Southeast Asian deforestation rates are among the world’s highest and threaten to drive many forest-dependent species to extinction. Climate change is expected to interact with deforestation to amplify this risk. Here we examine whether regional incentives for sustainable forest management will be effective in improving threatened mammal conservation, in isolation and when combined with global climate change mitigation.Using a long time-series of orangutan nest counts for Sabah (2000–10), Malaysian Borneo, we evaluated the effect of sustainable forest management and climate change scenarios, and their interaction, on orangutan spatial abundance patterns. By linking dynamic land-cover and downscaled global climate model projections, we determine the relative influence of these factors on orangutan spatial abundance and use the resulting statistical models to identify habitat crucial for their long-term conservation. We show that land-cover change the degradation of primary forest had the greatest influence on orangutan population size. Anticipated climate change was predicted to cause reductions in abundance in currently occupied populations due to decreased habitat suitability, but also to promote population growth in western Sabah by increasing the suitability of presently unoccupied regions.We find strong quantitative support for the Sabah government’s proposal to implement sustainable forest management in all its forest reserves during the current decade; failure to do so could result in a 40 to 80 per cent regional decline in orangutan abundance by 2100. The Sabah orangutan is just one  example of a forest-dependent species that stands to benefit from sustainable forest management, which promotes conservation of existing forests. It has been long recognized that deforestation, conversion of primary or secondary forests to agricultural and other land-use types, is the biggest threat to insular Southeast Asia’s biodiversity Pongo pygmaeus is threatened with extinction from habitat loss and degradation (http://iucnredlist.org/apps/redlist/details/17975). Its population has decreased by over 50% in the past 60 years The Bornean orangutan In contrast to the rest of Southeast Asia, the deforestation rate in Sabah  has slowed in recent years. Sabah was among the first places to develop industrialized deforestation on Borneo. By the 1980–90s, with the use of heavy machinery, the rate of deforestation peaked at an estimated 1.37% pa http://www.itto.int). After a successful SFM trial in Deramakot forest reserve initiated in 1997, six major forest reserves are now under SFM, approximately 77.5 km2 or 20.2% of Sabah’s Permanent Forest Estate In an attempt to conserve dwindling timber resources, the Sabah Forestry Department has committed to implementing Sustainable Forest Management (SFM) across all their commercial forest reserves. Compared to conventional timber extraction techniques that damage non-target trees and seedlings Reducing Emissions from Deforestation and Forest Degradation (REDD) is a Payment for Environmental Services scheme that attaches financial value to carbon stored in forests, offering incentives for developing countries to reduce emissions from deforestation and forest degradation P. p. morioWith an estimated 11,000 individuals, Sabah is considered the stronghold for the Bornean orangutan subspecies To examine how forest management and climate change might affect the Sabah orangutan population, we modelled their distribution and abundance using a Species Distribution Model (SDM) and projected it onto regional land-cover and global climate change projections. Our framework improves on bioclimate-envelope models by linking them to dynamic land-cover projections, allowing us to evaluate the relative influence of climate and land-cover change in a scenario analysis. Due to its spatially-explicit nature, this framework allows managers to evaluate the current-day and longer-term importance of individual habitat patches for population persistence. We predicted that the Sabah orangutan population would fare best under the scenario characterising minimum land-cover change and maximum climate change mitigation, hypothesising that land-cover change will further degrade primary forest and that climate – particularly temperature – in currently occupied habitat will change, rendering the habitat less suitable. We also predicted that land-cover change would have a greater influence on orangutan population abundance than climate change because the rate of change in the later is currently faster than the latter, but that their combined impact would be higher than when considered in isolation.P. p. morio populations across Sabah, the Kinabatangan Orangutan Conservation Project has counted orangutan nests along approximately 3366 km of aerial transects through 19 forest reserves and parks over six years  (To monitor major 2009–10) . Details2 grid cells, chosen because it is the average female orangutan territory size 2 resolution . To reduce temporal autocorrelation between repeat surveys, we used the average nest count in each cell calculated over the entire monitoring period. The final dataset had 1180 gridded aerial nest count cells. All spatial data processing was done in R (www.r-project.org) using packages raster and sp.To guard against spatiotemporal autocorrelation we calculated average spatiotemporal orangutan habitat suitability. To reduce any influence of spatial autocorrelation, we aggregated counts to 2.5 kmFailing to survey over-degraded and unforested areas with few or no nests introduces a systematic survey bias that will prevent a statistical model from distinguishing those areas as unsuitable. Consequently, we selected a number of unsurveyed point locations to represent “pseudoabsences”. These were selected at random from outside forest reserves, to distinguish them from “true absences” . To counter uncertainty in whether the pseudoabsences  were true absences, we used a statistical threshold to identify model predicted presences that down-weights the influence of pseudoabsences . We chose to retain both elevation and slope because orangutan prefer lowland forests (elevation) and because we expected forest in difficult terrain to remain standing (slope)  Habitat variables were expected to influence orangutan densities, and (iii) Climate variables were expected to delineate orangutan preferred average seasonal climatic conditions.We identified a number of variables considered important predictors of orangutan presence and abundance, and collated spatial data to represent them . We proc (slope) . We clasOver 43% of the aggregated orangutan nest counts were zero, and this increased to 73% when pseudoabsences were included. We modelled these zero-augmented data as originating from a hurdle process characterizing two separate processes: (1) presence across the landscape depending on whether or not habitat is suitable, and (2) abundance in suitable habitat. We assumed a hurdle approach because it has been shown to outperform alternatives lr) = 0.01, bag fraction (bf) = 0.7, first-order interactions (tc) = 2 and number of trees in the range ntlr, bf and nt control the contribution of each term to the regression model, the proportion of data randomly selected to build the regression model and the number of regression terms, respectively. To determine the optimum nt, we compared the out-of-sample predictive performance of models fitted with increasing nt where predictive performance was calculated as mean prediction deviance over 10 cross-validation subsets (Dcv). We used the model with nt that minimized DcvR using package gbm and dismo.We built a boosted regression tree SDM because they readily fit non-linear and interacting processes and have been used to model hurdle processes lrtcbf = 0.7. Again, we varied ntoptimized model was that with the combination of fitting parameters that minimized Dcv. Finally, we compared the optimized saturated model to models simplified by removing the least influential predictors until the change in Dcv exceeded the original cross-validation prediction deviance SE, repeating this for the binomial and Poisson parts separately and together To optimize the predictive performance of the saturated hurdle model, we fitted the binomial and Poisson parts separately using all possible fitting parameter combinations in the space defined by mse) and relative mean prediction error (rmpe) for the Poisson part We calculated model explanatory performance for the Binomial part of the hurdle model using confusion matrix-based statistics Kappa and AUC, and mean square error (c.f. 5 km for the main dataset) using GLM and Iterative Re-weighted Least Squares in R package MASS.We built the SDM using all available nest count survey data and so had no unused data with which to validate its predictions. Instead, we validated our model by examining the relationship between our nest count predictions and an independent aerial nest count data set Ai) and orangutan density (Dou) is given by Dou  =  exp(4.730+ (0.980 × ln(Ai)))/(p×r×t), where p = 0.9 is the proportion of nest building individuals, r = 1.084 is the daily rate of nest production and t = 286.3 days is the nest decay rate We converted nest count predictions to abundances for scenario testing. The relationship between the number of nests per km of aerial transect , a coupled gas-cycle/aerosol/climate model, was used to generate the climate anomalies that were an ensemble of seven GCMs chosen on the basis of their skill in reproducing seasonal rainfall (1980–1999) at global and regional (Southeast Asia) scales Both SFM scenarios were tested under two contrasting global climate change scenarios: (1) a no-climate-policy reference scenario , and (2) a corresponding policy (stabilization) scenario  rmpe) of 24% (tc = 3). The explanatory power of the binomial part was robust , as was the Poisson part . Model residuals were largest where the model estimated a nest count .The optimized saturated hurdle model explained 68% of the deviance in aerial orangutan nest counts and had an overall relative mean prediction error  of 24% . The Poist count  and theiThe model correctly predicted that the current-day orangutan population is restricted largely to forested areas in eastern Sabah, primarily in Kinabatangan Wildlife Sanctuary, Malua Biobank, Kulamba, Ulu Segama and Deramakot forest reserves and Tabin Wildlife Reserve . The opty = 0.288+0.311x, IWLS fit: y  =  −0.03+0.309x; Our model validated well, as evident by a strong positive linear relationship with independent data The 2000–10 annual deforestation rate in Sabah was 1.18% pa. The rate in unprotected forest (1.26) was higher than in forest reserves (1.13) . These dBy 2100, Sabah mean annual temperature was forecast to increase by 2.5 and 1.2°C under the Ref and Pol scenarios respectively. In contrast, mean seasonal precipitation  was forecast to remain relatively stable under both scenarios. A notable weakness in projecting SDM is their uncertainty when extrapolating to unobserved conditions. Our precipitation forecasts were always within the observed current-day range  but mean temperature exceeded maximum observed temperature by 2060. By 2100, the Ref temperature forecast for eastern Sabah was up to 2.2°C above the maximum observed temperature  will ensure Sabah’s orangutan population does not decline in the long term, and that the population will fare best under a climate change mitigation scenario. Spatially, our model predicted that eastern forest reserves currently support highest orangutan numbers but that, even if they remain forested under complete SFM implementation, their suitability for orangutan will gradually decline as temperature increases under Ref, and to a lesser extent Pol, climate change scenarios. Instead, currently unoccupied western forests might become a future orangutan climate refuge.2 emissions at around 450 ppm might cause the orangutan population to decline by an additional 46% by 2100 Click here for additional data file.Figure S2Relationship between nest count predictions and an independent orangutan nest count dataset. Plot showing the ordinary least squares (OLS) and robust iterative re-weighted least squares (IWLS) fits when predicting our SDM model predictions with data published in (TIFF)Click here for additional data file.Figure S3Land-cover class gains and losses and projected changes between 2010–2100. Plots showing (a) the observed gains and losses of cells in each land cover class, and (b) the projected changes in each land class between 2010–2100 in forest reserves and unprotected forests for the current-day SFM scenario. Absolute gains and losses in least-widespread classes were negligible compared to changes in degraded land and forest cover. Consequently, projected changes in these land-cover class were inconsequential compared to projected gains and losses in degraded and forest land cover, respectively.(TIFF)Click here for additional data file.Figure S4Land-cover change projections. Maps showing land cover change projections at 2011, 2041, 2071 and 2100 under (a) current-day SFM and (b) complete SFM implementations.(TIFF)Click here for additional data file.Figure S5Predictor importance for predicting 2010 observed land-cover. Plots showing the relative importance of spatial predictors in predicting observed 2010 land cover in (a) forest reserves and (b) unprotected forests.(TIFF)Click here for additional data file.Figure S62 mitigation scenario.Per cent wet season precipitation delta maps under each CO Per cent wet season precipitation delta maps at 2041, 2071 and 2100 relative to 2010 for Ref and Pol scenarios.(TIFF)Click here for additional data file.Figure S72 mitigation scenario.Per cent dry season precipitation delta maps under each CO Per cent dry season precipitation delta maps at 2041, 2071 and 2100 relative to 2010 for Ref and Pol scenarios.(TIFF)Click here for additional data file.Figure S82 mitigation scenario.Degree Centigrade temperature delta maps under each CO Degrees Centigrade temperature delta maps at 2041, 2071 and 2100 relative to 2010 for Ref and Pol scenarios.(TIFF)Click here for additional data file.Table S1Spatial predictors. Spatial predictors used to build the Species Distribution Models and notes on their processing.(DOC)Click here for additional data file.Table S2Land cover change predictors. Table of variables considered important predictors of land cover change.(DOC)Click here for additional data file.Table S3Accuracy statistics for the random forest models of 2010 land-cover predictions. Prediction error rates were low for the most-widespread land-cover classes but high for the least-widespread classes  because they constituted less than 5% of the land cover.(DOC)Click here for additional data file.


This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 10 In the UK, several v-CJD cases have been identified in patients that received blood or blood-derived products prepared from incubating asymptomatic donors. Since there is no screening test to identify infected donors, procedural risk reduction measures remain the only protection against v-CJD transfusion risk. These measures rely, in part, on the assumptions that (i) the level of infectivity in blood is low and (ii) the risk of blood borne transmission is directly correlated with the infectious titer of blood and blood products. Using a transmissible spongiform encephalopathy (TSE) animal model, we have provided evidence that despite a very low infectious titer in blood as measured by inoculation into brain, the transfusion of 0.2 mL of blood from asymptomatic infected donors is sufficient to transmit the disease with a 100% efficacy. We further demonstrated that this high efficiency of disease transmission is crucially dependant on the viability of the transfused white blood cells rather than on their infectious titer. These findings provide new insights into the pathogenesis of TSE diseases and require revision of some of the key assumptions of the v-CJD blood borne risk assessments. In 1996, a new form of CJD, named variant CJD (v-CJD) was identified in humans 50 per mL and that both plasma and leukocytes were infectious Current measures for prevention of v-CJD transmission by blood products rely on risk assessments based on infectious titers reported for the blood of rodent models of TSE i.e. the administration of large numbers of living cells and a large volume intravenously to the recipient.However, the IC inoculation route is unlikely to reflect the specifics of the transfusion exposure route; In early 2000, transmission of both experimental BSE and natural scrapie were reported to occur following transfusion of whole blood collected in asymptomatic incubating sheep In this study, using a TSE sheep infection model, we investigated the ability of blood and blood components to transmit the disease when administered by the transfusion route and measured their relative infectious titer by IC inoculation in transgenic mice (tg338) over-expressing ovine PrP. Our findings show that the disease is transmitted with high efficiency by transfusion of infected blood and blood components and requires revision of some of the key assumptions used for assessing the risk of blood borne transmission of v-CJD.All animal experiments have been performed in compliance with our institutional and French national guidelines, in accordance with the European Community Council Directive 86/609/EEC. The experimental protocol was approved by the INRA Toulouse/ENVT ethics committee.Sc detection. The PrP genotype was obtained for every animal by sequencing Exon 3 of the Prnp gene as previously described VRQ/VRQ sheep were produced in a biosecure unit from a flock that was sourced originally from New Zealand by Veterinary Laboratory Agency in the United Kingdom (UK). This TSE free flock is a unique source of VRQ/VRQ animals that are considered free of classical scrapie Blood was collected in vigil animals from jugular vein into Macopharma MSE3500 collection bags. To separate plasma, buffy coat and red cells, blood units were centrifuged at 3640 rpm for 12 minutes (including acceleration and deceleration) at 20°C in a Jouan LR5-22 centrifuge. For each sample, packed cell volume and cell numeration were measured using IdeXX Vet-station automat and Sysmex XT2000i. White blood cells (WBC) were obtained from buffy coat fraction, by osmotic lysis of erythrocytes with ACK solution . Briefly, buffy coat (1 volume) was gently mixed (by rolling tubes) with ACK (1 volume) and incubated for 5 min RT. WBC were then washed 3 times with 50 mL PBS to remove erythrocytes debris and plasma.WBC fixation was performed using paraformaldehyde (PFA)/PBS solution. WBC cells were re-suspended in a 50 mL conic tube using 10 mL of PBS and 10 mL of a PBS/PFA 4% solution was added . Tubes were then rolled on bench during 20 seconds and incubated for 10 min on ice. Cells were then washed four times in PBS (50 mL) and their morphology (lack of clumps and cellular integrity) was checked by direct microscopic examination and flow cytometry.Recipient sheep were anesthetized by IV administration of Ketamine/Diazepam (2 mg and 0.1 mg per kg respectively) and a catheter (16G) was installed in the jugular vein. This catheter was used for all transfusions and intravenous administrations of blood and blood products. Whole blood (200 mL), plasma (100–150 mL) or WBC (re-suspended in 100 mL 5% glucose) were transfused/intravenously administered by gravity in individual recipients over a 30–40 min period.50 IC in tg338) in a 20 mL volume. Each inoculum was then mixed with 200 mL of freshly collected whole blood by 30 minutes of continuous rocking before transfusion back into the same donor sheep. In the whole blood titration by transfusion route, the required whole blood volumes were sampled from the collection bag using a sterile syringe and either transferred into a new pouch or gently re-suspended in a sterile 5% glucose solution (QSP 200 mL), before administration (gravity) to recipients. In the WBC experiment, WBC (fresh or fixed) were gently re-suspended in a 200 mL sterile pouch of isotonic (5%) glucose before infusion (gravity) over a 20–30 min period. All transfusions were performed within the 6 hours following blood collection.In the whole blood spiking experiment, the titrated PG127 brain homogenate was diluted in isotonic (5%) glucose solution to obtain the desired number of Infectious Dose 50 as measured by IC inoculation in tg338 mice (IDSc) immunohistochemistry investigation while the other was snap frozen at −80°C for WB PrPSc detection.Sheep were housed in a category 2 animal facility. Donor and recipient sheep were kept separated. Contact controls (n = 5) were housed in the same pen as transfusion recipients throughout the experiment. None displayed any TSE symptoms. Orally inoculated sheep (donors) and transfused sheep (recipients) were monitored daily. When displaying symptoms that could impair their ability to feed, the animals were euthanized by an intravenous injection of T61 (Intervet). Transfused animals that had not displayed clinical signs after 400–450 days were also euthanized. Upon completion of the experiment, the contact controls were also euthanized. All animals were systematically necropsied and a panel of tissues  over-expressing the VRQ allele of the ovine prion protein (PrP) Bioassay experiments were carried out in transgenic mice  and inoculated neat (20 µl IC) in tg338 (n = 18). No acute toxicity was observed at inoculation during this experiment. The infectious titer per mL of blood (number of ID/mL) was estimated by the limiting dilution titration (application of Poisson model) described by 338 mice (20 µL/mouse). The infectious titer  were determined by the Spearman-Kärber method WBC corresponding to known quantity of whole blood was high speed homogenized for 90 seconds  in 5% glucose solution. The resulting suspension was then titrated by end point dilution by intracerebral (IC) inoculation of 1/10 successive dilutions into groups of 6 tg50 per mL was considered in average equivalent to 0.693 ID.According to Fischer Sc extraction. Immunodetection was performed using Sha31 monoclonal antibody conjugated to horseradish peroxidase  which recognizes amino acids 145–152 (YEDRYYRE) of the sheep PrP sequence. Peroxidase activity was detected using ECL substrate (Pierce). Immunohistochemistry was performed as previously described A Western blot kit  was used following the manufacturer's recommendations. For each sample, 250 µL of 10% tissue homogenate was subjected to PrP50 as measured by end point dilution titration in tg338 mice by the IC route) able to transmit TSE infection in sheep by transfusion. This was done by transfusing groups of two TSE free VRQ/VRQ recipient sheep with whole blood that had been spiked with successive 1/10 dilutions of a brain homogenate prepared from scrapie affected sheep (PG127 isolate). This brain homogenate had been previously titrated by end point dilution in tg338 mice, and had a titer of 106.6 ID50 IC/g 3 ID50 IC in tg338 mice  at a late stage of the incubation period . 200 mL, 20 mL, 2 mL and 0.2 mL of each donor's fresh whole blood were transfused into TSE free VRQ/VRQ recipients . It therefore suggested that in scrapie infected sheep (close to symptomatic phase) the infectious load per mL of blood should be higher than 103.6 ID50 IC in tg338.These results, when considered together, indicate that the transfusion of 0.2 mL of whole blood transmitted scrapie more efficiently than the IV administration of 10To confirm this estimate, whole blood from three donors (D1 to D3) and one TSE free control (C1) were each inoculated intracerebrally into eighteen tg338 mice (20 µL per mouse). An incomplete attack rate was observed in the inoculated mice  and infePlasma, red blood cells and WBC were prepared from the same blood samples. Plasma and RBC were inoculated undiluted intracerebrally into eighteen tg338 mice. Only a single infection was observed (D1 plasma). The infectivity titers in plasma and red blood cells expressed as the equivalent volume of whole blood were computed to have a 0.95 probability of contributing less than 8.6 and 2.7 ID IC in tg338 per mL of blood respectively.50 IC in tg338 per mL of blood, i.e between 4.5 and 13.4 ID per mL .The infectivity in WBC was measured by end point dilution titration by IC inoculation of ten folds serial dilutions in tg338 mice . Titers The infectious titers of whole blood in sheep incubating scrapie were comparable with those previously reported in hamster and mouse models In an attempt to resolve this discrepancy we investigated the relative abilities of whole blood, plasma and white blood cells to transmit the disease by the transfusion route in sheep. Over 700 mL of blood was collected, few days (D4) to three weeks (D7) before clinical onset, from each of four orally challenged VRQ/VRQ sheep (D4 to D7) , 6. 500 Whole blood and fresh WBC produced clinical scrapie in all recipients. The incubation periods of recipient sheep receiving whole blood or WBC were similar suggesting comparable virulence of both products . In contWhereas all recipients that received fresh WBC succumbed to clinical disease, the IV administration of PFA fixed WBCs resulted in TSE infection of only two of four recipient sheep  and in iUsing TSE infection in sheep as a model, we have shown that the intravenous administration of a few hundred microliters of blood is sufficient to infect a transfusion recipient. This high efficiency of transmission by blood transfusion contrasts with the very low infectious titer measured by IC inoculation of blood into transgenic mice expressing ovine PrP.This discrepancy shows that the infectious titer as determined by IC inoculation of rodents does not accurately predict the ability of blood and blood-derived products to transmit the disease when administered by the intravenous route. Importantly, this finding indicates that assessments of transfusion transmission risks from v-CJD that are based on IC measurements in rodents may seriously underestimate the risk.Our results indicate that freshly prepared WBCs have a comparable capacity to whole blood to infect transfusion recipients. However WBC fixation with PFA strongly inhibited their ability to transmit the disease intravenously. Tissue fixation with aldehydes is classically described to reduce by several log10 the TSE infectivity titer (as measured by IC inoculation in rodents) in vitro prion infection models in which cell-mediated infection was reported to be more efficient than infection with cell homogenates or PFA fixed cells 0/0RAG-1 mice following IV administration whereas no transmission could be observed following the IV administration of splenic cell homogenates to the same mouse model This view is consistent with results obtained in In the PG127 scrapie sheep model, plasma was less able than whole blood and fresh WBCs to transmit the disease by the transfusion route. This observation is consistent with data obtained from similar transfusion experiments carried out in BSE infected sheep and in Chronic Wasting Disease infected cervids In this study, labile blood products containing viable WBC presented the greatest risk of transmitting Prion disease. This finding strongly supports the continuation of universal leuco-reduction as currently applied in the EU countries and Canada to reduce, amongst other potential adverse effects, the risk of v-CJD transmission Finally, our results also raise some concerns about the use of the ‘spiking’ models for investigation of blood-borne TSE transmission risk


The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209. The lymphatic sinuses in human lymph nodes (LNs) remain poorly characterized, despite exquisite studies of the lymphatic network in rodent LNs + APC population that occupied many of the lymphatic sinuses, as well as being occasionally present in the paracortex Hence we set out to map a range of markers for LECs in human LNs, and determine which lymphatic sinuses were labeled with each marker. We were also interested in mapping which markers were shared between LECs and sinus APCs. As well as LYVE1, we were interested in better defining which of these cell populations expressed CD209. In our previous work we had characterized a CD209Here we define an exclusive marker that detects all the lymphatic structures composed of LECs in the human LN. For the first time we also describe the distinct distribution of two different populations of LECs forming lymphatic sinuses within human LNs. Finally we establish which markers are capable of readily distinguishing between sinus-type APCs and LECs.We obtained LNs from living donors undergoing surgery and donors post-mortem , snap frozen in liquid nitrogen and sectioned using a cryostat. Sections 5 µm thick were fixed with ice-cold acetone and blocked with 0.25% casein, then incubated with the primary antibodies listed in Table S2 . The priThe slides were mounted using Prolong Gold (Invitrogen). Sections were visualized with a Leica DMRE fluorescent microscope equipped with the epi-fluorescent filters: UV, 450–490 nm, 530–560 nm and 590–650 nm. Images were acquired at room temperature using 5x/0.15 numerical aperture (NA), 10x/0.30 NA, 20x/0.50 NA and 40x/0.7 NA Leica objectives, a Leica DC500 Digital camera or SPOT camera  and analySIS FIVE software . Images were generated using Cytosketch  and figures were formatted using Photoshop .To establish a complete map of LECs within human LNs, we stained for nuclear expression of the transcription factor Prospero-related homeobox 1 (PROX1), previously described as a definitive and exclusive marker of LECs in adult tissues + cells were also detected within the hilum , although there did appear to be some variability in the expression level of this receptor between sinuses, including between sinuses in the same region e.g medulla . In almost all LNs, LYVE1, the LEC marker routinely used in murine LNs, was expressed by the LECs of the paracortical and medullary sinuses ; however+ APCs in human LNs We previously used the antibody DCN46 to identify CD209+ cells, LECs could readily be identified as a CD144+ mesh Click here for additional data file.


Canis lupus) and 2) compare the genetic affinities of modern breeds to regional indigenous village dog populations. The Y chromosome markers indicated that village dogs in the two regions corresponded to reciprocally monophyletic clades, reflecting several to many thousand years divergence, predating the Neolithic ages, and indicating long-indigenous roots to those regions. As expected, breeds of the Middle East and East Asia clustered within the respective regional village dog clade. Australian dingoes also clustered in the Southeast Asian clade. However, the European and American breeds clustered almost entirely within the Southeast Asian clade, even sharing many haplotypes, suggesting a substantial and recent influence of East Asian dogs in the creation of European breeds. Comparison to 818 published breed dog Y STR haplotypes confirmed this conclusion and indicated that some African breeds reflect another distinct patrilineal origin. The lower-resolution mtDNA marker consistently supported Y-chromosome results. Both marker types confirmed previous findings of higher genetic diversity in dogs from Southeast Asia than the Middle East. Our findings demonstrate the importance of village dogs as windows into the past and provide a reference against which ancient DNA can be used to further elucidate origins and spread of the domestic dog.Modern genetic samples are commonly used to trace dog origins, which entails untested assumptions that village dogs reflect indigenous ancestry or that breed origins can be reliably traced to particular regions. We used high-resolution Y chromosome markers (SNP and STR) and mitochondrial DNA to analyze 495 village dogs/dingoes from the Middle East and Southeast Asia, along with 138 dogs from >35 modern breeds to 1) assess genetic divergence between Middle Eastern and Southeast Asian village dogs and their phylogenetic affinities to Australian dingoes and gray wolves ( Canis lupus) 12,000–40,000 years BP, and that they spread rapidly throughout Eurasia and the Americas at the end of the last ice age Archaeology and DNA studies indicate that dogs evolved from or share a recent common ancestor with the gray wolf  limit the practical utility of this approach. An alternative approach is to assess the genetic divergence between relatively large samples of extant village dogs from multiple regions to infer population ages. Specifically, if populations reflect primarily indigenous ancestry, their genetic divergence should reflect thousands of years' isolation, whereas if they are heavily admixed with modern Western breeds, they should reflect little genetic divergence. Mitochondrial DNA (mtDNA) sequences are commonly used in phylogeographic studies of plants and animals, including dogs 2–104 generations.DNA markers on the non-recombining region of the Y chromosome (NRY) have been used to great advantage in studies of several domestic species and humans Our first objective was to determine whether village dogs from the Middle East and Continental and Island Southeast Asia were indigenous to those regions or, alternatively, secondary products of a post-Victorian expansion of Western breed dogs. The second objective was to determine whether modern breeds could be traced to either of these putative indigenous village dog populations. We used highly resolved Y-chromosome SNP-STR haplotypes to assess the approximate minimum age and genetic similarity of these village dog populations. To address uncertainty in STR mutation rates, estimates of divergence time were calibrated using Australian dingoes and Bali dogs, both of which are known to have been isolated for several thousand years based on independent evidence All procedures involving animals were reviewed and approved by the University of California, Davis, Animal Care and Use Committee (Protocol No. 16643).Canis mesomelas) and a dhole  from captivity. Buccal swabs were collected from dogs. Most village dogs (mainland and Island Southeast Asia) were captured in the course of spay-neuter programs  from the Middle East and Southeast Asia, 15 Australian dingoes , 45 desert-bred Salukis , and 93 male breed dogs from 35 additional breeds or mixtures of breeds. Blood, tissue, or buccal swabs were obtained from the wild canids, including gray wolves from Iran (n = 3), China (n = 1), and the Yukon, Canada (n = 3), along with a black-backed jackal  and CTTATATGCATGGGGCAAACC (reverse) and Big Dye sequencing chemistry . Males were genotyped using 5 dinucleotide-repeat STRs from the NRY, including 650−79.2, 650−79.3, 990−35 DNA was extracted from buccal swabs using a standard protocol Male samples were genotyped at 11 NRY SNP loci Mitochondrial DNA sequences were used both as a means of assessing the similarity of our samples to the nearby samples of Pang et al. We constructed phylogenetic networks based on Y-SNPs, Y-STRs, and the combination of both markers. The Y-SNP networks were used to coarsely characterize the deeper phylogenetic structure in village dogs, whereas the Y STR networks provided far greater resolution with respect to recent divergence in the same village dogs and enabled us to directly compare 818 previously published breed dog haplotypes r = 2 and є = 0. We first constructed median-joining networks The Y chromosome networks were constructed using program Network 4.50 In principle, use of coalescent models and Markov chain Monte Carlo (MCMC) approaches can be used to estimate population (or clade) splitting times n = 200) and Southeast Asia (n = 231) were sequenced at the mtDNA locus. This analysis revealed a total of 54 HVI haplotypes, of which 17 were novel (Genbank Accession Nos. HQ287728–HQ287744). The 15 Australian dingoes from Fraser Island all had indigenous dingo haplotypes  and Southeast Asia . The 402-bp equivalents of all of the widespread ancestral haplotypes previously identified as universally occurring haplotypes (UTs) were found in at least one of the two populations (n = 12) than the Middle East (n = 4) (n = 612 village dogs) than the Middle East (n = 37 village dogs) in the past Village dogs from the Middle East (ulations . In totaulations  were fou (n = 4) , S4, eve−8 per year It is noteworthy that village dogs from Bali (n = 94) exhibited a lower haplotype diversity than dogs from other islands in Southeast Asia or sampling sites in the Middle East . This waThe more ancient phylogenetic relationships of the Southeast Asian and Middle Eastern village dog patrilines were revealed by the NRY SNPs in 300 village dogs and dingoes, along with 7 gray wolves, and an out-group of 1 dhole and 1 black backed jackal. The 8 resulting haplotypes  fell intThe 5 NRY STRs revealed considerably more Y chromosome diversity, including 95 haplotypes in the 300 village dogs and dingoes (and 5 more in the 7 wolves). The diversity of village dog STR haplotypes also was higher than previously found in a much larger sample of breed dogs . AlthougCombining the two marker types corrected inconsistencies due to ambiguous positioning of distinct STR haplotypes, revealed a greater mutational distance between the Southeast Asian and Middle Eastern clades, equalized the apparent divergence of both dog clades from the basal wolf clade, and revealed that several Middle Eastern village dog haplotypes clustered with the wolf clade, suggestive of recent wolf introgression in Middle Eastern village dogs. The 4 haplotypes exhibited by Australian dingoes clustered more closely with Southeast Asian village dogs, including a basal haplotype shared with some village dogs. The haplotypes of Bali dogs also clustered within the Southeast Asian clade. The haplotypes of Southeast Asian village dogs clustering in the Middle Eastern clade were linked by long branches, indicating ancient derivation. Additionally, the SNP-STR network revealed a diversity of STR haplotypes corresponding to each of the SNP haplotype. Only SNP haplotypes 7 and 11 shared a STR haplotype, indicating that the SNP separating these haplotypes was likely a recent mutation. Thus, the SNP- STR data set provided a powerful basis for assessing ancient vs. recent divergence between village dog populations and for comparing to the haplotypes of breed dogs to infer their proximate origins.−4 per year, fell within the expected range for mutation of human Y STRs , each haplotype was rooted to its most proximate common ancestral node  breeds, 3 from 2 East Asian breeds, 31 from a Middle Eastern breed, desert-bred (Bedouin) Salukis from Israel, and 28 of various mixed breeds . The Saln = 818; n = 60) from these 818 breed dogs clustered primarily in the Southeast Asian village dog clade, including all but 12 European and American breed dogs  sharing 2 haplotypes  that are representative of their indigenous ancestry. Thus, our first question in this study was whether contemporary village dogs in Asia (including parts of Southeast Asia and the Middle East) primarily exhibited indigenous ancestry or, alternatively, whether they reflected admixture among recently created breeds . Second, because breeds are so commonly used to trace ultimate origins e.g., , we wishOur findings indicated that Middle Eastern and Southeast Asian village dog populations must have originated either from a common gene pool thousands of years before present or from distinct groups of wolf or wolf-like founders, but are clearly not the product of a post-Victorian expansion of dog breeds. First, the monophyly of Y chromosome clades associated with the two populations, and their long and similar divergence from wolves, suggest that most of the extant Y chromosome diversity evolved after these populations were established. The numbers of mutations separating haplotypes and their ancestral nodes in each of these clades provided a measure of time to most recent common ancestor, which also reflected the minimum time since separation. The time estimates, while necessarily imprecise, were qualitatively robust because they were based on calibration to known-age indigenous, insular dog populations of Bali and Australia. These Island populations were known to be founded on the order of 3,000–5,000 years BP, based on archaeological evidence Having established that village dogs used in this study reflect indigenous populations, it was possible to compare ancestry of breeds with respect to these indigenous populations. Although we could not trace modern breeds to a precise location of origin because we did not sample village dogs from a large intervening portion of Asia, the considerable genetic distance between these village dog populations, even if ends of a continuum, enabled inference about the relative regional affinities. As expected, Middle Eastern breeds clustered with Middle Eastern village dogs and East Asian breeds clustered with Southeast Asian village dogs. However, putative European and American breeds also clustered in the Southeast Asian clade, which ran counter to expectations. It would not have been surprising to find some Eastern influence in Western breeds, as many breeds developed during the Victorian dog-fancy era either were initiated from East Asian stock If Western breeds do not derive from their putative regions of origin, this warrants reconsideration of some previous conclusions about dog origins. In particular, Savolainen et al. On the other hand, our findings with both mtDNA and Y chromosome analyses provided strong confirmation of higher diversity previously observed in Southeast Asia than the Middle East Up to now, use of ancient DNA approaches to the study of dog origins have been limited to mtDNA, which has been most useful in showing that modern dogs do not necessarily reflect the ancient ones inhabiting those same regions Figure S1Sampling locations throughout the Middle East and Southeast Asia. Yangtze River is indicated by blue line. Israeli desert-bred Saluki , Iranian village dogs , Southeast Asian village dogs .(TIF)Click here for additional data file.Figure S2Y chromosome SNP-STR haplotype network depicting haplotype names. Network of dog (circles) and wolf (squares) NRY SNP-STR haplotypes, including 300 village dogs, 124 breed dogs, and 7 wolves. Haplotype names are beside their respective haplotype. Size of circle is proportional to sample size, except that the larges circle represents 18–50 individuals. NRY SNP-STR subclades corresponding to numbered SNP haplotypes in (TIF)Click here for additional data file.Table S1a.List of Sequenom PCR primers developed for SNP loci, which correspond to Natanaelsson et al. 2006(DOC)Click here for additional data file.Table S2a.Sequenom SNP extension primer sequences and expected extension products, developed for SNP loci, which correspond to Natanaelsson et al. 2006(DOC)Click here for additional data file.Table S3Frequency of 402 bp mtDNA haplotypes of village dogs sampled in 7 populations. Haplotypes are named as the lowest-numbered previously named synonymous 582 bp haplotype Savolainen et al. 2002a and Pang et al. 2009b or are novel in this study (haplotype names beginning with “V”); see (DOCX)Click here for additional data file.Table S4Seventeen novel mtDNA haplotypes, location of sample, and number of substitutions differing from the nearest published haplotype. Sequences were deposited in Genbank .(DOC)Click here for additional data file.Table S5Number of individuals (n), number (No.) of mtDNA haplotypes, and rarified haplotype richness (corrected for differing sample sizes to n = 10) for 402 bp mtDNA D-loop haplotypes of village dog sampling locations in the Middle East and Southeast Asia.(DOC)Click here for additional data file.Table S6a).Y chromosome SNP haplotypes as resolved from 11 “Ydog” loci Click here for additional data file.Table S7Imputed Y-SNP haplotypes corresponding to the 23  Y-STR haplotypes for which 1 or more of the 11 SNPs failed (indicated by “-”) or for which no SNP genotyping was attempted. All but one incomplete SNP haplotype corresponded to the same failed locus and the same haplotype ambiguity (7 or 11), suggesting a mutation in the priming region associated with this clade. The SNP haplotypes are indicated in (DOCX)Click here for additional data file.Table S8Allelic composition of NRY STR haplotypes and corresponding SNP haplotypes. An asterix identifies a SNP haplotype with one or more positions imputed (see a.uted see . Previou(DOCX)Click here for additional data file.Table S9a), and gray wolves.Frequency of NRY STR and SNP haplotypes observed in Middle Eastern and Southeast Asian village dog populations, Australian Dingoes, breed dogs (including published ones An asterix identifies a SNP haplotype with one or more positions imputed (see uted see .(DOCX)Click here for additional data file.Table S10a, breed class, and haplotype name of STR haplotypes included in NRY STR and SNP-STR analyses.Breed, number (No.) of individuals, putative region of breed origin Parentheses indicate individuals and haplotypes found in breed dogs that were genotyped in the present study and used in SNP-STR analysis; otherwise entries refer to published datab.(DOCX)Click here for additional data file.


Genetic analyses in zebrafish identify a novel physical signaling mechanism that drives formation of invadopodia-like structures and promotes cell invasion in vivo. meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process.The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell invasion in the intestine of zebrafish  meltdown form invadopodia-like protrusions and invade the tissue stroma in response to unregulated contractile tone in the surrounding smooth muscle layer. The invadopodia-like protrusions that form in response to this physical signal are required for epithelial cell invasion in this in vivo model, and they can be induced when unregulated smooth muscle contraction is induced by oxidative stress. These findings provide the first direct evidence that invadopodia play a role in tissue cell invasion in vivo and identify a novel inducible physical signaling mechanism that can drive this process.The epithelial cells lining the digestive tract are separated from the connective tissue stroma by a thin layer of extracellular matrix called the basement membrane. During cell invasion, as occurs during cancer metastasis, epithelial cells breach the basement membrane and invade the tissue stroma. The proteases used by invasive cells to degrade basement membrane in vitro are localized in specialized plasma membrane protrusions known as invadopodia. It is not known, however, whether invadopodia are required for cell invasion in vivo or what triggers their formation. Here, we show that epithelial cells in the intestine of the zebrafish mutant  Physical signaling mechanisms are increasingly recognized as playing an important role in regulating the growth, differentiation, and morphology of vertebrate tissues v-SRC , in which intestinal architecture is disrupted by a mutation in myosin heavy chain 11 (myh11), the gene encoding the principle myosin present in smooth muscle mlt, and their inhibition rescued the mutant phenotype, thus linking mlt to established models of cell invasion. Here we present in vivo evidence that the mlt mutation transforms Myh11 into a constitutively active contractile protein, and that unregulated actomyosin interactions increase the basal level of smooth muscle contractile tone in the larval intestine. The physical signal arising from increased contractile tone activates a feed forward redox signaling loop that induces invadopodia-like protrusions in the epithelium, and we show that this requires Tks5, a Src substrate that is a component of the ROS generating Nox complex and is required for formation of invadopodia and podosomes in mammalian cells mlt mutants normally have no detectable phenotype, however treatment with ROS generators induced both the protrusions and cell invasion. Together, these findings identify a novel inducible in vivo signaling mechanism that non-cell autonomously drives formation of invadopodia-like protrusions and cell invasion.In previous work, we showed that cell invasion can be modeled in a zebrafish mutant, mlt invasive phenotype is first visible at 74 h post-fertilization (hpf), shortly after the onset of myh11 expression in the developing intestinal circular smooth muscle layer surrounding the epithelium mlt, the intestinal tube has an irregular contour in comparison to the wild type intestine , a metalloproteinase associated with invadopodia, as well as Mmp2, a metalloproteinase that is activated by Mmp-14 mlt intestinal epithelium, we derived a zebrafish transgenic line, Tg(miR194:Lifeact-GFP), in which a GFP-labeled peptide that binds F-actin . Immunostainings confirmed that Cortactin, a protein present within invadopodia and that is required for their formation, was present in the actin-rich mlt protrusions (n = 18 of 19 cells with protrusions). These findings argue that the protrusions present in the mlt intestinal epithelial cells are invadopodia homologs.In tissue culture models, degradation of the basement membrane by invasive cells is driven by metalloproteinases associated with invadopodia, plasma membrane protrusions that are enriched in actin, and the actin binding protein Cortactin mlt protrusions we compared the cellular localization of an ectopically expressed Mmp14a-mCherry fusion protein in mlt and wild type intestinal epithelial cells. This approach was necessary because none of the antibodies we tested in immunostainings detected endogenous Mmp-14 protein in the intestine. Confocal analyses showed that the Mmp14a-mCherry fusion protein preferentially localized to the basal portion of mlt epithelial cells with protrusions .To further characterize the mlt epithelial cells are closely related, if not identical to invadopodia and or podosomes, and furthermore, that the protrusions are required for invasion. Because podosomes have not been described in epithelial cells and are associated with cell migration rather than invasion, hereafter we refer to the mlt protrusions as invadopodia-like protrusions.Together, these findings argue that the membrane protrusions present in v-srcmlt intestinal epithelial cells, we expressed a fusion construct encoding Src with C-terminal mCherry in the epithelium. Confocal microscopy showed that Src-mCherry was present at the apical and lateral membrane of WT epithelial cells (n = 40 cells examined in eight larvae), but was concentrated at the basal cell membrane of invasive mlt epithelial cells that formed invadopodia-like protrusions (n = 25 single cells in five larvae).Invadopodia have first been described in cells transformed with the Rous Sarcoma Virus oncogene, mlt mutant larvae (n = 40 cells in eight larvae). caSrc-mCherry (or caSrc-GFP) induced formation of actin-rich protrusions at the basal surface of the wild type epithelial cells by 78 hpf . However, none of the cells with protrusions were invasive in either 74 hpf larvae, or larvae followed to 5 d post-fertilization. The 5 d post-fertilization (dpf) transgenic larvae also had far fewer protrusions than the 74 hpf larvae . This mutant form of Src induces invadopodia formation in non-transformed cells y 78 hpf . Anti-laf larvae , indicatmlt. Mutant larvae and larvae expressing the caSrc transgene were treated with three established mammalian Src inhibitors: SU6656, PP2, and Src-I1 . PP2 blocked formation of the invadopodia-like protrusions induced by caSrc (n = 0 protrusions detected in 34 larvae), but it had no effect in mlt (n = 20 larvae). In contrast, the Src-I1 inhibitor had a pronounced effect on both the formation of the invadopodia-like protrusions in response to caSrc (0 protrusions detected in 33 larvae) and on invasion in mlt (n = 21 mlt larvae). The effect of Src-I1 was comparable to the Tks-5 knockdown . The response of mlt to Src-I1 is similar to the effect of Src knockdown in invasive breast cancer cells We next tested whether Src contributed to invasion in d Src-I1  [29]. SU compare  and S1. mlt protrusions formed in response to mechanical cues from the unregulated mutant Myh11. To test this idea, we derived transgenic mlt larvae expressing fluorescent reporters in smooth muscle Tg) and recorded intestinal peristalsis using time-lapse confocal microscopy. Contractile force in smooth muscle arises from the interaction of Smooth muscle actin (Sma) and myosin (Myh11) filaments that are anchored to the cortical cytoskeleton n = 6 larvae examined). By contrast, slow tonic smooth muscle contraction that distorted tissue architecture was evident in 76 hpf mlt mutants (n = 6 larvae examined). These findings indicated that the mutant myosin had non-regulated contractile activity that was not detected in our original in vitro motility assays Mechanical signaling modulates the formation and activity of invadopodia mlt larvae at 72 hpf (n = 6 mlt and 6 wild type larvae), as previously reported Invasive cells are already present in t 72 hpf , howevermlt we examined levels of smooth muscle regulatory proteins in the mutant and wild type intestine using Western blot analyses. The enteric nervous system regulates intestinal peristalsis by controlling levels of the phosphorylated form of the smooth muscle regulatory Myosin light chain (p-Mlc) that binds Myh11 mlt were similar to wild type at both time points , when invasion is first detected (n = 6 independent experiments). Premature h-CaD phosphorylation in mlt is consistent with the idea that resting smooth muscle tone is increased in the intestine of the mutant larvae.We next examined levels of the high molecular weight isoform of Caldesmon (h-CaD), a smooth muscle–specific actin binding protein that regulates contractile tone independently of p-Mlc mlt we inhibited translation of the mRNA encoding zebrafish Sma mlt invasive phenotype at 74 hpf  as well as epithelial expansion and cysts that occur at later stages. Western blots confirmed the efficacy of the Sma knockdown and that it did not alter levels of Myh11  of which 66% were predicted to be mlt/+; 12 of 12 genotyped larvae were mlt/+) mlt/+ larvae) mlt mutants, neither h-CaD-deficient nor L-NAME-treated heterozygotes developed epithelial cysts or other features of the advanced homozygous mlt phenotype, thus indicating that these phenotypic features require sustained smooth muscle contraction.We next asked whether enhancing contractile tone was sufficient to induce invasion in heterozygous  72 hpf; . h-CaD k 72 hpf; , similarmlt we measured tissue elasticity in intestines isolated from wild type and mutant larvae using a force displacement assay mlt larvae and their wild type siblings throughout the range of indentations tested (n = 6 mlt and 21 wild type larvae). When remodeling is first detected (74 hpf), compliance was slightly increased at the surface of the intestine but comparable at greater depths (n = 7 mlt and 12 wild type larvae examined). Values compatible with intestinal stiffening (reduced compliance) were never recorded.Matrix stiffening promotes cell invasion in breast cancer models, in part through activation of Focal Adhesion Kinase (FAK) mlt using immunostainings mlt  mutants. Among the group of upregulated genes were three that neutralize reactive oxygen species (ROS) (glutathione peroxidase (gpx), thioredoxin, and glutathione/thioredoxin reductase) and several members of the AP-1 family of transcription factors , RNA in situ hybridization , thus arguing that their expression is non-cell-autonomously activated in response to smooth muscle tension. These findings are noteworthy because there is extensive evidence linking ROS and AP-1 factors to cancer cell invasion through their regulation of expression and activation of metalloproteinases To identify signaling pathways activated by increased smooth muscle tension in  factors , which adization , and S6,dization . Fluoresmlt we treated 72 hpf larvae with the ROS quenchers, N-acetylcysteine, and Tiron. This treatment did not rescue the mlt invasive phenotype, but it also did not affect epithelial gpx expression (unpublished data), suggesting that the neutralizing compounds could not access the ROS or that the inhibition was not strong enough to inhibit redox signaling.To test if redox signaling was required to induce invasive epithelial remodeling in mlt phenocopy in homozygous wild type larvae and both were lethal during prolonged exposure. However, within 3 h of exposure both compounds induced epithelial invasion in 72 hpf mlt heterozygotes that was comparable to early homozygous mutants (mlt/+ larvae (n = 22) but absent from all +/+ wild types (n = 17). Invasion was histologically confirmed in all mlt (n = 12) larvae examined but was not present in wild types (n = 9). The treated heterozygotes did not develop the cysts and epithelial expansion typical of the late mlt phenotype (unpublished data). Together, these findings suggest that redox signaling is involved in the initial remodeling of the mutant intestine, but on its own it is not sufficient to cause the more pronounced architectural changes seen in more advanced homozygous mutants.To determine whether oxidative stress was sufficient to induce the formation of the invasive phenotype, we treated 72 hpf larvae with intracellular ROS generators menadione and LY83583  mutants . Invasivmlt we next asked whether menadione induced formation of invadopodia-like protrusions in the heterozygous epithelium. Live confocal imaging of menadione-treated heterozygous larvae showed protrusions that were identical to those present in homozygous larvae (n = 10 larvae). By contrast, the protrusions were not seen in menadione-treated wild type larvae (n = 10 larvae) or heterozygous larvae that did not receive menadione (n = 10 larvae). Interestingly, caSrc not only induced invadopodia-like protrusions in wild type epithelial cells, but also activated expression of the redox responsive gene gpx, as occurs in mlt (n = 12 larvae). These findings establish further links between Src, redox signaling, and formation of the invadopodia-like protrusions in mlt.Having established a role for redox signaling in mlt heterozygotes to menadione was that activation of redox signaling increased smooth muscle contractile tone, similar to the effect of h-CaD knockdown or Nos inhibition , as in mlt homozygotes .To test this hypothesis, we asked whether menadione induced h-CaD phosphorylation in dissociated heterozygous smooth muscle cells. Intestines were dissected from heterozygous rn blots  showed tmlt smooth muscle cells arises from an ROS-activated epithelial signal (Tg(beta-Actin:grx-roGFP2)  . This ree larvae  and S6.  mutants  and S6. mlt mutants had a pronounced response to menadione when treatment began at 74 hpf, however invasion was not detected when larvae were treated at later stages (5 dpf). We hypothesized that this was caused by a change in the responsiveness of the epithelium to redox signaling. To test whether the responsiveness of the 5 dpf epithelium to menadione was modified in a tumor model, we generated transgenic larvae that express an activated human KRAS allele in the intestinal epithelium (G12V)Tg(miR194:eGFP-KRAS). The KRAS transgenics were also homozygous for a loss of function allele of the Wnt regulator axin1KRAS allele causes intestinal epithelial cell hyperplasia in zebrafish larvae apc mutants have severe developmental delay, the axin1 mutants were used to study the combined effect of activated KRAS and enhanced Wnt signaling in older mlt heterozygotes. Prior to treatment with menadione, 5 dpf axin1 mutants that express the mutant KRAS allele (KRAS-axin larvae) and were heterozygous for the mlt mutation had intestinal epithelial hyperplasia without evidence of invasion. Remarkably, within 5 h of treatment, menadione generated a dramatic invasive response in the KRAS-axin larvae that were also heterozygous for mlt, whereas it had no effect in larvae that were homozygous for the wild type myh11 allele (KRAS-axin larvae and 0 of 8 untreated KRAS-axin larvae).Heterozygous mlt mutants is caused by a mutation of the smooth muscle myosin heavy chain gene. Initially, we attributed the non-cell-autonomous mutant phenotype to the constitutive actin-independent ATPase activity of the mutant Myh11 protein, rather than its non-regulated contractile function, because contraction was not detected in an in vitro motility assay mlt mutants. Together, these findings are consistent with the idea that invasive transformation of the mlt intestinal epithelium is triggered by a physical signal from the adjacent smooth muscle layer, and that on its own, the unregulated ATPase activity of the mutant myosin (which is independent of actin) is not sufficient to induce invasion.In previous work, we reported that invasive remodeling of the developing intestinal epithelium of zebrafish mlt from these cancer progression models in several ways. First, invasive remodeling of the mlt intestinal epithelium occurs in the absence of any oncogenic stimuli. Second, invasion in mlt is triggered by increased smooth muscle tone rather than qualitative or quantitative changes in matrix proteins. Third, we did not detect tissue stiffening or increased levels of activated Fak in mlt, thus arguing that force arising from smooth muscle tension activates a distinct mechanotransductive signaling mechanism than matrix stiffening mlt intestine Physical signaling mechanisms are increasingly recognized as important for organ development and disease mlt are not transformed, they share some features with cancer cells in that they are highly proliferative and not fully differentiated mlt larvae rescued by transient Myh11 knockdown have only a modest invasive response to tension mlt heterozygotes also did not respond to menadione, and they formed fewer invadopodia in response to Src than undifferentiated cells. Together, these findings argue that epithelial progenitor cells, and by analogy cancer cells, have an intrinsic capacity for invasive transformation in response to mechanical signals. Supporting this, the invasive response of mature epithelial cells in heterozygous 5 dpf larvae was restored by activation of oncogenic signaling pathways known to play a role in colorectal cancer in humans.Although invasive cells in mlt form before the onset of cell invasion, and immunostainings show that the protrusions are located adjacent to degraded segments of the basement membrane, through which the invasive cells migrate into the tissue stroma. The mlt protrusions are enriched in the invadopodia proteins Actin, Cortactin, and Src, and their formation requires Tks5, a Src substrate required for invadopodia formation in cancer cells. Trafficking of Mmp14, a metalloproteinase that associates with invadopodia in vitro, was also preferentially distributed to the basal region of mlt epithelial cells with protrusions. The latter finding is consistent with our previous work showing upregulated epithelial expression of Mmp14 and Mmp2 in mlt, and that metalloproteinase inhibition rescues invasion In vitro studies link cancer cell invasion to the matrix-degrading function of invadopodia, however it is not known whether invadopodia play a role in cell invasion in vivo The mechanical properties of cell culture substrates can modify the number and activity of invadopodia that form spontaneously in cancer cells Src signaling is sufficient to induce invadopodia formation in non-transformed mammalian cells mlt, as the well-characterized Src inhibitor, Src-I1, blocked matrix degradation and invasion in mutant larvae. Interestingly, Src inhibition did not disrupt formation of the invadopodia-like protrusions. The response of mlt larvae to Src inhibition therefore resembles the effect of Src knockdown in invasive breast cancer cells mlt could be explained by either their inability to access the intestine at larval stages (SU6656) or their different mechanisms of action. Src-l1 competitively inhibits ATP and substrate binding to Src, whereas PP2 only competes for substrate binding mlt, however both Src-I1 and PP2 are specific inhibitors of mammalian Src Although activated Src did not induce invasion in wild type epithelial cells, our findings indicate that Src is required for invasion in mlt to Src, we could not detect a higher level of activated Src (phospho-Tyrosine 417) in the mlt intestine by Western blot (unpublished data). One explanation for this is that Src and related kinases are already highly activated in larvae due to their function in controlling proliferation and cell polarization mlt could therefore involve the relocalization of activated Src to the basal plasma membrane. Alternatively, invasion may require the de novo activation of a relatively small localized pool of cytoplasmic Src. Supporting this idea, force application to the plasma membrane of cultured cells can rapidly activate cytoplasmic Src Although our data link invasion in mlt based on the enhanced expression of ROS sensitive genes, the requirement for Tks5, a component of the ROS producing NAD(P)H oxidase (Nox) complex, and the invasive response of heterozygotes to exogenous oxidative stress induced by ROS generators. Redox signaling has previously been linked to cancer cell invasion and metastasis through formation of invadopodia and activation of EMT regulators mlt redox signaling pathway is triggered by a non-cell-autonomous physical signaling mechanism that requires the interaction of cells within adjacent tissue cell layers. A related redox signaling pathway has been implicated in vascular remodeling in mammals mlt, contraction of smooth muscle in this model is triggered by mechanical stress applied to the adjacent cell layer (the endothelium) We identified a role for redox signaling in MYH11, similar to mlt, were reported in human colorectal cancers MYH11 mutations were found more frequently in the epithelium, which does not normally express MYH11, than in smooth muscle MYH11 mutations in colorectal cancer therefore could not be resolved by these association studies. The findings presented here argue for a primary effect of these mutations in smooth muscle rather than the epithelium. As the invasive remodeling in mlt larvae occurs without prior tumor formation, we do not consider mlt to be a model for how cancers develop, but rather a model for how cancer cells react to external physical force. Thus, mlt can be used to study how epithelial tumors progress from a localized lesion to invasive cancer. Indeed, in one study the presence of the MYH11 mutations was associated with the invasive transformation of benign adenomatous polyps Recently, heterozygous somatic activating mutations in mlt larvae to oxidative stress is an important finding from this study because it provides an example of how tissue architecture can be altered in the appropriate genetic context. Based on our model, we argue that activating mutations of MYH11 or other human smooth muscle genes could cause invasion of existing cancer in the setting of oxidative stress that is intrinsic to cancers or occurs in the setting of inflammatory conditions that promote cancer formation The response of heterozygous G12V (a generous gift from Steven Leach) in the intestinal epithelium was driven by a 2 kb promoter fragment from the zebrafish miR194 gene. Expression of GFP in smooth muscle was driven by a promoter fragment from the zebrafish sm22-alpha gene beta-actin gene axin1 mutants All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the animal welfare committee at the University of Pennsylvania School of Medicine. Larvae were raised at 28°C in E3 medium 3 dpf old larvae were anesthetized with 0.1 mg/ml Tricaine, fixed in 4% PFA/PBS, washed in PBST (PBS+0.1% Tween), dehydrated in methanol, and stored at −20°C. For whole mount staining with anti-laminin and anti-cytokeratin antibodies, larvae were washed in PBST and permeabilized by a 15-min Proteinase K digestion (100 ug/ml in PBST). They were then rinsed in PBST and postfixed in 4% PFA/PBST. The skin above the trunk and intestine was removed using fine forceps. Larvae were stained with antibody in 10% goat serum/PBST. The laminin antibody (Sigma #L-9393) was used at 1∶50 or 1∶200 dilution; the cytokeratin antibody  was used at 1∶100 dilution. The Cortactin antibody  was used at 1∶100. For FAK staining, larvae were dehydrated in −20°C acetone for 30 min followed by staining in 2% BSA/PBST. Anti-phospho-Fak (Y397)  was used at 1∶200. Secondary antibodies were labeled with Alexa 568 or 488 (Molecular Probes/Invitrogen). Histological analyses of the mounted specimens were performed as described Confocal scans of live larvae and sections of immunostained larvae were performed using a Zeiss LSM710 laser scanning microscope. For time lapse microscopy, 3 dpf larvae were anesthetized with 0.1 mg/ml Tricaine mlt and wild type intestinal epithelial cells, single confocal scans were imported into the ImageJ software. Crops of single cells were copied to a new file and mean mCherry fluorescence was measured in the apical 2/3 and basal 1/3 of each cell. To measure ROS levels in epithelial cells transgenic fish expressing Grx1-roGFP2 were crossed to heterozygous mlt fish and their offspring treated with menadione as described below. Larvae mounted for confocal microscopy were excited with 408 and 488 nm lasers, and the emission at 500–530 nm was calculated  in E3 media for 3 h (3 dpf larvae) or 5 h . Larvae were bathed for 3 h in 10 mM l-NAME (Sigma N-5751) dissolved in E3/10 mM Tris pH 7.2.Whole mount in situ hybridization was performed as described acta2-morpholino was from Open Biosystems  was designed against the smooth-muscle-specific exon of Caldesmon (based on acc. # BC158175), which was identified from intestinal smooth muscle cDNA. Injection of ∼20 pg caused an in-frame deletion of this exon but had no effect on transcript of the low molecular weight isoform tks5 was a generous gift from Danielle Murphy and Sara Courtneidge and was injected as described Morpholinos were injected as described mlt larvae were measured using a microprobe indenter device t test for data sets with unequal variance was performed to determine the significance of differences between Young's moduli of wild type and mlt intestines samples.The elastic moduli (Young's modulus) of intestines isolated from wild type and RNA was isolated from trunk sections of 74 hpf larvae that encompassed the mid- and posterior intestine. Six pairs of mutants and wild type siblings were analyzed (25–35 trunks per sample). RNA was recovered using Trizol (Invitrogen) and Qiagen RNeasy columns. Probes were hybridized to the Affymetrix zebrafish genome array (Affymetrix #900487). The array data were evaluated by importing affymetrix *.cel files into genespring v.7.3.1, and expression intensities were calculated using gcrma for each probe set. Gene expression data were validated by quantitative RT-PCR using sybr green and by RNA in-situ hybridization.tata-box binding protein cDNA (tbp) was used as internal standard. PCR amplification and data analysis were performed as described t test for data sets with unequal variance.RNA was recovered from intestines manually dissected from larvae using Trizol and Qiagen RNeasy columns. For each PCR amplification RNA from 15 or more larvae was pooled. An amplified fragment from the glutathione peroxidase: 5′ gctgttcagcctggactttt; 3′ QPCR cgttgctgagtttggactttt; 3′ in situ ctcagatgaacgagctgcacjun B: 5′ tctgttgggttacggtcaca; 3′ QPCR cgtctggatgatgagcctct; 3′ in-situ ggaccttctgcttgagttgcggggacaagtttgtacaaaaaagcaggctgccaccatgggtggagtcaagagtaa; 3′ ggggaccactttgtacaagaaagctgggtcgaggttttctccgggttggta; 3′ Y528F ggggaccactttgtacaagaaagctgggtcgaggttttctccgggttggaattgtggttc.src (including attB sites): 5′ 6 hpf (shield stage) zebrafish embryos were sterilized by two 10-min washes with 0.1% sodium hypochlorite and raised in sterile embryo medium. Intestines were dissected from sterilized embryos at 74 hpf and dissociated in 0.25% trypsin/EDTA. Cells were collected by centrifugation for 5 min at 800 g and treated with 1.5 µM menadione for 1 h in DMEM supplemented with 10% FBS, 100 ug/ul pen/strep, and 0.2% amphotericin B. Treated cells were centrifuged and immediately fixed in sample buffer and stored at −80°C for Western blots.Figure S1tks5 knockdown rescues epithelial invasion in the intestine of mlt mutant larvae. (A–C) Lateral images of live 5 dpf larvae. Control morpholino injected mlt larvae show cystic expansion of the intestinal epithelium  (B), while only a small number of cysts can still be detected in the tks5 morpholino injected mlt larvae . The majority of the posterior intestine in these larvae resembles WT . Findings confirmed histologically .(PDF)Click here for additional data file.Figure S2Tg(miR194:Lifeact-GFP) with mosaic expression of a caSrc-mCherry transgene. Only a small number of invadopodia-like protrusions are detected . (B) Quantification showed that invadopodia-like protrusions were detected in 89% of caSRC-mCherry positive cells in 3 dpf larvae (n = 28 cells in 5 larvae), while they were present in only 9% of cells in 5 dpf larvae (n = 64 cells in 5 larvae). Error bars, standard deviation.Src induces less invadopodia-like protrusions in mature intestinal epithelial cells than in epithelial progenitor cells. (A) Sagittal confocal scans through the intestine of a 5 dpf (PDF)Click here for additional data file.Figure S3sma) knockdown rescues mlt mutants. (A–C) Western blot showing reduced Sma protein in the intestine of 74 hpf and 4 dpf Sma MO injected larvae  compared to a control MO . Myh11 protein levels are unaffected by the Sma knockdown (lanes 1–4). Each lane contains protein extracted from the intestines dissected from 30 larvae. This blot is representative of two independent experiments.Smooth muscle actin ((PDF)Click here for additional data file.Figure S4mlt heterozygotes. Lateral views of 5 dpf WT (A) and mlt/+ (B) larvae that had been injected with a splice-blocking morpholino (MO) that specifically targets h-CaD. Intestinal morphology is normal in the WT larva, whereas there is cystic expansion of the mlt/+ intestine. Histological analyses confirmed invasive expansion of the mlt/+ intestine (mlt/+ intercross (n = 240) of which 66% were predicted to be mlt/+. 12 of 12 genotyped larvae were mlt/+.h-CaD knockdown triggers invasion in ntestine . Invasio(PDF)Click here for additional data file.Figure S5mlt larvae compared with WT. beta-Actin serves as loading control. (B–D) Histological sections show no activation of FAK by phosphorylation  in the epithelium of WT or mlt larvae at 74 hpf. Basement membrane detected with anti-laminin staining (red) . (E) Lower power image showing expected p-FAK (green) in the myoseptum of a WT larva.Invasive remodeling occurs without changes in matrix composition or FAK activation. (A) Western blot showing normal levels of Type 1 Collagen (Col1) and Fibronectin (Fn) in the intestine of 74 hpf (PDF)Click here for additional data file.Figure S6mlt intestine. (A–D) Whole mount in situ hybridization shows strong expression (blue) of the AP-1 gene junB and the ROS activated gene gluthatione peroxidase (gpx) in the intestine of 74 hpf mlt  but not WT  larvae. n = 15 mlt and wild type larvae examined.  Histological cross-sections of whole mount specimens processed for fluorescent RNA in situ hybridization show strong junB expression (green) in mlt intestinal epithelial cells with only low level expression in smooth muscle cells (labeled red by myh11 expression).  Similarly, gpx expression (green) can only be detected in the mlt intestinal epithelium. n = 12 mlt and 12 wild type larvae examined. (I) Quantitative RT-PCR shows increased junB expression in the intestine of mlt homozygotes. junB expression is also increased in menadione treated wild type larvae (+/+), and to a greater degree in mlt heterozygous larvae . (J) Western blot showing phosphorylation of several components of the Map-Kinase signaling pathway in intestines dissected from mlt larvae at 74 hpf and 96 hpf. ERK is prematurely activated at 74 hpf in mlt. p38 (Mapk), Jnk, and Sapk are strongly activated in mlt but not WT larvae at 74 hpf and 96 hpf. beta-Actin, loading control. Western blots are representative of between two and four independent experiments.AP-1 transcription factors, ROS responsive genes, and MAP-Kinase signaling are activated in the (PDF)Click here for additional data file.Figure S7gpx expression in WT larvae that express caSrc and homozygous mlt larvae. (A–C) Whole mount RNA in situ hybridization for gluthatione peroxidase expression . gpx expression is low in the WT intestine (A), whereas it is markedly elevated in the mlt intestine  Mosaic expression of a caSrc transgene induces gpx expression in the wild type intestine . Interestingly, expression of caSrc in the pronephric duct (via the miR194 promoter) also induces gpx expression . n = 12 larvae.Comparable induction of (PDF)Click here for additional data file.Movie S1Epithelial actin cytoskeleton in the wild type larval intestine. Sagittal 3-D renderings through the posterior intestine of a 5 dpf wild type Lifeact-GFP transgenic larva. Lifeact-GFP is principally located in the brush border of the epithelial cell apical plasma membrane.(AVI)Click here for additional data file.Movie S2Invadopodia-like protrusions arise from the basal intestinal epithelial cell plasma membrane of mlt larvae. Sagittal 3-D renderings through the posterior intestine of a 5 dpf mlt Lifeact-GFP transgenic larva. In mlt epithelial cells Lifeact-GPF labels basal invadopodia-like protrusions in addition to the apical plasma membrane.(AVI)Click here for additional data file.Movie S3mlt epithelial cells. Sagittal confocal scans through the intestine of mlt larva expressing Lifeact-GFP in the intestinal epithelium (Tg(miR194:Lifeact-GFP)), beginning at 72 hpf. Initially, Lifeact-GFP (green) bound to Actin is only present in the epithelial cell apical brush border. As development proceeds, Actin-rich invadopodia-like protrusions form at the basal epithelial cell membrane and persist for several hours. Time lapse movie; 1 s corresponds to 15 min real time; duration is 5 h.Formation of actin-rich invadopodia-like basal membrane protrusions in (AVI)Click here for additional data file.Movie S4Formation of actin-rich invadopodia-like protrusions precedes invasion of mlt epithelial cells. Sagittal confocal scans through the intestine of mlt larva expressing Lifeact-GFP in the intestinal epithelium (Tg(miR194:Lifeact-GFP)), beginning at 76 hpf. Actin rich protrusions are present at the basal membrane, and the protrusions frequently precede the development of invasive lesions (arrows). Time lapse movie; 1 s corresponds to 15 min real time; duration is 5 h.(AVI)Click here for additional data file.Movie S5Formation of actin-rich invadopodia-like protrusions precedes invasion of mlt epithelial cells. Sagittal confocal scans through the intestine of mlt larva expressing Lifeact-GFP in the intestinal epithelium (Tg(miR194:Lifeact-GFP)), beginning at 74 hpf. Actin-rich protrusions are present at the basal membrane, and the protrusions frequently precede the development of invasive lesions (arrows). Time lapse movie; 1 s corresponds to 15 min real time; duration is 5 h.(AVI)Click here for additional data file.Movie S6Tg(miR194:Lifeact-GFP)), beginning at 74 hpf. Actin bound Lifeact-GFP is localized to the epithelial cell apical brush border membrane . 1 s corresponds to 15 min real time, duration is 5 h.Lifeact-GFP localization in the WT intestinal epithelium. Sagittal confocal scans through the intestine of WT larva expressing Lifeact-GFP in the intestinal epithelium ((AVI)Click here for additional data file.Movie S7Tg). Time lapse movie; 1 s corresponds to 9 min real time.Peristaltic circular smooth muscle contractions in the 76 hpf WT intestine. Sagittal confocal scans through the intestine of a 76 hpf WT larva expressing GFP (green) in smooth muscle cells and mCherry (red) in the intestinal epithelium ((MOV)Click here for additional data file.Movie S8mlt intestine. Sagittal confocal scans through the intestine of a 76 hpf mlt larva expressing GFP (green) in smooth muscle cells and mCherry (red) in the intestinal epithelium ). Slow tonic contraction of the intestinal smooth muscle disrupts intestinal architecture. Time lapse movie; 1 s corresponds to 9 min real time.Constitutive smooth muscle contraction in the 76 hpf (MOV)Click here for additional data file.Movie S9Tg). Time lapse movie; 1 s corresponds to 9 min real time.Absence of smooth muscle contraction in the 72 hpf WT intestine. Sagittal confocal scans through the intestine of a 72 hpf WT larva expressing GFP (green) in smooth muscle cells and mCherry (red) in the intestinal epithelium ((MOV)Click here for additional data file.Movie S10mlt intestine. Sagittal confocal scans through the intestine of a 72 hpf mlt larva expressing GFP (green) in smooth muscle cells and mCherry (red) in the intestinal epithelium ). Epithelial cell invasion is already present at this stage. Time lapse movie; 1 s corresponds to 9 min real time.Absence of smooth muscle contraction in the 72 hpf (MOV)Click here for additional data file.Table S1mlt (1.5-fold or greater). Array signal ratio (mlt:WT) shown in Column C; data are mean of six experiments. Gene expression ratio (mlt:WT) as measured by quantitative PCR, Column D (74 hpf) and E (80 hpf). EST annotation of microarray probe .Transcriptional profiling identifies genes with increased expression in (XLS)Click here for additional data file.