do not follow the cancer stem cell model The cancer stem cell model predicts that experimental conditions facilitating stem cell self-renewal will increase or maintain TIC frequency, whereas those stimulating differentiation will reduce their frequency. Consequently, we propagated freshly-isolated primary tumor cells in either serum-free medium, which facilitates the survival and self-renewal of human or mouse mammary epithelial stem/progenitor cells as non-adherent mammospheres We also compared the frequency of TIC in three additional independent mammary tumors with that in tumorspheres derived from these same tumors. The tumorspheres were passaged 3–4 times during a 2–3 week period to limit any potential genetic changes in the sphere-resident cells that might alter TIC frequency. Mice transplanted with dilutions of the dispersed tumorsphere-derived cells were sacrificed 2–4 months after cell transplantation. in vitro culture of TIC-rich primary tumor cells in serum-containing medium for a relatively short period drastically reduced TIC frequency during a time when the tumor cell population expanded.To assess any effect of culturing primary tumor cells in serum-containing medium on TIC frequency, we propagated freshly isolated primary tumor cells from 3 independent tumors that occurred in the MMTV-Neu strain in this medium. In pilot experiments we found that the primary tumor cells adhered and proliferated resulting in a net expansion of the cell population for 4–6 serial passages, but the total tumor cell population subsequently declined with each successive passage and these cultures could not be sustained beyond 7–9 serial passages. In consequence, we limited propagation of the primary tumor cells to 3 serial passages at 4-day intervals, a protocol similar to that used to propagate tumorspheres. We observed that independent of the tumor source between 10,000 and 100,000 tumor cells that had been propagated in serum-containing medium were required to elicit a new tumor after transplant into syngeneic female mice  to separate hematopoietic, endothelial and stromal cells from tumor cells prior to their transplantation, whereas all previous studies employed FACS to enrich for tumor cells We used Matrigel as a vehicle to enhance TIC engraftment, whereas none of the other studies used this agent. Matrigel reportedly facilitates the engraftment of mouse mammary epithelial stem and progenitor cells and may have similarly stimulated the engraftment of TIC and progenitor-like tumor cells in our transplantation experiments The procedure we used to prepare dispersed tumor cells differed from those of previous studies; indeed none of the published studies used the same conditions to dissociate tumor cells from tumor tissue. Previous work employed only collagenase 8 cells prior to the removal of tumor cell aggregates by filtration through a 40 µm sieve; ∼95% of these cells were viable. After filtration through the sieve ∼20% of the cells were recovered in the filtrate; ∼95% of these cells were viable. The latter cell yield is in keeping with that reported previously from the mammary glands of mice using the Pullan and Streuli protocol It is conceivable that the conditions we used to prepare tumor cells may have led to the selective recovery of TICs after filtering the bulk tumor cells through a 40 µm sieve to remove undigested tumor fragments and tumor cell aggregates. The yield of tumor cells from a 1–2 gram tumor arising in the MMTV-Neu (N202) strain approximated ∼ 2–3×10It is also not clear whether the extent of progression of the tumors analyzed in the various studies influenced TIC frequency. Multiple different TICs may be present during the early stages of tumorigenesis, but clonal evolution may occur during tumor progression resulting in the selection of a TIC population with an increased propensity to self-renew and a decreased capacity to differentiate The question arises as to whether the mammary tumors occurring in the transgenic mice we examined follow the cancer stem cell model especially in light of their high TIC fraction. All the mouse breast cancer models studied previously with the sole exception of the MMTV-Neu transgenic mice have been suggested to follow this model In keeping with the cellular heterogeneity of the tumors in the mouse models, the use of antibodies to mammary epithelial stem cell surface markers together with FACS have permitted sorting mammary tumor cells into tumorigenic and non-tumorigenic fractions. A very minor tumorigenic cell population can be separated from the bulk, non-tumorigenic tumor cell fraction using FACS in the MMTV-Wnt-1 model, analogous to the MMTV-β-catenin model we studied In light of the very high TIC frequency in the mouse mammary tumors we studied, the unavailability of markers that might facilitate fractionation of TIC from non-tumorigenic cells in the MMTV-Neu model in vitro and cannot rule out the possibility that TICs simply do not survive in serum-containing medium.We found that the TIC frequency of the mammary tumors in the MMTV-Neu model could be maintained by culturing freshly isolated tumor cells in chemically-defined, serum-free medium, but that propagating these cells for a similar period and passage number in serum-containing medium reduced the TIC fraction by roughly 4 orders of magnitude. It is noteworthy that only 1% of primary tumor cells form tumorspheres, and that this fraction of sphere-forming cells does not vary during serial passage in vitro in either growth condition for only 12 days it also seems unlikely that genetic changes account for their wide difference in TIC frequency.It seems unlikely that TIC were diluted by the expansion of non-tumorigenic cells in serum-containing medium because the net increase in total cell number was roughly 10-fold, whereas the decline in TIC frequency was 10,000 fold or greater. The possibility that fibroblasts present in the primary tumor cell preparations overgrew the epithelial tumor cells during their propagation in serum-containing medium seems unlikely because the vast majority of the cells growing in serum-containing medium appeared to be epithelial in morphology independent of their passage history. Because the tumor cells were cultured in vitro may afford an opportunity to identify TIC biomarkers and TIC-targeted therapeutic agents, especially in view of the technical limitations inherent in sourcing human breast TIC to this end. Indeed we have used the mouse mammary tumorspheres and corresponding normal mouse mammospheres to identify TIC-selective compounds At this juncture is not clear what specific factors account for the difference in TIC fraction between tumors arising in the same mouse models reported by different laboratories, or whether the various models studied follow the cancer stem cell model. Whereas resolving these matters have therapeutic implications and consequently needs to be critically examined, the high TIC frequency in mouse mammary tumors and the capacity to maintain TIC-rich or TIC-depleted tumor cell populations Our results demonstrate that mouse mammary tumors, including those arising in transgenic strains thought to conform to the cancer stem cell model, can comprise a very high TIC frequency that approach 50% in some tumors. Moreover, we show that a similarly high TIC frequency can be maintained by propagating the tumor cells in serum-free medium as non-adherent tumorspheres. By contrast, culturing the tumor cells for an identical period in serum-containing medium resulted in a substantial reduction in TIC frequency. These various tumor cell sources provide a rich source of tumorigenic or non-tumorigenic tumor cells for comparative analyses.Figure S1A), Histology of a primary mammary tumor from an MMTV-Neu transgenic mouse. (B), Histology of a tumor seeded by transplant of tumor cells from the tumor shown in panel A that had been propagated in vitro in serum-containing medium. H&E staining of tumor sections illustrates the cytoarchitecture characteristic of Neu-induced tumors. Scale bar (inset) represents 40 µm in all panels.Tumors arising from transplant of tumor cells propagated in serum-containing medium recapitulate the histology the parental tumor. ((TIF)Click here for additional data file.


DNA methylation and repressive histone modifications cooperate to silence promoters. One mechanism by which regions of methylated DNA could acquire repressive histone modifications is via methyl DNA-binding transcription factors. The zinc finger protein ZBTB33  has been shown in vitro to bind preferentially to methylated DNA and to interact with the SMRT/NCoR histone deacetylase complexes. We have performed bioinformatic analyses of Kaiso ChIP-seq and DNA methylation datasets to test a model whereby binding of Kaiso to methylated CpGs leads to loss of acetylated histones at target promoters.Our results suggest that, contrary to expectations, Kaiso does not bind to methylated DNA in vivo but instead binds to highly active promoters that are marked with high levels of acetylated histones. In addition, our studies suggest that DNA methylation and nucleosome occupancy patterns restrict access of Kaiso to potential binding sites and influence cell type-specific binding.We propose a new model for the genome-wide binding and function of Kaiso whereby Kaiso binds to unmethylated regulatory regions and contributes to the active state of target promoters. Genes are epigenetically regulated by a combination of histone modifications and methylation of CpG dinucleotides near their promoters ,2. PromoIn addition to repressive histone methylation modifications, active versus inactive promoters can also be distinguished by the acetylation status of histone H3. Active promoters are marked by both acetylated lysine 9 and acetylated lysine 27 on histone H3 (H3K9Ac and H3K27Ac). In fact, H3K9Ac is the modified histone most highly associated with transcriptional activity, as shown in the integrative analysis of a large number of datasets by the ENCODE consortium . AlthougA model has been proposed in which Kaiso binds to methylated DNA in a promoter region, recruits components of the NCoR histone deacetylase complex, and represses transcription Figure B. This mWe began by downloading the sequenced tags for all Kaiso ChIP-seq datasets from the UCSC browser (from the ENCODE consortium); two replicate Bowtie-mapped ChIP-seq datasets were downloaded for GM12878, K562, A549, HepG2, HCT116, and SK-N-SH cells. Peaks were called for each replicate in each cell type using Sole-Search . AlthougAfter determining the quality of the Kaiso ChIP-seq datasets, the next step was to select a defined peak set for further analysis. Because of the nature of the ChIP-seq assay, there is not a discrete number of peaks for any transcription factor. Rather, as more reads are acquired, more and more peaks are identified. The peaks that are only identified when large numbers of reads are used tend to be much smaller than the median height of the peaks identified when less than 10 million reads are analyzed. The issue of small peaks is especially problematic when two ChIP-seq replicate datasets (each of which having more than 10 million reads) are merged. As shown in Figure As described above, Kaiso has been shown to bind to methylated DNA and has been suggested to function as a transcriptional repressor via recruitment of HDACs via interaction with the NCoR and SMRT complexes ,10,15-18To investigate the mechanism(s) by which Kaiso functions on a genome-wide scale, we first determined the location of the high-confidence Kaiso ChIP-seq peaks in GM12878 cells relative to transcription start sites using the Sole-Search location analysis tool. We found that approximately 70% of high-confidence Kaiso peaks bind within 1 kb of a transcription start site of a Refseq gene Figure A. A simiTo characterize the epigenetic profiles of the Kaiso binding sites, tag density plots were created to identify enrichments of specific histone modifications proximal to Kaiso binding sites. Tag density plots provide a more accurate view of the relationship of different ChIP-seq datasets than do simple peak overlap analyses. This is because all tags are used in these analyses, eliminating any user-selected cut-offs. ChIP-seq datasets for the various histone modifications, Pol2, CTCF, and Sin3A in GM12878 cells were downloaded from the UCSC browser and compared to the Kaiso peak genomic coordinates. Consistent with the overlap analysis of Kaiso peaks and Pol2 peaks shown above, Pol2 ChIP-seq tags were highly enriched at Kaiso peaks Figure A. AnalysAs a third method of characterization of the Kaiso binding sites, we determined if Kaiso binds to promoters having high levels of DNA methylation. Using whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) data from GM12878 cells, we determined the DNA methylation profile of the sequences surrounding the Kaiso binding sites. As shown in Figure If Kaiso binds to a short region of highly methylated DNA that is not exactly in the middle of a peak having an overall low level of DNA methylation, then it is possible that a relationship between Kaiso binding and DNA methylation is obscured when the analyses are centered on the middle of the genomic regions identified by the peak-calling program. To alleviate this concern, the DNA methylation analyses can be centered on the Kaiso binding motif. Kaiso has been shown to bind to a methylated motif TCTCGCGAGA in vitro and in vivo , and an The analyses of Kaiso binding sites described above were performed using GM12878 cells, which is an EBV immortalized normal lymphoblastoid cell line. Because Kaiso binds to promoter regions, it is possible that the lack of correlation between Kaiso binding and DNA methylation is simply due to the fact that most CpG island promoters are hypomethylated in normal cells. In contrast, in cancer cells although the majority of the genome is hypomethylated, many promoters become hypermethylated. Thus, it is possible that there are more methylated TCTCGCGAGA motifs in cancer cells, providing a platform for Kaiso to bind to methylated DNA. To determine if Kaiso binds to methylated DNA in a cancer cell, we analyzed Kaiso binding sites in the myeloid leukemia cell line, K562. Kaiso was determined to be similarly expressed in GM12878 and K562 by Western blot and by RNA-seq data (see Methods). We first identified a set of Kaiso binding sites that were present in both replicates of the K562 Kaiso ChIP-seq datasets. The number of these sites was much higher than the number of GM12878 Kaiso binding sites and therefore we performed further inspection of the K562 peaks. We noted that a much lower percentage of the K562 Kaiso peaks contained the Kaiso motif than did the GM12878 peaks (12% versus 43%), suggesting that the K562 peak set may contain a large number of false positive peaks. K562 cells are cancer cells and have a number of highly-amplified genomic regions. Although our peak-calling program has features that remove many of the false positive peaks due to genomic amplifications ,12, we oTo determine if Kaiso binds to methylated promoters in K562 cells, we selected the set of Kaiso peaks that contained a CGCG motif and determined the methylation percentage of each of the CpG dinucleotides. In this case, we used RRBS data for DNA methylation analysis because WGBS data is not available for K562 cells. The genomic coverage is lower in the K562 data than in the GM12878 data; the number of CpGs with 3× coverage or greater in the K562 RRBS dataset is 1 million, compared to 45.6 million in the GM12878 methylation data for which we could use a combination of WGBS and RRBS. Therefore, with a 3× coverage cut-off, we could only analyze 1,069 CGCG motifs in the K562 Kaiso peaks. Of these, 28 peaks contained medium levels of DNA methylation and only two peaks contained high levels of methylation  the median tag height for the cell type-specific binding sites is lower than for the sites occupied by Kaiso in both GM12878 and K562 cells  may not be bound by Kaiso in K562 cells if the promoter is highly methylated and densely packed by nucleosomes. To address whether differences in DNA methylation are responsible for cell type-specific binding to promoters in GM12878 versus K562 cells, we determined the methylation status in GM12878 cells of the K562-specific Kaiso peaks and the methylation status in K562 cells of the GM12878-specific peaks Figure . We founLastly, we have used RNA-seq data from GM12878 and K562 to examine the relationship between Kaiso binding and gene expression. We identified the genes nearest to the Kaiso binding sites that were in common between the GM12878 and K562 Kaiso ChIP-seq datasets and the genes that had Kaiso bound to their promoter regions only in GM12878 or only in K562 cells. We then determined the expression levels of these three sets of genes in both GM12878 and K562 cells  [Two replicate Bowtie-mapped ChIP-seq datasets were downloaded for GM12878, K562, A549, HepG2, HCT116, and SK-N-SH from the UCSC browser. All Kaiso ChIP-seq experiments were carried out in the Myers Lab using Santa Cruz Biotechnology antibody sc-23871. The antibody has been validated to be specific to Kaiso as required by ENCODE standards , using Whttp://hgdownload.soe.ucsc.edu/admin/exe/macOSX.i386/. HOMER [To determine the DNA methylation status of the genomic regions corresponding to Kaiso peaks, a BigBed file for GM12878 WGBS data was downloaded from the UCSC genome browser and was converted to BED format using the BigBedtoBed script available at /. HOMER  was thenhttp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCaltechRnaSeq/). A union file was created by combining all transcripts for individual genes with multiple transcripts. For both cell types, all replicates were combined for analysis. All reads were aligned to the hg19 reference genome using the bowtie aligner, and those uniquely mapped were used to calculate the expression level as reads per kilobase per million reads (RPKM), using the following formula:The RefSeq gene annotation files for RNA-seq data in GM12878 and K562 were downloaded from the UCSC genome browser , the Mann-Whitney rank test was applied.Bp: Base pairs; ChIP: Chromatin immunoprecipitation; EBV: Epstein-Barr virus; HDAC: Histone deacetylase; HOMER: Hypergeometric Optimization of Motif EnRichment; MNase: Micrococcal nuclease; RPKM: Reads per kilobase per million reads; RRBS: Reduced representation bisulfite sequencing; WGBS: Whole genome bisulfite sequencing.The authors declare that they have no competing interests.AB carried out the analyses and interpretation of ChIP-seq and MNase-seq data and drafted the manuscript. LY performed analyses of the DNA methylation, YW performed LONUT alignments and BELT peak calling, ZY performed the analysis of RNA-seq data, VXJ coordinated LONUT and BELT ChIP-seq data analysis and RNA-seq data analysis, and PJF conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors have read and approved the final manuscript.Chromosomal locations and peak scores for Kaiso peaks sets in all cell lines.Click here for filePeak selection for Pol2 in GM12878 cells. Shown are the peaks called using Sole-Search for the merged replicate datasets of Pol2 in GM12878 (panel A). The parameters used in the Sole-Search peak calling were as follows: permutation of 5, fragment length of 150, alpha value of 0.0010, an FDR of 0.0010, and a peak merge distance of 0. The top 20% of the called peaks were used (tag height > 50). The arrow indicates the position at which the peak list was truncated. Panel B shows the location analysis for Pol2 peaks that were used in our analysis (red) and those that were discarded (blue).Click here for fileHOMER Annotation of Kaiso peak files in GM12878 and K562, including locations of Kaiso motifs.Click here for fileChromosomal location and percent methylation of CGCG motifs within GM12878 and K562 Kaiso peaks.Click here for fileMotif analysis of subsets of K562 Kaiso peaks identified in highly amplified genomic regions. As shown, the Kaiso motif is not highly represented in the removed peaks. The adjusted high-confidence peak set is used for analyses in this paper. However, the genomic coordinates of the retained and removed peaks are provided in Additional file Click here for filePeak files used in the removal of amplified regions in K562 cells.Click here for fileMethylation of Kaiso peaks in K562. Snapshot of the region on chromosome 6 containing the highest methylated CGCG motif identified within Kaiso K562 high-confidence peaks. The ChIP-seq track for Kaiso is shown in black, with called peaks represented by black bars below the track. ChIP-seq tracks for Pol2 and histone modifications are shown in blue. The inset shows a zoom in of the region bound by Kaiso containing a methylated CGCG motif. Red bars in the RRBS track represent methylated cytosines.Click here for fileMotif analysis of cell type-specific peaks lacking a Kaiso motif. (A) K562 Kaiso peaks that lack a Kaiso motif were searched for known motifs for other TFs using Homer. (B) GM12878-unique distal Kaiso peaks that lack a Kaiso motif were searched for known motifs for other TFs using Homer.Click here for filePlot of DNA methylation versus peak rank. The methylation status of each CGCG motif within Kaiso peaks was calculated using WGBS and RRBS data  and RRBS , and plotted relative to the score of the peak containing the motif. The left panel shows high-confidence Kaiso peaks in GM12878 and the right panel shows high- confidence Kaiso peaks in K562.Click here for fileMethylation of Kaiso peaks identified by LONUT. (A) Comparison of methylation levels within peaks called by different programs. Peaks were called by BELT using non-uniquely mapped tags (left) and uniquely-mapped tags (middle) that were mapped using LONUT. These peaks were compared to Sole-Search-called peaks (right) for methylation of CGCG motifs. Red arrows indicate CGCG motifs identified in the promoters of the PHF8 and PRDX4 genes. (B) Genome browser snapshot of the region surrounding the PHF8 gene, where a CGCG motif is 73% methylated in GM12878 cells. ChIP-seq tracks for Pol2 and histone modifications are shown in red and the Kaiso ChIP-seq track is shown in black.Click here for fileMTA2 promoter showing no DNA methylation and active promoter marks in HeLa cells. Genome browser snapshot showing the MTA2 gene. The region analyzed for Kaiso-binding and DNA methylation in Yoon et al. is represented by the black box under the red arrow. RRBS tracks for several cell types are shown in green, red, and yellow, but is absent in all cell lines for the region in question. ChIP-seq density tracks for Pol2 and histone modifications in HeLa cells are shown in grey-scale.Click here for file


Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs) and the Somatic Gonadal Precursor cells (SGPs). The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. To reach the SGPs, the PGCs must traverse the midgut wall and then migrate through the mesoderm. A combination of local repulsive cues and attractive signals emanating from the SGPs guide migration. We have investigated the role of the hedgehog (hh) pathway gene shifted (shf) in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. We show that production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, we present evidence indicating that ectopic expression of hmgcr in the nervous system promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification.The  Drosophila melanogaster results in embryonic gonad formation consisting of primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs). Moreover, it likely involves a complex series of attractive and repulsive cues. Molecular and genetic analysis has been performed to elucidate the nature of the attractive cue(s) and components that guide germ cells to the SGPs in the mesoderm. One current model proposes that 3-Hydroxy-3-Methylglutaryl Coenzyme A reductase (Hmgcr), synthesized in the SGPs, potentiates signaling downstream of Hedgehog (Hh) ligand also emanating from the SGPs. The model pivots on the novel activity of an established morphogen, Hedgehog, to function as a chemoattractant for the migrating germ cells. A variety of ‘loss-’ and ‘gain-of-function’ strategies manipulating different components of this signaling pathway have been successfully employed in support of the proposed model.The molecular mechanisms underlying directed cell migration have been studied extensively in different biological contexts. Germ cell migration provides an effective model to study the dynamics of in vivo cell migration. The process of germ cell migration in  In this model, most of the Hh-Np that reaches the apical surface is recaptured and internalized by an endocytic pathway that depends upon dynamin and Rab5. Instead of being targeted for lysosomal degradation, the Hh-Np endosomes are recycled by a Rab8 dependent mechanism into exocytic vesicles that are targeted to the basolateral membrane of the hh expressing cells. It is possible that Disp, with its sterol sensing domain, plays a role in selecting Hh-Np containing endocytic vesicles for recycling to the basolateral membrane. Disp is found co-localized with Hh-Np in endocytic vesicles near the apical surface and along the basolateral membrane. It is also in an immunoprecipitable complex with Hh-Np. Two proteins previously implicated in facilitating the response of cells receiving the Hh signal, the Dally-like protein (Dlp) and Interference Hh (Ihog), also seem to participate in the targeting and/or release of Hh-Np from the basolateral membrane.Recent studies by Callejo et al. hh expressing cells hmgcr), contains a transmembrane sterol-sensing domain like disp. HMGCoA reductase is responsible for the synthesis of mevalonic acid, which is a precursor for isoprenoid and sterol biosynthesis, and is a key regulatory target in both biosynthetic pathways −hmgcr mutant embryos, Hh-Np is not properly released from sending cells and instead accumulates in punctate aggregates along the basolateral membranes. Unlike vertebrates, flies are unable to synthesize cholesterol from mevalonic acid hh signaling because it is a precursor for the synthesis of the isoprenoid geranylgeranyl-pyrophosphate by geranylgeranyl diphosphate synthetase (qm). Like hmgcr, qm mutations impede the release of Hh-Np from hh expressing cells hh signaling, a key target for geranylation is the G protein γ subunit 1 (Gγ1) Other proteins have been implicated in the basolateral release and transmission of Hh-Np from Many of the factors implicated in the basolateral release and subsequent transmission of Hh-Np in polarized epithelium also function in the signaling pathway(s) that directs migration of primordial germ cells (PGCs) to the somatic gonadal precursor cells (SGPs) during mid-embryogenesis hh or other hh pathway genes like hmgcr, qm and gγ1 function in migration remains unresolved and controversial shifted (shf) gene. shf encodes an extracellular protein that is the Drosophila ortholog of the vertebrate Wnt Inhibitory Factor-1 (WIF-1) wnt signaling by capturing Wnt and anchoring it to the heparan sulfate proteoglycan hh pathway. The Shf protein is enriched in the basolateral ECM, and it facilitates the long distance transmission of Hh-Np after it has been released from the basolateral membranes of signaling cells. In shf mutants, the basolateral accumulation and subsequent spreading of Hh-Np in the wing disc is disrupted, while apical accumulation appears to be relatively normal. These defects in Hh-Np transmission result in a reduction in the expression of hh targets in the anterior compartment of the wing.While it is generally agreed that an attractive signal(s) produced by the SGPs is needed to guide PGC migration, the identity of the attractant(s) and how hh signaling in the wings of shf mutants can be suppressed by overexpression of hmgcr. Since hmgcr plays a pivotal role in generating the PGC attractant shf seen in the wing could also be of significance in PGC migration. Here we have first tested whether shf functions in PGC migration using both ‘loss’ and ‘gain’ of function strategies. We have then used genetic ‘epistasis’ experiments to link shf and the hh pathway gene gγ1 to the functioning of the hmgcr→qm isoprenoid biosynthetic pathway in PGC migration. Finally we use co-expression experiments to show that hmgcr potentiates the transmission of hh-GFP out of the nervous system and into the mesoderm, where it associates with PGCs.Guerrero and colleagues shf is expressed during embryogenesis, shf mutant embryos have no discernable patterning defects. The likely reason for this is that Hh-Np needs to spread only 3–4 cell diameters in the embryonic ectoderm shf is nevertheless able to promote hh signaling during embryogenesis we asked if ectopic shf can upregulate the hh→wingless (wg)→engrailed (en) autoregulatory loop. In this loop, hh expressing cells in each parasegment signal to the neighboring anterior cells to activate wingless (wg) expression. Wg in turn signals back to the row of hh expressing cells and upregulates en expression. To direct shf expression in hh sending cells we used an hh-Gal4 driver, while we used a patched-Gal4  driver to express shf in hh receiving (wg expressing) cells. hh-Gal4 driver (panel B). While driving Shf in wg expressing cells using ptc-Gal4 also appears to upregulate the En stripes, the increase over wild type is less pronounced than with the hh-Gal4 driver. To confirm that shf can potentiate hh signaling in the embryo we examined the accumulation of Wg in hh-Gal4:UAS-shf embryos. hh receiving cells is upregulated by expressing shf in hh sending cells.Even though shf could have a role in PGC migration, in which case it should be expressed in the mesoderm. Previous studies indicate that shf mRNA is maternally deposited and is uniformly distributed in blastoderm stage embryos shf mRNA is expressed in a segmentally repeating pattern in the ectoderm. This is shown in the stage 10 embryo in shf expression in the mesoderm (arrow) can also be detected at the posterior. The mesodermal expression is shown more clearly in the stage 12–13 embryo in panel B. Expression in the mesoderm appears to be highest in segments A4–A8, which is the region of the embryonic mesoderm that gives rise to the SGPs, while it is lower in the more anterior segments, which give rise to fat body cells. The upregulation of shf expression in more posterior segments would fit with recent studies of Zhai et al. shf is regulated by the Bithorax complex gene, Abdominal-B. It should be noted that though shf seems to be preferentially expressed in the posterior mesoderm during the period when PGCs are migrating, its pattern of expression is much broader than hmgcr, which at this stage is only expressed in a subset of mesodermal cells, the SGPs While long distance signaling might not be essential for patterning of the embryonic ectoderm, attractants produced by the SGPs must travel from the posterior mesoderm to the midgut in order to direct migrating PGCs. If Hh-Np functions as a PGC attractant, then shf is needed for proper PGC migration. For this purpose we examined three different shf alleles, 2shf, x33shf and EY03173shf. 2shf has a missense mutation in the third EGF repeat, replacing a conserved cysteine (Cys374) with a serine. 2shf flies are viable and fertile; however, their wings display the typical shf wing phenotype (i.e. a narrowing of the L3–L4 intervein space) x33shf mutant has a 630 bp deletion and is considered a null allele x33shf flies show slightly stronger shf wing and eye phenotypes than 2shf. Finally EY03173shf is a P-element insertion into the middle of the shf transcription unit. It exhibits the shf wing and eye phenotypes and is semi-lethal.We next asked whether shf embryos. However, as shown in trans-heterozygous 2shf/x33shf combination, migration phenotypes are evident by the end of stage 13. At this point in wild type embryos, all of the PGCs have migrated from the midgut to the mesoderm and are aligned with the SGPs. By comparison, in shf mutants, a subset of the PGCs lingers behind on the outside surface of the midgut. Interestingly, a similar though more pronounced lingering phenotype is observed in hmgcr embryos. By the time of gonad covalence, we find that over 40% of the shf embryos have 5 or more PGCs scattered in the posterior as compared to two or less for wild type (shf mutants (not shown).We found that the early steps in PGC development from their formation through stage 12 are unaltered in ild type . Similarshf in PGC migration, we tested the effects of ectopic expression. Previous studies have shown that misexpression of genes that clearly cannot encode the attractant itself  and gγ1) can disrupt the migration of PGCs towards the SGPs To further substantiate a role for shf also belongs to this special group of genes, we used hh-GAL4 and ptc-GAL4 drivers to ectopically express shf. Since shf ectopic expression using the hh-GAL4 driver was more effective in promoting hh signaling in the embryonic ectoderm than the ptc-GAL4 driver, we anticipated that any effects on PGC migration would likely be stronger with hh-GAL4. ptc-GAL4:UAS-shf embryos had three to four mislocalized PGC, the vast majority resembled wild type  but are not expressed and/or not required in the nervous system.In order to misdirect migrating PGCs, the attractant generated in the nervous system by ectopic hmgcr, have a greater effect on the attractant from the SGPs than on the ectopic attractant. However, it also possible that the factors encoded by these genes might be more limiting for the production or transmission of the ectopic attractant in the nervous system than they are in the SGPs. In this case, reducing their dose should suppress rather than enhance the effects of ectopic hmgcr on PGC migration. For these experiments we selected shf and a second gene gγ1 whose relationship to hmgcr is well defined. The Gγ1 protein must be geranylated in order to be active, and in hh signaling it is an important target for the hmgcr→fpps→qm isoprenoid biosynthetic pathway. hmgcr are mitigated when the embryos are heterozygous for two different gγ1 alleles. Whereas about 60% of the elav-GAL4,UAS-hmgcr embryos have 10 or more scattered PGCs, this number drops to less than 25% for both gγ1 alleles. This finding would argue that gγ1 must also function downstream of hmgcr in the production or transmission of the ectopic attractant. This conclusion would fit with previous studies which showed that ectopic expression of a dominant negative Gγ1 protein that can't be geranylated and poisons the heterotrimeric GαGβGγ1 complex disrupts PGC migration. We next tested shf. Like gγ1 the number of embryos with 10 or more scattered PGCs is reduced, going from 60% to 20%. This finding would argue that production or transmission of the attractant induced at ectopic sites by hmgcr expression depends upon a second hh signaling gene shf.For signaling genes whose expression is not restricted to the SGPs or the mesoderm, reducing their dose could, like hmgcr and hh pathway genes in the production of an ectopic PGC attractant in the nervous system, we examined the effects of co-expression. For this purpose females carrying a recombinant chromosome containing both elav-GAL4 and UAS-hmgcr were mated to males carrying UAS transgenes for shf, ttv and gγ1. As a positive control we used UAS-hmgcr, as we already had evidence that its effects on germ cell migration were dose dependent. hmgcr. Adding a second copy of UAS-hmgcr essentially eliminates the intermediate migration phenotypes, and almost all embryos have greater than 10 scattered PGCs. ttv, gγ1 and shf in the nervous system also exacerbates the effects of ectopic hmgcr; however, none is equivalent to an extra copy of hmgcr.To further explore the functional connections between hmgcr with wild type Hh-Np or the C-terminal truncation, Hh-N, which lacks the cholesterol modification. In polarized epithelial cells ectopic hmgcr would be expected to promote the basolateral release of Hh-Np and enhance its long distance transmission hmgcr should have less of an effect on the release or transmission of the partially modified Hh protein Hh-N, which is thought to signal most effectively apically. While it is not clear how the subcellular organization of the secretion machinery in neurons might be related to that in polarized epithelial cells, the hmgcr-dependent PGC attractant must nevertheless be released from neuronal cells in a manner that enables it to move readily into and then through the underlying layer of mesodermal cells so that it can contact the migrating PGCs. If a key function of hmgcr in generating the ectopic attractant in neurons is to enhance the release and transmission of Hh into the underlying mesodermal layer then the same distinction should hold in PGC migration. In this case, co-expression of wild type Hh-Np would be expected to exacerbate the hmgcr induced migration defects, while co-expression of Hh-N should have more modest effects. On the other hand, if the germ cell attractant generated by ectopic hmgcr is completely independent of hh, then co-expression of either Hh ligand should contribute nothing to the hmgcr induced PGC migration defects.We next co-expressed hh or hh-N expression in the nervous system on PGC migration. UAS-hh transgene in the nervous system using an elav-GAL4 driver has a greater effect on PGC migration than a UAS-hh-N (3nd chromosome) transgene encoding the truncated and partially modified Hh-N protein. We next examined the effects of co-expressing Hmgcr with either Hh-Np or Hh-N. rd) and Hmgcr leads to only a small increase in the number of mismigrated PGCs. To confirm this finding, we tested a second UAS-hh-N transgene (2nd chromosome). It gave comparable results. In contrast to the UAS-hh-N transgenes, a transgene expressing wild type Hh-Np collaborates with hmgcr, substantially augmenting the effects of ectopic hmgcr on PGC migration. In fact, the effects of the Hh-Np transgene are just about equivalent to that seen with two copies of hmgcr.To test these predictions we first compared the effects of driving twi-GAL4 driver. A similar result is obtained when the elav-GAL4 driver was used to direct Hh-GFP expression in the embryonic nervous system  the differences between hh-GFP and hh-GFP/hmgcr embryos become more pronounced. This is illustrated in hh-GFP and hh-GFP/hmgcr embryos; panels C and D show views of the surface of stage 14 embryos; while panels E and F show cross sections of stage 14 embryos. The same embryos, together with a series of plots of the average pixel density across several ∼3 cell wide sections (as indicated), are shown in To determine whether Hmgcr promotes the transmission of Hh into the underlying tissue, we compared the Hh-GFP distribution in embryos expressing this fusion protein in the nervous system with or without Hmgcr. We first detect Hh-GFP in the developing PNS in stage 9–10 embryos, while it subsequently comes on in the developing CNS in stage 11–12 embryos. At these early stages the level of Hh-GFP is relatively low, and the differences in Hh-GFP distribution between hh-GFP and hh-GFP/hmgcr embryos are apparent. First, the overall level of Hh-GFP is elevated in the hh-GFP/hmgcr embryos. In scans of several similarly staged and oriented embryos there was a ∼1.8–2.0 fold difference in the Hh-GFP signal between hh-GFP and hh-GFP/hmgcr. Since different elav-GAL4 transgenes (one on the 2nd and the other recombined with UAS-hmgcr on the 3rd) were used to drive UAS-hh-GFP expression, one explanation for this difference is that the recombined elav-GAL4 transgene drives a higher level of expression. To test this we used the two elav-GAL4 transgenes to express GFP carrying a nuclear localization signal. As shown in hmgcr stabilizes or otherwise promotes Hh accumulation. Consistent with this explanation, we found that the level of endogenous Hh-Np in the ectoderm increased to a similar if not greater extent when hmgcr was ectopically expressed using a hh-GAL4 driver hh-GFP embryos. In contrast, Hh-GFP is found not only in the nervous system but also in the adjacent ectoderm  hmgcr seems to play a rather similar role in the release/transmission of Hh-Np from hh sending cells, in this case through the geranylation of Gγ1, an important and controversial question is whether the functioning of the hmgcr→qm biosynthetic pathway in hh signaling has any connection to the generation of the PGC attractant. We have addressed this question by determining if the PGC migration defects induced by hmgcr expression in the nervous systems depend upon Gγ1 and Shf. We find that mutations in both gγ1 and shf dominantly suppress the migration defects induced by ectopic hmgcr. In contrast, reducing the dose of the hmgcr gene dominantly enhances the migration defects induced by hmgcr expression in the nervous system. This later finding is expected since reducing hmgcr activity in the SGPs should further compromise the ability of the attractant generated by the SGPs to compete with the attractant generated in the nervous system. The former findings show that the production/activity of the attractant generated in the nervous system by ectopic hmgcr depends on both gγ1 and shf. By themselves, these results do not exclude the possibility that gγ1 and shf only collaborate with hmgcr when it is ectopically expressed in the nervous system while they are not actually needed for the hmgcr-dependent production of the attractant by the SGPs. However, this scenario seems unlikely. For one, there are PGC migration defects in gγ1 and shf mutant embryos. For another, the Gγ1 protein must be geranylated to function in PGC migration hmgcr, ectopic expression of gγ1 and shf in the mesoderm and ectoderm perturbs PGC migration.Previous studies have established that a rate limiting step in generating the PGC attractant either by the SGPs or by other tissues and cell types is the biosynthesis of geranylgeranyl-pyrophosphate by geranylgeranyl diphosphate synthetase  produced by the SGPs. If Hh-Np is the relevant endogenous PGC attractant, then even subtle deficiencies in the activity of the chimeric Hh-GFP ligand would be expected to compromise its ability to compete with the wild type protein. It would also follow that it should be possible to “rescue” ectopic Hh-GFP by enhancing its activity. This is the case. While hh-GFP is not very active on its own, it is able to collaborate with hmgcr when co-expressed in the nervous system.Though an imperfect mimic of Hh-Np, we have taken advantage of a chimeric Hh-GFP fusion protein to analyze the effects of Hmgcr on the transmission of Hh from cells in the embryonic nervous system. We found that Hh-GFP is less effective than Hh-Np (and even Hh-N) in competing with the PGC attractant produced by the SGPs when it is ectopically expressed using the hmgcr in hh producing cells in the ectoderm increases the overall level of Hh protein and enhances its transmission to adjacent cells hmgcr in the nervous system—Hh-GFP levels are elevated, while its transmission into and through the underlying mesodermal cell layer is appreciably enhanced. These hmgcr dependent effects, particularly on the movement of Hh-GFP from the neuroectoderm into the underlying mesoderm, would also provide a plausible explanation for why this biosynthetic enzyme plays such a pivotal role in PGC migration even though it is not directly responsible for the synthesis of the PGC attractant. In the period when PGCs are migrating through the mesoderm, the SPGs are the only cells in the embryo simultaneously expressing both hmgcr and hhhh expressing cells elsewhere in the mesoderm or in the ectoderm. This would provide a mechanism for ensuring that SGP derived Hh-Np out-competes Hh-Np produced elsewhere. Taken together, these findings support the idea that Hh-Np expressed in the SGPs functions as a PGC attractant. With the caveat that the activities of Hh-GFP are not identical to Hh-Np, the fact that Hh-GFP accumulates on the surface and around the PGCs further bolsters this suggestion. Moreover, in a subset of the PGCs Hh-GFP is closely associated with bulges or protrusions that could potentially be of relevance to the process of migration.In previous studies we have shown that expressing hh signal could actually translate into directed movement. The endpoint of the signaling cascade in the canonical pathway is the transcriptional activation of target genes, including the hh receptor ptc. However, transcription is likely not involved in this instance, as ptc reporters are not activated in PGCs hh dependent axonal guidance and fibroblast migration are independent of transcription and involve instead the coupling of Smo activation to pathways that mediate the reorganization of the cytoskeleton hh signaling to the PGCs, changes in the cytoskeleton and directed movement. Another unresolved question is whether SGPs produce any other PGC attractants. Although no other plausible candidates have been identified, our experiments do not exclude the possibility that there are other PGC attractants, even including an attractant(s) whose activity, like Hh-Np, is potentiated by the hmgcr isoprenoid biosynthetic pathway.On the other hand, a number of critical questions remain. For one, it is not clear how reception of the The embryo stainings were performed essentially as described in 21. Vasa (from Paul Lasko) and Hh (from Tom Kornberg) antibodies are rabbit polyclonal antibodies. Both were used at a 1∶500 dilution. Engrailed and Wingless antibodies are mouse monoclonal antibodies and were used at 1∶10 dilution. ß-Galactosidase antibody was either a rabbit polyclonal purchased from Cappel (used at 1∶1000 dilution) or a mouse monoclonal antibody from Developmental Studies Hybridoma Bank (used at 1∶10 dilution). GFP antibody is a rabbit polyclonal purchased from Torrey Pines Biolabs (used at 1∶1000 dilution). For con-focal analysis a magnification of 40× was used in almost all the instances, and images were collected using identical settings for the control and experimental samples. Multiple pairs of wild type (sibs) and mutant embryos were imaged in each case, and representative examples are presented.Shf stocks including mutants and UAS transgenes were obtained from Seth Blair UAS-Hh-GFP fusion stocks were a kind gift of Isabel Guerrero UAS-Hh-Np , UAS-Hh-N: two different stocks on the second (from Tom Kornberg) and third chromosome (from Phil Beachy) respectively, gγ1 mutant stocks, N159gγ1 and gγ1k0817, were obtained from Fumio Matsuzaki, while UAS- gγ1 stock (gγ1) was kindly provided by the Olson lab UAS and Gal4 stocks used for the misexpression studies were from the Bloomington stock center. hairy-Gal4, elav-Gal4, nanos-Gal4, patched-Gal4, UAS-ß-galactosidase, hh-Gal4/TM6 Ubx-LacZ. In most experiments, males carrying two of the copies UAS transgene were mated with virgin females carrying two copies of the Gal4 transgene. The resulting progeny embryos were fixed and stained for subsequent analysis. elav-Gal4, UAS hmgcr recombinant UAS-qm stock Figure S1shf elevates level of Wingless (Wg) accumulation. Females homozygous for the UAS-shf transgene were mated with hh-GAL4/TM6 Ubx-LacZ males. Embryos generated from the cross were collected and coimmunostained with Wg (imaged in red) and β-galactosidase  antibodies. (A) control UAS-shf/Ubx-LacZ. (B) hh-GAL4/UAS-shf.Overexpression of (TIF)Click here for additional data file.Figure S2elav-GAL4 driver inserted on the 2nd chromosome Click here for additional data file.Figure S3-GFP and hh-GFP/hmgcr embryos. Embryos in Distribution of Hh-GFP in stage 13 hh(TIF)Click here for additional data file.Figure S4hh-GFP and hh-GFP/hmgcr embryos. Embryos in Distribution of Hh-GFP in stage14 (TIF)Click here for additional data file.Figure S5hh-GFP and hh-GFP/hmgcr embryos. Embryos in Distribution of Hh-GFP in stage 14 (TIF)Click here for additional data file.Figure S6nd and recombined 3rd chromosome elav-GAL4 transgenes drive equivalent levels of expression of a UAS-GFP(NLS) reporter. Females homozygous for the recombined UAS-hmgcr, elav-GAL4 3rd chromosome or the 2nd chromosome elav-GAL4 driver (elav-GAL4/CyO Actin GFP) were mated to UAS-GFP(NLS) males. The resulting embryos were fixed and stained with anti-GFP antibody. (A) elav-GAL4 (2nd)/UAS-GFP(NLS). (B) UAS-hmgcr,elav-GAL4 (3rd)/UAS-GFP(NLS). The signal intensity in each nuclei was estimated by doing pixel counts over a fixed area using ImageJ software. The average pixel intensity per nucleus for elav-Gal4/UAS-GFP(NLS) was 69.8  whereas for UAS-hmgcr,elavGal4 embryos was 66 .The 2(TIF)Click here for additional data file.Text S1hh misexpression perturbs PGC migration are contradicted by experiments reported in et al.Developmental Biology. The material presented in Does ectopic Hh perturb germ cell migration? The data in (PDF)Click here for additional data file.


Genetic deletion of mouse genes has played a crucial role in our understanding of embryonic eye development. Transgenic, tissue specific Cre recombinase expression in various eye structures has facilitated these experiments. However, an early expressing, temporally-regulated, optic vesicle-specific Cre line has not been available. In this report, we detail the generation and analysis of a knock-in, inducible Cre line designed to drive recombination specifically within the Rx expression domain. Crossing this line with a reporter line demonstrates that recombination can be mediated within the early optic vesicle and throughout retinal development. Recombination can also be mediated in the Rx-expressing, ventral diencephalon and future posterior pituitary gland. Furthermore, it was demonstrated that dietary doxycycline could effectively modulate Cre activity. This line has the potential to facilitate conditional knock-out experimentation to study early retina and/or posterior pituitary development. The field of developmental biology has greatly benefitted from the use of an evolving set of genetic tools. Utilizing conditional, recombination-based strategies, mouse genes can be deleted in a specific cell type and in some instances with temporal control. This is accomplished through the use of mouse lines that express the bacteriophage gene Cre-recombinase (Cre) from cell type-specific promoters Pax6 enhancer and promoter elements to drive Cre expression throughout lens development rx3 regulatory elements that drives Cre expression at the onset of retinal formation The study of early mouse eye development has been aided by several Cre-recombinase transgenes. For example, much has been learned from lens induction and morphogenesis of the lens pit using the Le-Cre line, which utilizes portions of a Rx is a transcription factor initially expressed approximately at E8.0 in patches of the anterior neural plate that will eventually invaginate to form the optic vesicles To determine whether the Rx-tTA: TetOp-Cre allele drives Cre expression in the developing retina, Rx-tTA: TetOp-Cre transgenic mice were crossed with the Z/EG reporter line that expresses EGFP in cells where recombination occurs The Rx gene is expressed in the ventral diencephalon in early embryonic development and is required for normal development of the posterior pituitary In addition to the eye and posterior pituitary, expression of Cre was also consistently observed in the developing atrial myocardium of the heart . At E8.5Rx knock-out mice (n = 3). Because a doxycycline diet prevents embryonic defects under several different doxycycline regimens and embryonic stages . With these data, we recommend minimizing the period of doxycycline withdrawal that permits analysis at a given stage.Although none of the Rx-tTA: TetOp-Cre knock-in positive progeny fed with a constant diet of doxycycline n = 14) had any observable phenotype, we noticed that when doxycycline is never fed to pregnant dams the pups were runted, lacked all observable eye structures and resembled the c stages , 3, 4, w had any The Rx-tTA: TetOp-Cre knock-in mouse line was designed to enable the analysis of specific genes in the developing retina from the onset of its development. This line may be especially useful for genetically ablating genes required for retinal induction and/or early morphogenesis. This report also demonstrates the utility of this line to carry out genetic experiments in the posterior pituitary as well. It is important to point out that the Cre activity in the heart might be a complicating factor as genes required for heart development might disrupt cardiac function and cause embryonic death precluding analysis of the eye or pituitary at desired stages. Fortunately the inducible nature of this line could limit this effect.All experiments were performed in accordance with institutional guidelines under Institutional Animal Care and Use Committee (IACUC) approval at Cincinnati Children's Hospital Research Foundation (CCHRF). IACUC at CCHRF approved the study described in this manuscript with Animal Use Protocol number 0B12097.Design, construction of the Rx-tTA: TetOp-Cre targeting vector, and the subsequent steps to generate Rx-tTA: TetOp-Cre heterozygous mice were performed by the Gene Targeted Mouse Service Core at the University of Cincinnati. In short, the vector was designed in order to insert a 5.0 kb cassette containing IRES-tTA and Cre recombinase driven by the TetO promoter in the 3′-UTR (104 bp from the stop codon) of the Rx gene. The cassette was flanked by 3.4 kb and 2.7 kb of homologous arms, respectively. A neomycin resistance gene and a thymidine kinase gene were included for positive and negative selection. The construct was electroporated into mouse ES cells derived from a 129Sv strain, and ES cell clones with the correctly targeted allele were identified by PCR and confirmed by Southern blot analysis. Correctly targeted ES cell clones were injected into blastocysts to generate chimeras, which were then bred to obtain ES cell-derived offspring, as determined by the presence of agouti coat color. Agouti mice were further analyzed by PCR for the presence of targeted allele. Rx-tTA: TetOp-Cre heterozygous mice were crossed to flp recombinase mice to remove the neo cassette to obtain neo- mice, which were then further bred with wildtype mice to remove the flp transgene . The Rx-tTA: TetOp-Cre knock-in will be available to the research community upon acceptance of the manuscript.In accordance with institutional policies, mice were housed in a pathogen free vivarium. Pregnant females were identified by the presence of a vaginal plug and the embryos were designated as stage E0.5. When experimentally indicated, animals were fed doxycycline-containing mouse chow  instead of the normal stock diet. Embryos were dissected from the uterine horn at the indicated stages, fixed in 4% paraformaldehyde, infiltrated with sucrose, and embedded in OCT (Tissue-Tek). The Z/EG line has previously been described Embryonic cryosections were placed on glass slides, rinsed with PBST, blocked in 4% powdered milk, and incubated for 1 hour with a 1∶1000 dilution of phalloidin  and Hoechst  to visualize F-actin and nuclei, respectively.