Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual’s response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after therapeutic radiation. Management of radiological causalities that could occur from natural calamities, failures in operational safety mechanisms of nuclear power plants or even a terrorist attack require immediate intervention from emergency responders and medical personnel. The damage caused by a meltdown can be catastrophic as it could release large amounts of radioactivity that quickly affects the environment and the health of surrounding population. Recent events involving the Fukushima Daiichi nuclear reactor have shown the unfortunate and immediate dangers posed by accidental radiation exposure. Nuclear exposure management protocols include rapid dose assessment for the affected population and identification of the individuals who require immediate medical attention. Development of robust biomarkers based on an individual’s biological response is crucial for accurate assessment of the level of exposure and making important medical decisions. A personalized assessment will allow evaluation of an individual’s physiological response to radiation damage. The calculated LD50 for humans exposed to total body irradiation is in the range of 4.0 to 4.5 Gy and the dose range at which supportive care will be effective is narrow. Therefore, development of biomarkers for fast and accurate dose assessment is critical. Moreover, an individual’s response varies depending on many confounding factors such as immune status, age and genetics. These factors will ultimately determine a person’s apparent response to exposure, and in some cases victims may not immediately exhibit visible signs of radiation damage. Therefore, physical dosimetry alone or the available protein markers such as cytokines have limitations to accurately estimate the dose and response of an individual.Acute effects  will manifest themselves as Hematopoietic, Gastrointestinal (GI) and Cerebrovascular syndromes. Studies have shown that individuals exposed to an intermediate dose (5–8 Gy) could die within a few weeks due to GI syndrome. Lower doses (2–5 Gy) that are not immediately lethal but compromise the hematopoietic system can increase susceptibility to infection and death within months if supportive care is not provided on time Over the last several years, there have been attempts to estimate the radiation dose exposed using hematological, biochemical and cytogenetic parameters microRNAs (miRNAs) are non-coding RNAs of 19–22 nucleotides that were originally identified as regulators of gene expression by inducing cleavage of their target mRNA or blocking translation through base pairing to partially complementary sequences Mus musculus. Male inbred mice  were co-housed (five per standard cage) and fed ad libitum. Mice were exposed to total body gamma radiation (TBI) using GammaCell@40 irradiator (Cesium −137 Source) at a dose rate of 1.1 Gy/min). For each radiation dose  and time point (24 and 48 hrs) a minimum of five animals were used. Control animals were sham-exposed. For investigating the effect of fractionated dose, 15 animals were exposed to X-rays (in 2 Gy fractions) from a RS-2000 Biological Irradiator at a dose rate of 1 Gy/min. All the animal experiments were done with strict adherence to the institutional guidelines established and approved by the Ohio State University Animal Care and Use Committee (Permit number: 2011A00000029).For animal studies involving acute single dose exposure, we used 8–9 week old Blood was collected by submandibular bleeding or by cardiac puncture. Following coagulation (1 hr at room temperature), serum was separated using microtainer tubes (BD Biosciences) by centrifugation at 10,000 g for 10 min, and then frozen at −80°C. RNA was extracted using the Qiagen miRNA easy kit following the manufacturer’s protocol. miRNAs were isolated from serum samples collected from 4–5 animals for each time points, and samples with high levels of hemolysis were excluded from analysis. In a typical isolation procedure, 100 µl serum was used. After lysis using QIAzol reagent, 4–20 pg synthetic oligonucleotides (spike-in oligos) Osa-miRNA-414, Cel-miRNA-248, At-miRNA-159a (Integrated DNA Technologies) were added prior to extraction. RNA was eluted in 100 µl water and concentrated to 20 µl and 3 µl was used for each assay for profiling using nanoString Technologies’ multiplexed nCounter platform. The platform incorporates fluorescent barcodes together with a digital readout for single-molecule imaging The digital multiplexed nanoString nCounter mouse miRNA expression assay (nanoString Technologies) was performed with 10–30 ng total RNA isolated from a net volume of 20 µl serum as input material. Small RNA samples were prepared by ligating a specific DNA tag (miR-tag) onto the 3′ end of each mature miRNA according to the manufacturer’s instruction. These tags serve several purposes: they normalize the wide range of melting temperatures (Tms) of the miRNAs, provide a template to facilitate the use of the nanoString dual probe system, enable single base pair discrimination and specificity of highly homologous miRNA family members, and identify each miRNA species. Excess tags were removed by restriction digestion at 37°C. Hybridizations were carried out by combining 5 µl of each miRNA-miRTag sample with 20 µl of nCounter Reporter probes in hybridization buffer and 5 µl of nCounter Capture probes  overnight at 65°C for 16–20 hrs. Excess probes were removed using two-step magnetic bead based purification on the nCounter Prep Station (nanoString Technologies). Abundances of specific target molecules were quantified on the nCounter Digital Analyzer by counting the individual fluorescent barcodes and assessing the target molecules. For each assay, a high-density scan encompassing 600 fields of view was performed. The data was collected using the nCounter Digital Analyzer after taking images of the immobilized fluorescent reporters in the sample cartridge with a CCD camera.miRNA data analysis was performed using the nSolver software analysis, freely available from nanoString Technologies. The serum miRNA profiling data was normalized using the average signals obtained from three spike-in oligos, and miRNAs that gave significant hybridization signals were used for downstream analysis. ANOVA was performed with a cutoff p-value of 0.05 to identify a set of miRNAs that had the highest difference in means across samples. Coefficient of variance across samples was also performed with a cutoff of 0.4 and overlapping sets of miRNAs from the above two methods were selected as the most significant set. R software was used for the analysis.The digital multiplexed nanoString nCounter mouse miRNA expression assay was performed on total RNA isolated from 20 µl of serum usually containing a total amount of 10–30 ng of RNA. The nCounter multiplex platform is capable of detecting approximately 600 mouse specific miRNAs, four housekeeping genes and three non-mammalian miRNAs: Osa-miRNA-414, Cel-miRNA-248 and At-miRNA-159a. During RNA isolation, synthetic oligonucleotides (spike-in oligos) corresponding to Osa-miRNA-414, Cel-miRNA-248 and At-miRNA-159a were included as controls allowing the normalization of samples. The amounts of spike-in oligos were optimized by comparing their counts with that of endogenous miRNAs in serum samples. The optimal amount of spike-in oligos for normalization was identified to be 0.5–2 pg in each reaction (data not shown). The inclusion of probes hybridizing to the house keeping genes enabled us to further identify and separate preparations with cellular RNA contaminations. The optimized method allowed detection of changes in serum miRNAs that are specific to changes in physiological and treatment conditions, such as response to radiation. The purity and integrity of the RNA recovered from serum samples was validated on an small RNA bioanalyzer . miRNAs The nCounter expression profiling conducted on total RNA isolated from mice serum samples identified 88 miRNAs with high confidence. miRNA-451 was found to be the most abundant in serum preparations, contributing to 22–23% of total miRNAs . miRNA-1Using the nCounter multiplex assay, miRNAs in serum samples from control and irradiated animals collected 24 hrs after 1, 2, 4, 6 and 8 Gy total body irradiation (TBI) were compared. In order to minimize experimental error, irradiation, serum collection, RNA isolation, miRNA profiling, and normalization were done in parallel with controls and treatment groups. Samples with traces of cellular RNA contamination (with counts of 30 or above for any of the four housekeeping genes) were excluded from the analysis. Samples with high levels of hemolysis observed visually or based on relative abundance of miRNA-451 (>23%), miRNA-16 (>13%) correlating with increase in miRNA-25, miRNA-106b, let-7 g and miRNA-93 were also excluded from analysis.p-value of 0.05 to identify a set of miRNAs that had the highest difference in means across samples. Next, the coefficient of variance was calculated with a cutoff of 0.4 , 48 hrs (4×2 Gy = 8 Gy) and 72 hrs (6×2 Gy = 12 Gy) was analyzed for changes in miRNAs using the multiplex nCounter platform . Serum cThe current study has identified several evolutionarily conserved miRNAs responsive to acute radiation in a dose range relevant to accidental radiation exposure or clinical radiation therapy. Identification of serum abundant radio-responsive and non-responsive miRNAs together with spike-in oligos provide a panel of markers and controls for developing radiation biodosimeters. This will aid rapid diagnostic screening to identify individuals who are at risk of developing acute radiation syndromes. Accurate dose evaluation is critical for making medical decisions and timely administration of mitigators to prevent or reduce the acute and late effects. Individual miRNAs such as miRNA-150 alone or in combination with other markers have the potential to estimate the dose to which the individual was exposed. The majority of serum miRNA markers did not respond to radiation, but the hierarchical clustering has identified several markers, potentially originating from blood cells, exhibiting dose- and time- sensitive responses to acute single or fractionated dose. In this study, 24 and 48 hr time points were used, which are realistic time frames to collect blood samples in a scenario involving mass causality from radiation exposure. miRNA-150 depletion kinetics indicate that the response is fast and robust with a near complete depletion in 48–72 hrs with 8 Gy acute dose and 8–12 Gy fractionated dose. The evaluation of the kinetics of depletion of miRNA-150 during three days of fractionation, using a schedule followed in a clinical setting, signifies the translational potential of this marker. In addition to chemo-based approaches, fractionated total body irradiation is used for conditioning in patients undergoing bone marrow transplantation. At the same time, management of hematopoietic injury is a major clinical question in both chemo and radiation based cancer therapies. Recent studies show that miRNA-150 control hematopoiesis We propose miRNA-150 as a sensitive marker for damage to the hematopoietic system, which is the most radiosensitive organ/system. The biodosimetry potential of miRNA-150 is evident from its time and dose dependent depletion, correlating with lymphocyte depletion kinetics Cells are likely in a constant state of releasing miRNAs through secretory vesicles Although the comparison of miRNA in body fluids has tremendous translational potential, especially for early prediction of diseases and evaluation of response to treatments, there were major issues associated with quantification of low abundant extracellular RNA. Since miRNAs are in low abundance in body fluids, PCR based assays do not provide good quantification with acceptable Ct values. Therefore, previous studies relied on templates that were subjected to pre-amplification


Purpose. In neocortical epilepsies not satisfactorily responsive to systemic antiepileptic drug therapy, local application of antiepileptic agents onto the epileptic focus may enhance treatment efficacy and tolerability. We describe the effects of focally applied valproate (VPA) in a newly emerging rat model of neocortical epilepsy induced by tetanus toxin (TeT) plus cobalt chloride (CoCl2). Methods. In rats, VPA (n = 5) or sodium chloride (NaCl) (n = 5) containing polycaprolactone (PCL) implants were applied onto the right motor cortex treated before with a triple injection of 75 ng TeT plus 15 mg CoCl2. Video-EEG monitoring was performed with intracortical depth electrodes. Results. All rats randomized to the NaCl group died within one week after surgery. In contrast, the rats treated with local VPA survived significantly longer (P < 0.01). In both groups, witnessed deaths occurred in the context of seizures. At least 3/4 of the rats surviving the first postoperative day developed neocortical epilepsy with recurrent spontaneous seizures. Conclusions. The novel TeT/CoCl2 approach targets at a new model of neocortical epilepsy in rats and allows the investigation of local epilepsy therapy strategies. In this vehicle-controlled study, local application of VPA significantly enhanced survival in rats, possibly by focal antiepileptic or antiepileptogenic mechanisms. The effective treatment of focal neocortical epilepsies not satisfactorily responsive to systemic antiepileptic drug (AED) therapy remains a considerable clinical challenge. Surgical removal of the epileptogenic zone is not an ideal alternative option to pharmacotherapy if eloquent brain areas are involved. Moreover, oral administration of AEDs at dosages high enough for adequate seizure control may be associated with intolerable systemic side effects. Emerging therapeutic strategies aiming at enhanced efficacy and tolerability therefore include the local application of AEDs directly onto the individually identified epileptic focus. To systematically evaluate such a local epilepsy therapy in animals, we have recently constructed particular valproate (VPA) polycaprolactone (PCL) implants and determined their VPA release kinetics .2) powder to the cortex, the authors had to deal with a considerable attrition in the number of rats. In contrast to cobalt-based focal epilepsy models, Nilsen et al. [The development of novel antiepileptic agents and therapeutic strategies in humans relies on the preclinical application of adequate animal models . Howeven et al. . reporte2. Preliminary results with this novel methodological approach encouraged us to examine the antiepileptic and possibly antiepileptogenic effects of VPA applied via PCL implants at the neocortical site treated with TeT and CoCl2. We here report on the methods and results of a vehicle-controlled pilot study. We hypothesized that the local application of VPA onto a neocortical epileptic focus, induced by TeT and CoCl2, might reduce interictal epileptic activity in intracranial EEG as well as video documented seizure frequency and severity. Consequently, a reduction of seizure-related deaths could possibly be expected in rats focally treated with VPA.We therefore intended to develop a new rat model of focal neocortical epilepsy by combining a triple injection of TeT in the primary motor cortex together with the cortical application of CoClAll animal procedures were carried out according to the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the local ethics committee .μL .TeT was provided by Quadratech Diagnostics Ltd  at a working concentration of 100 ng/2 with a diameter of about 4 mm.Cobalt(II) chloride hexahydrate  was dissolved in water at portions of 15 mg each. Evaporation of this solution in standard Eppendorf tubes  resulted in round slices of CoClElectrodes consisted of teflon-coated silver wires  with deinsulated tips. PCL implants  containing either VPA or sodium chloride (NaCl) 10% w/w were produced by a novel 3D-bioplotting technology , leading to continuous local VPA release [Ten male Sprague-Dawley rats  at a mean age of 100 days and a mean weight of 250 g on the day of surgery were included. Animals were kept in standard animal facilities (one rat per cage after surgery) under controlled temperature of 21°C, with a 12-h light/dark cycle and free access to food and water.Rats were anesthetized with 5 vol% isoflurane and placed in a small animal stereotaxic frame assembly . Further surgery was performed with 1.5–2.5 vol% isoflurane and 1-2 L/min oxygen. After exposing the skull, hydrogen peroxide was applied, using its haemostatic and antiseptic effect to prepare the operating field. During surgery, 5 mL NaCl 0.9% were injected subcutaneously (s.c.) for compensation of a possible fluid loss. Directly after surgery, 0.015 mg buprenorphine was given s.c. for analgesia. All coordinates given below for local TeT injection or electrode position relate to the bregma and were determined according to a stereotaxic atlas of the rat brain .A hole (5 mm diameter) was carefully countersunk with a mill in the skull above the right primary motor cortex . Care was taken not to injure the cortical surface. While drilling, the mill was constantly cooled with saline solution. The meninges were removed with a dural hook. Drying out of the cortical tissue was prevented by regular administration of saline solution. μL each was injected stereotaxically 1 mm below the neocortical surface , using a 2 μL Hamilton syringe . A total amount of 225 ng TeT in three volumes of 0.75 2 was laid onto the motor cortex and slightly pushed down by a PCL implant (containing either VPA or NaCl) by which the bore was sealed.Afterwards, a 15 mg round slice of CoClFor electrocorticography (ECoG) und electromyography (EMG) recordings, six electrodes were placed in each rat . Three iAll electrodes were linked to a 6-pin plug connector  with two anchor bars. To assure permanent fixation of the electrodes, the operating field was sealed with dental acrylate .For simultaneous recording of the postoperative behaviour of the rats, of the ECoG and of the EMG, rats were placed in individual transparent cages. The cranial electrode assembly was connected to an MPA8I preamplifier , a slip-ring , and a PGA32 amplifier , coupled to an analog-to-digital converter CED Power1401 . Signals were amplified (×5000), filtered (band-pass of 1 Hz–5 kHz), and digitized at a sampling rate of 5–10 kHz. Data were stored and then visually analyzed in a referential montage to REF using the CED Spike2 software, version 5 . The behaviour of the rats was monitored simultaneously with a digital high definition video camera recorder  and synchronized with the ECoG and EMG traces. From the first day after surgery, animals were scheduled for video-ECoG monitoring every three days for a period of three weeks and at least once a week thereafter. Each monitoring session lasted at least two hours. ECoG sections disrupted by relevant movement or other artifacts were excluded from the evaluation.ECoG and video recordings were visually analyzed by an epileptologist (blinded regarding treatment until day 21 after surgery) and an assistant trained in ECoG interpretation in rats. The analysis focused on a qualitative and quantitative evaluation of both interictal and ictal activity and seizure semiology. Efforts were made not to mistake artifacts for epileptic activity. Interictal epileptic activity was defined as unambiguous epileptiform potentials characterized by single or multiple spikes followed by a slow wave and clearly interrupting background activity with respect to amplitude and frequency. By definition, ictal seizure patterns consisted of runs of repetitive spiking or rhythmic activity with a dynamic evolution in frequency and topography. Seizure patterns without any overt clinical manifestation as assessed by simultaneous video recordings were defined as subclinical. After identification, epileptiform potentials and subclinical and clinical seizures were counted. Additionally, seizure duration was determined, and semiology was correlated with ictal ECoG topography.2 into the tissue, the PCL implants had direct contact to the neocortex and released their content continuously towards the tissue. The assignment of the individual rats to local VPA (n = 5) or NaCl (n = 5) was random.The PCL implants closed the bore above the right primary motor cortex and contained either VPA or NaCl 10% w/w. After regional diffusion of CoClP value less than 0.05 was considered significant.Rat survival as a function of treatment was determined as Kaplan-Meier estimates, and differences in the survival curves were evaluated with this method. There were no censored data. A 2 rats randomized to the NaCl group died within one week after surgery . In contrast, the TeT/CoCl2 rats treated with VPA survived significantly longer (P < 0.01), with postoperative death occurring on days 7, 11, and 12 or sacrifice after more than 5 months (n = 2). A plot of the Kaplan-Meier estimate of the survival function is shown in All TeT/CoCln = 3), exitus took place during status epilepticus  or was probably seizure-related . Neither in the VPA nor in the NaCl group any relevant complication had occurred along with the invariable surgical procedures. When witnessed  and were systematically analyzed. The interanimal variability of the total duration of the individually analyzed ECoG recordings is explained by differences in the extent of artifacts and different survival times. The screening of video-ECoG recordings beyond 30 postoperative days in two rats of the VPA group did not yield any subclinical or clinical seizures or a relevant further change of interictal epileptic activity. In the resting ECoG, a physiological theta rhythm more or less intermingled with alpha and delta activity was prevailing in all animals. 2 rats with available ECoG recordings showed clear-cut interictal epileptiform potentials. Examples of their interindividually varying specific appearance are depicted in μV.All TeT/CoCl2. In all other rats, epileptiform potentials were predominantly located at electrodes outside the TeT/CoCl2 application site or were distributed diffusely. In one animal , the leading interictal epileptic focus was identified at the electrode within the right motor cortex treated with TeT and CoClTheir frequency of occurrence varied interindividually but constantly reached a peak between days 5 and 7 (range: days 1–10) after surgery . In this2-treated rats surviving the first postoperative day developed epilepsy, by definition. The latency between TeT/CoCl2 application and epilepsy onset lay in the range between days 1 and 5 but could not be defined precisely as video-ECoG monitoring was noncontinuous. Therefore, the first unprovoked seizure in a given rat may have been missed. In the two long-term surviving rats of the VPA group, the latest seizures sampled during repeated monitoring epochs occurred on days 8 and 11, respectively. Definite electroencephalographic seizure patterns were documented during sampling in 2 out of 3 rats of the NaCl group and in 4 out of 5 rats of the VPA group. They occurred spontaneously beyond the acute surgery and toxin administration period and were often clearly associated with clinical seizure symptoms in all affected rats. Thus, at least 75% of the TeT/CoClECoG seizure patterns consisted of repetitive spiking or rhythmic activity (most commonly in the delta or theta range) with clear evolution in rhythm frequency and spatial distribution and with durations between 2 and 189 seconds. Their specific appearance varied among individual animals. Examples of individual subclinical and clinical ECoG seizure patterns are shown in 2 application site only twice . Predominantly, ECoG seizure patterns involved initially the ipsilateral electrode outside the TeT/CoCl2 application site or were distributed diffusely at all intracranial electrodes from the beginning onwards. Seizure onset, as determined by the three depth electrodes, was unequivocally localized at the TeT/CoClThe frequency of subclinical ECoG seizure patterns and patterns associated with clinically overt seizures varied interindividually. Typically, there was a peak between postoperative days 5 and 8 . One rat of the VPA group, additionally, exhibited intermittent ictal left-sided facial twitching  survived without local application of VPA more than three weeks. Continuous video-ECoG monitoring in this rat documented frequent unprovoked clonic seizures for 12 days after surgery  no interictal epileptic activity and no subclinical or clinical seizures were detected by video-ECoG during three weeks after surgery. In addition, in a sham-operated rat  because such calculations may be hampered by the risk of erroneously involving artifacts or mistaking physiological for epileptic activity. Driven by a clinical approach in the visual analysis of intracranial EEG and seizure semiology (similar to that applied for invasive video-EEG monitoring in humans), we were able to document frequent clear-cut interictal and ictal epileptic activity as well as recurrent spontaneous seizures in the major part of TeT/CoClAlthough some interanimal variability in electrocorticographic and behavioural seizure characteristics was observed, initial seizure semiology in all rats typically consisted of focal rhythmic clonic or tonic motor phenomena. In contrast to Nilsen et al. , we did 2 approach predominantly found at electrodes outside the TeT/CoCl2 application site? A prominent contralateral epileptic focus following unilateral frontal but not parietal cobalt application was also described by Dow et al. [2-induced necrotic process adjacent to the “focal” electrode. The implantation of multiple “focal” and “extrafocal”  depth electrodes, as established in our TeT/CoCl2 setup, might therefore be crucial for a reliable detection of interictal and ictal neocortical epileptic activity. Why were the epileptic discharges in our TeT/CoClw et al. . Notewor2-treated rats surviving the first postoperative day developed epilepsy with recurrent spontaneous neocortical seizures. Since Tamargo et al. [2 alone, though at higher dosage (20 mg instead of 15 mg), the additional triple injection of TeT in our novel approach may have increased epileptogenesis. On the other hand, after insertion of cobalt wires in the left motor cortex, Chang et al. [Despite a rather small number of compared animals (5 in each group), we have proven that the majority (75%) of TeT/CoClo et al.  reportedg et al.  observedg et al. . 2 model described here resembles that seen earlier in cobalt-treated rats [2 is not yet possible, as only rats treated with the antiepileptic drug VPA survived longer than one week. However, in an additional rat with neocortical administration of only 187.5 ng TeT and 7.4 mg CoCl2, but no local VPA treatment, distinct clinical seizures have occurred far beyond one week after surgery. This also argues against the assumption that the documented seizures might be directly provoked by the initially administered toxins. Instead, toxin-induced secondary epileptogenic processes leading to recurrent spontaneous seizures are more likely. As shown in a sham-operated rat without TeT/CoCl2, the surgical procedure (including anesthesia) alone does not induce interictal or ictal epileptic activity. Currently, it remains speculative whether a possible idiosyncratic or supra-additive epileptogenic pathomechanism is specifically induced by the combination of TeT and CoCl2. Overall, the course of the focal neocortical epilepsy induced in the TeT/CoClted rats –7, 10–132 and with continuous long-term video-ECoG monitoring are necessary. These studies must further validate our TeT/CoCl2 approach as suitable animal model and will determine whether additional TeT, in comparison with the CoCl2 component alone, provides decisive advantages with respect to the induction rate of focal neocortical epilepsy and its duration. By means of thorough histopathological analyses, we are also planning a systematic evaluation whether the degree of epilepsy in the evolving model correlates with the extent of morphological lesions induced by TeT plus CoCl2 or whether traumatic neocortical injury induced by surgery may play an additional role in epileptogenesis.We are aware that further studies with lower and presumably less lethal dosages of TeT and CoClin vitro; 88% of VPA were released after 4 days [In vivo, however, these high release rates may be lower since only one side of the implant contacts the brain surface. From a technical point of view, the implantation of VPA PCL devices in our new TeT/CoCl2 procedure can easily be integrated during surgery, thus ensuring a reliable local VPA release directly onto the neocortical epileptogenic focus. Since its introduction for the treatment of epilepsy in 1963, VPA has confirmed its particular antiepileptic efficacy in a broad spectrum of seizure disorders. It is a water soluble low molecular weight drug with several mechanisms of action . For cl2 rat model. The present randomized study, therefore, was aimed at examining the antiepileptic effects of local VPA in a current version of this TeT/CoCl2 model, using NaCl PCL-treated rats as controls. Animals of both the VPA and the NaCl group exhibited clear-cut interictal epileptiform potentials and neocortical seizures, as described above. However, statistical analyses of variables beyond the below-detailed survival rate were not possible as only few vehicle-treated rats were eligible for sufficient longitudinal video-ECoG recordings. Preliminary experiments in our laboratory had indicated that locally applied VPA potentially suppressed interictal epileptic activity in the emerging TeT/CoCl2 dose regime that allows prolonged survival of epileptic rats and enables a statistically valid comparison of immediate and long-term AED effects on interictal and ictal epileptic activity. Further investigations should also compare local versus systemic application of VPA and other AEDs in a finally elaborated TeT/CoCl2 model. A goal of further research will be, therefore, to establish a balanced TeT/CoClIn the present randomized study, local administration of VPA directly onto the epileptogenic focus clearly prevented early death which was obvious in control rats. Note that the Kaplan-Meier survival function from life-time data is much more meaningful than the percentage survival at a random time point. The differences in survival between the VPA and the NaCl group can neither simply be attributed to perioperative surgical complications nor to any differing surgical variables or postoperative conditions. The reasons for the enhanced survival due to focal application of VPA are yet unexplained. It might be suspected that a VPA-mediated prevention of lethal seizures played a role, as all witnessed deaths in our study occurred during status epilepticus or were seizure-related. 2 [2 are not rare. While Brener at al.  reported2 . Overallμg/mL. Walton and Treiman [μg/mL. To our knowledge, reports on effects of VPA in rat TeT models of neocortical epilepsy are not available. However, there is some evidence that VPA might exert an antiepileptic effect in cobalt-treated animals. Van Duijn and Beckmann  describe Treiman  reported2 intraperitoneal VPA at high doses (300 mg/kg) neither antagonized seizures nor decreased mortality [In a rat epilepsy model based on intraventricular injection of CoClortality . systemic application, it seems probable that the local VPA application in our experimental TeT/CoCl2 setup enhances the efficacy of this drug through targeted focal delivery. The concept of local therapy of neocortical epilepsy has already been studied earlier in rats using intracerebral phenytoin polymers adjacent to a CoCl2-induced epileptic focus [2. The resulting antiepileptic efficacy might have contributed to a lower incidence of fatal seizures and a prolonged survival in the VPA group. Thus, we speculate that the effect on survival reported in the present study might not be unique to valproate but potentially shared by other antiepileptic agents. While the antiepileptic effect of VPA in cobalt-treated rats appears mild only after ic focus . Also thic focus . Focal nic focus  used in However, further investigations are necessary in order to determine whether in addition a specific antiepileptogenic effect of VPA  might p2 approach towards a reliable animal model of focal neocortical epilepsy is a promising tool for video-ECoG-based preclinical studies to develop effective and well tolerated local pharmacotherapeutic strategies in neocortical epilepsies. Here, local VPA significantly enhanced survival in rats, possibly due to its antiepileptic/antiepileptogenic properties. Nevertheless, the neocortical TeT/CoCl2 epilepsy model needs further elaboration. The novel TeT/CoClIn order to evaluate the potential impact of local epilepsy therapy in humans, we are currently examining the effects of local administration of VPA onto circumscribed neocortical epileptic foci in patients scheduled for tailored epilepsy surgery after a presurgical work-up with subdural electrodes.


The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19–31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops  or intramuscular injection . IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ∼38-fold lower serum and ∼2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans.ClinicalTrials.govNCT00949572 The mucosal surface is the most common route of infection for a wide range of viral diseases and therefore inducing both mucosal and systemic immunity is a key objective of modern vaccines. The rich infiltration into the sublingual mucosa of antigen-presenting dendritic cells makes it an attractive route of immunization that avoids needles and targets the mucosal immune system The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Ethical Approval was obtained from the UK National Research Ethics Service, Wandsworth Research Ethics Committee reference 09/80803/77. Written informed consent was obtained from all participants after the nature and possible consequences of the study was explained. Clarification of the legal status of the study was obtained by submitting the protocol to the UK Medicines and Healthcare products Regulation Agency (MHRA) which confirmed it as a “Characterization Study” and not a Clinical Trial of an Investigational Medicinal Product (non-CTIMP/NIMP). Although not a clinical trial, we registered this study protocol on ClinicalTrials.gov (NCT00949572) prior to subject recruitment.We sought to characterize, and contrast, the nature and dissemination of the immune response to sublingual or intramuscular deposition of meaningful viral vaccine antigens in humans, and to compare this with published murine studies The target recruitment defined by the protocol was 18 healthy female volunteers  aged 25–35. Subjects were all recruited at one site, St George's - University of London, London. As this was a hypothesis-generating study no formal power calculation for sample size was performed. Inclusion criteria included: provide written informed consent; in good health determined by medical history, physical examination, hematology; available for the duration of the study; if of childbearing potential, must have a negative pregnancy test before each immunization; have not donated blood in previous 3 months; eligible for free medical treatment in the UK. Exclusion criteria included: already received HPV vaccine; recent or concurrent participation in another clinical research study; recent or planned use of any investigational or non-registered product; pregnant or breast-feeding; known or suspected ongoing cervico-vaginal disease, malignancy or abnormality; positive results for Human Immunodeficiency Virus or Hepatitis B/C infection; abnormality in hematology; acute or chronic pulmonary, cardiovascular, hepatic, hematologic, renal, blood or neurological disorders, immune dysfunction, autoimmune diseases, diabetes or malignancy; recent immunosuppressive therapy; medications via vaginal route; tongue or frenulum piercings or oral jewelry; recent receipt of blood products or immunoglobulin.Saccharomyces cerevisiae CANADE 3C-5 Strain 1895) by recombinant DNA technology and adsorbed on amorphous aluminum hydroxyphosphate sulphate adjuvant . All subjects received three immunizations with 0.5 mL (one standard dose) of vaccine at weeks 0, 4 and 16  vaccine Gardasil® (Sanofi Pasteur), which contains L1-based virus-like particles (VLPs) representing four HPV types: 20 µg each of HPV types 6, 18; 40 µg each of HPV types 16 and 11 per 0.5 mL dose. VLPs are produced in yeast cells . VLP formation was confirmed by electron microscopic analysis of negatively staining particles  adsorbed on copper grids coated with formvar (Sigma-Aldrich) and carbon.HPV11 L1 VLPs were not available and no responses to HPV11 were measured. HPV 6 and 18 L1 VLPs were a kind gift of Shantha Biotechnics Ltd, India. HPV16 L1 VLPs were generated using the Bac-to-Bac® Baculovirus Expression System (Invitrogen) wherein the recombinant bacmid DNA contained an HPV16 L1 gene with a 100% amino acid sequence identity to GenBank accession numbers DQ469930 and EU118173. Recombinant HPV16-expressing baculovirus stocks were used to infect Sf21 insect cells (Invitrogen) for 72 hours at 27°C before lysis  at room temperature in the presence of protease inhibitors . The cell lysate was then subjected to iodixanol gradient fractionation and gradient fractions were collected by bottom puncture and stored at −80°C. The L1 concentration and purity were visualized by SDS-PAGE stained with Colloidal Blue (Invitrogen) and analyzed using ImageJ software  containing penicillin–streptomycin. Each cell concentration was added as duplicate wells on the three antigens or uncoated parts of the plate after blocking. A 2 mg/mL PHA positive control was also added on each plate. After overnight incubation, the specific antibody-secreting cells were recognized by the goat anti-human IgG or IgA conjugated with alkaline phosphatase, and counted and analyzed by the AID EliSpot Reader System. The final results were standardized as positive cell number/1×105 PBMCs plated.The frequency of L1-specific spontaneously antibody-secreting plasmablasts was enumerated in PBMCs separated from heparinized whole blood by Ficol gradient centrifugation in an ELISPOT assay as described previously The concentration of serum, cervical or vaginal L1-specific IgG and IgA was measured by indirect ELISA as described previously The HPV16 pseudovirus neutralization assay As this was not a clinical trial, no randomization was performed and no safety data (adverse events) were solicited, no primary or secondary endpoints were specified. Subjects were allocated to one of two sequential cohorts: “IM” who received all immunizations via the intramuscular route (n = 6); and “SL” who received all immunizations as sublingual drops (n = 12). Subjects were not randomized as we wished to develop and evaluate B cell assays carried out on fresh blood samples by recruiting the first subjects into the intramuscular delivery group (and for whom measurable antibody secreting cells (ASCs) were likely to be seen). As the immunization routes could not be blinded the study was not blinded. There were no protocol deviations. As this hypothesis-generating study was not powered to detect significant differences between groups or between time points, no statistical testing was performed and descriptive statistics only are presented.Eighteen female subjects aged 19–31 years  were enrolled and completed the protocol. There were no protocol deviations .HPV16 L1 VLPs and whole vaccine  were used as a coating antigens. From previous studies using oral, nasal or intramuscular immunization The kinetics of the anti-HPV L1 VLP antibody response in serum and in cervico-vaginal secretions was measured before and at various time points after each immunization using an antigen-specific antibody binding ELISA, with purified L1 HPV16, 6 and 18 VLPs as coating antigens. As expected, IM immunization induced an increase in serum anti-HPV6, HPV18 and HPV16 L1 VLP IgG from baseline . IM immuThe detection of antibodies capable of neutralizing HPV16 pseudoviruses was examined using serum and cervical and vaginal samples taken at week 0 and 20 (4 weeks post last immunization) for all subjects . Some inParenteral immunization with vaccines containing HPV L1 VLPs and formulated using alum and/or TLR agonist adjuvants is highly effective at inducing both serum and mucosal antibodies, and conferring long-lasting protection against HPV infection by the homologous or related HPV genotypes We observed that sublingual immunization generally induced a similar pattern of immune responses to intramuscular, but at much lower magnitudes. An increase in serum anti-HPV16 L1 VLP IgG was detected after both IM and SL immunization . HoweverOne of the potential translational advantages proposed for mucosal immunization is that it appears to specifically induce mucosal immunity de novo in 2/12 subjects whose day 0 serum neutralizing antibody titer was below the cut-off in our assay, and boosted the day 0 titer in another subject. These observations are encouraging for potential future vaccine strategies based upon sublingual delivery. It is also possible that the bivalent vaccine, Cervarix®, which makes use of a TLR-4 agonist in the vaccine formulation and has been shown to induce higher levels of HPV-specific antibodies when administered parenterally, may have induced significantly higher antibody levels Due to the considerable volume of saliva produced despite covering the parotid ducts, it is highly likely that some sublingually administered VLPs would have been removed from the mucosal surface within minutes of application. To be effective, extensive optimization of sublingual delivery will be required to improve penetration of the sublingual mucosa of humans, perhaps by making use of mucoadhesives or other delivery systems designed to resist salivary degradation In conclusion, this preliminary translational human study indicates that, in marked contrast to murine studies, SL delivery of a representative virus vaccine antigen formulated with alum is only modestly immunogenic in humans. This route can, however, induce low level serum and mucosal antibodies, and functional serum neutralizing antibody. The observation that SL immunization could boost pre-existing serum neutralizing activity also points to the possible use of IM prime/SL boost schedules. For this approach to be advanced, the next steps require significant optimization of the SL delivery system for human use, and the investigation of optimal SL-IM prime-boost schedules. Once this is achieved the benefits of sublingual delivery on the character, magnitude and dissemination of responses should be compared in clinical trials.Protocol S1Trial Protocol.(PDF)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.


Yet treatment of such NB cell lines with retinoic acid results in growth control and induction of differentiation. This indicates that the signaling pathways that regulate cell growth and differentiation are not functionally lost but dysregulated. Agents such as retinoic acid normalize the signaling pathways and impose growth control and induction of differentiation. Recent studies in embryonic stem cells indicate that polycomb repressor complex proteins (PRC1 and PRC2) play a major role in regulating stem cell lineage specification and coordinating the shift from a transcriptome that supports self-renewal or growth to one that specifies lineage and controls growth. We have shown that in NB, the PRC2 complex is elevated in undifferentiated NB tumors and functions to suppress a number of tumor suppressor genes. This study will review the role of MYC genes in regulating the epigenome in normal development and explore how this role may be altered during tumorigenesis.It is well known that Neuroblastoma (NB) patients whose tumors have an undifferentiated histology and a transcriptome enriched in cell cycle genes have a worse prognosis. This contrasts with the good prognoses of patients whose tumors have histologic evidence of differentiation and a transcriptome enriched in differentiation genes. Tumor cell lines from poor prognosis, high-risk patients contain a number of genetic alterations, including amplification of MYCN, 1pLOH, and unbalanced 11q or gains of Chr 17 and 7, and exhibit uncontrolled growth and an undifferentiated phenotype in  Ever since MYCN was discovered to be the commonly amplified sequence in neuroblastoma (NB) tumors some 30 years ago, the mechanisms by which it contributes to NB tumorigenesis and strategies to target it have been intensively investigated . The finFunctionally MYC family genes  have been implicated in nearly every biologic process evaluated. Evidence indicates that MYC and MYCN are functionally interchangeable with MYCN having a more restricted spatial and temporal role during development . In thisFigure 1). Based on studies using naked DNA, two prevailing views emerged as to how MYC affects transcription: (1) a direct mechanism in which MYC/MAX dimers upon binding to E-boxes recruit higher order chromatin complexes that activate or repress transcription or (2) an indirect mechanism in which MYC/MAX dimers compete with other bHLH-Zip transcription factors for E-box binding to alter target gene activity. In searching for a common MYC target signature investigators have proposed numerous target signatures in different cellular contexts, but none have proven true in all cellular contexts (MYC is a member of a basic helix-loop-helix transcription-leucine zipper (bHLH-Zip) factor family whose members dimerize with MAX and bind to specific DNA sequences called E-boxes . Nucleosomes are then joined by a stretch of linker DNA (20 bp). When DNA is tightly complexed to histones, transcription is silenced. Activation of gene transcription requires loosening of DNA–histone interactions to enable access by transcription factors, the melting of the DNA that enables access of the basal transcription machinery including RNA polymerase II (Pol II) and the sliding of the nucleosomes to enable transcriptional elongation. The accessibility of DNA to DNA binding transcription factors is dynamically regulated by post-translational modifications to histone “tails” such as methylation (methyltransferases and demethylases), acetylation (acetyltransferases and deacetylases), phosphorylation (kinases and phosphatases), ADP-ribosylation, and ubiquitination .The necessity to compact some 2 m worth of DNA into the 10–20 μm nucleus of a cell has resulted in the evolution of a dynamic packaging system in eukaryotes that allows for the regulated sequestration or exposure of stretches of DNA. DNA (about 146 bp) is wrapped twice around an octamer of histone proteins  and stabilized by histone H1, forming the nucleosome, the basic unit of chromatin .When MYC function was first being described over 20 years ago, it was noted that MYC preferentially binds to “open” nucleosomes and DNase sensitive loci that we now know characterize euchromatin . EuchromSaccharomyces cerevisiae as members of the SAGA and NuA4 HAT complexes, respectively, and have been shown to have similar function in mammals . Concordantly, in embryonic stem cells (ESCs) MYC has been shown to elicit its transcriptional induction through the mammalian NuA4 complex and in neural stem cells MYCN and GCN5 have similar transcriptional signatures . Although TBP has no HAT activity, its binding to MYC suggests MYC is able to recruit Pol II machinery to activate transcription . Therefore, through its binding partners MYC is able to regulate the chromatin landscape to exert its function.Further work revealed that, via TRRAP, MYC was able to recruit histone acetyltransferases (HATs), GCN5 and Tip60 to its targets. GCN5 and Tip60 were first identified in gnatures . Both GCgnatures . In addiFigure 2D). GCN5 and Tip60 function to promote transcription by their acetyltransferase activity, but it is not limited to histones. It has also been demonstrated that MYC is a substrate of acetyltransferase activity. GCN5, Tip60, and CBP/p300 all acetylate MYC . As MYC’s bHLH domain is required for its transactivation function and binding to INI1, INI1 appears indispensable for proper MYC function. Furthermore, intact SWI/SNF complexes are also needed for MYC function. BRG1 is a functional component of the SWI/SNF complex and its ATPase-defective mutant abrogates histone remodeling activity and blocks transcription of MYC targets  and functioning to amplify transcription of actively transcribed genes  suppressible MYCN SHEP NB model system and ChIP hybridizations to gene promoters (ChIP-Chip) revealed that MYCN binding associates with euchromatic histone marks H3K4me and H3K9Ac . The cored genes . An inteed genes . This imde novo factors to bind to DNA to initiate its transcriptional function , only OSK can bind to distal enhancer regions at an early time during reprogramming. Subsequently MYC is recruited to the TSS to stabilize chromatin binding and promote transcription. Furthermore, OSK proteins are all able to bind to one side of the DNA helix, suggesting they are able to bind to heterochromatic regions. MYC does not bind to heterochromatic regions. MYC is predominantly found on euchromatic regions indicating it requires a prior transcriptional stimuli or function .de novo transcription, the question arises as to how MYC can promote the induction of transcription initiated by reprogramming factors. Regulation of gene transcription in complex chromatin models is still evolving and occurs in at least five steps: (1) initiation, (2) promoter pausing, (3) mRNA capping, (4) promoter escape, and (5) transcript elongation. A clue to MYC function during initiation lies with MYC binding to TBP , p-Ser5-Pol II. Genome-wide analyses of Pol II binding demonstrated that p-Ser5-Pol II commonly resides at promoters or is paused 20–40 bases downstream of the promoters of many non-expressed genes. The capping machinery is recruited to the CTD of Pol II and results in the addition of a methylated guanine at the 5′-triphosphate of the nascent mRNA strand. MYC also binds to TFIIH and the increased p-Ser5-Pol II increases capping associated methylation. Capping is required for the recruitment of the translation machinery and mRNA binding to ribosomes. The acetylated lysine binding bromodomain containing protein BRD4, recruits P-TEFb complex which phosphorylates serine 2 on the CTD region of Pol II (p-Ser2-Pol II) causing elongation of mRNA transcripts and promoter escape  results in cells with a relative nuclear size twice that of the parental or control-transfected (8B) cell lines which contain a single copy of MYCN and do not express MYCN mRNA . Decreased euchromatic regions have also been identified in tumor models when MYC is knocked down . In NB, the combined treatment of RA and Interferon-gamma (INF-γ) has been shown to dramatically decrease cell growth and induce differentiation , via SIN3 a transcriptional corepressor, to target loci to induce transcriptional silencing . Thus thntiation . It was ntiation . The preAnother model of MYCN-mediated transcriptional repression invokes the association of MYC binding to the MIZ and SP1 transcriptional activators resulting in the recruitment of HDACs. This has been demonstrated for two genes associated with good prognosis and differentiation in NB, NTRK1(TrkA), and p75(NGFR). Upon induction of MYCN, there is increased HDAC1 and decreased acetylated H3 binding over the TSS of these promoters. The functional presence of MYCN was found to attenuate their promoter activity . One canMost recently MYC has been found to transcriptionally amplify epigenetic modifiers with transcriptional repressive activities. For example, in ESCs MYC stimulates all components of the polycomb repressive complex 2 (PRC2), including embryonic ectoderm development (Eed), suppressor of zeste 12 (Suz12), and histone methyltransferase enhancer of zeste homolog 2 (EZH2), in ESCs . PRC2 fuFurthermore, in NB cell lines and tumor samples, MYCN stimulates B cell-specific Moloney murine leukemia virus integration site (BMI1) expression. BMI1 is a member of the PRC1 complex whose targets are often hypermethylated suggesting epigenetic silencing . As partGiven MYCN’s role in stimulating expansion of neural progenitors, it is not unexpected that differentiation genes like NTRK1 or p75(NGFR) are repressed by MYCN. A global survey by ChIP-Chip of ESCs for MYC and Miz-1 binding sites indicated that almost 30% of Miz-1-regulated genes could be repressed by MYC. These included homeobox genes, developmental proteins and genes involved in regulation of apoptosis. In NB tumor samples, within the MYCN gene signature set, 30% of the down-regulated genes in the set are neuronal tissue-specific genes while only 2% of such genes are up-regulated . This isSince MYC has been implicated in oncogenic transformation, researchers and clinicians have sought to target MYC and its oncogenic functions. A common mode of function of therapeutic interest has been MYC’s chromatin remodeling and transcriptional activities. It was rationalized that since MYC binds and recruits coactivators containing the bromodomain and extraterminal (BET) members of human bromodomain proteins , which associate with acetylated histones, it could be functionally targeted . This inFigure 4; Figure 4A) or a small molecule inhibitor of EZH2, 3-Deazaneplanocin A  led to decreased growth and induction of neurites. Mechanistically this was also accompanied by induction of cell death  which was due to increased levels of activated caspase 3/7 activity  since the apoptotic effects of DZNep could be reversed by a caspase 3/7 inhibitor . The finding that DZNep inhibited the growth of NB tumor xenografts is promising . However, all mice still succumbed to tumors suggesting that combination therapies may be required to more efficaciously block tumor growth.As MYC and MYCN have been shown to induce EZH2 expression and activity  it is reMYC’s dysregulation of epigenetic writers, readers, or erasers thus provides a novel avenue that can be therapeutically developed since many of these proteins have targetable enzyme activities .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Well adapted to tropical conditions, it is also believed trypanotolerant according to its putative AFT ancestry. However, such origins needed to be verified to define relevant husbandry practices and the genetic background underlying such adaptation needed to be characterized.The Senepol cattle breed (SEN) was created in the early XXpolled locus underlying the absence of horns and ii) one footprint on BTA20 within the slick hair coat locus, involved in thermotolerance. Annotation of these regions allowed us to propose three candidate genes to explain the observed signals .We genotyped 153 SEN individuals on 47,365 SNPs and combined the resulting data with those available on 18 other populations representative of EUT, AFT and Zebu (ZEB) cattle. We found on average 89% EUT, 10.4% ZEB and 0.6% AFT ancestries in the SEN genome. We further looked for footprints of recent selection using standard tests based on the extent of haplotype homozygosity. We underlined i) three footprints on chromosome (BTA) 01, two of which are within or close to the slick locus.Our results do not support the accepted concept about the AFT origin of SEN breed. Initial AFT ancestry (if any) might have been counter-selected in early generations due to breeding objectives oriented in particular toward meat production and hornless phenotype. Therefore, SEN animals are likely susceptible to African trypanosomes which questions the importation of SEN within the West African tsetse belt, as promoted by some breeding societies. Besides, our results revealed that SEN breed is predominantly a EUT breed well adapted to tropical conditions and confirmed the importance in thermotolerance of the  Over the past decades, population genetic approaches associated with archeological evidence have provided better insights into the origin of modern livestock breeds including domestication processes, migration routes and their relationships In addition, high density SNP dataset has highlighted chromosomal regions and genes targeted by artificial selection in dairy and/or beef cattle and by natural selection in West African cattle Such a global approach is applied hereby on the Senepol cattle breed (SEN), a completely polled breed originating from the Virgin Islands, now widespread in several tropical regions. The SEN breed is particularly well adapted to tropical conditions, SEN animals being able for instance to maintain a normal body temperature during heat stress and appearing as heat tolerant as Brahman Zebu (BRA) cattle According to the St. Croix (US Virgin Island) breed society, SEN was created by Henry Nelthropp’s family at the beginning of 1900s on the St. Croix Island Trypanosoma . To our knowledge, trypanotolerance has never been rigorously assessed in SEN, although failure to verify it could have dramatic consequences both for local breeders and for local breed diversity if importation of SEN animals were carried out towards West Africa. Moreover, the official version of the SEN origins, which represents the only argument to suggest a possible trypanotolerance of the SEN breed, has been disputed As an important consequence, many technical documents about SEN breed report that SEN individuals are considered to have inherited from their presumed NDA ancestry trypanotolerance characteristics, i.e. the ability displayed by West AFT breeds such as NDA to “survive, reproduce and remain productive under trypanosomiasis risk without trypanocidal drug” Our study was primarily aimed at providing a detailed assessment of the genetic origins of the SEN using high density SNP data. To that purpose, a total of 153 SEN cattle were genotyped on the Illumina Bovine SNP50 chip and the resulting data were combined with those already available on 18 other breeds In order to characterize the SEN genetic history, we first combined the SNP data obtained on the 147 SEN animals  tree based on allele sharing distance (ASD) separated individuals according to their population of origin . Breeds An individual Principal Component Analysis (PCA) was then carried out using all available SNP information . The firSTF <0.021), and ii) no inbreeding within the whole SEN population (|ISF|<0.01)  a low level of differentiation among populations from the four different Venezuelan states represented (S|<0.01) . This waSTF between SEN and EUT breeds were lower than those between SEN and ZEB or AFT breeds .Following Gautier & Naves (2011) iHS and/or significant Rsb scores, three of which  map to BTA01 and one (#4) to BTA20. According to our somewhat stringent criteria, region #4 was significant for the three tests performed , region #1 was significant for two tests (iHS and ZEB/SENRsb) and both regions #2 and #3 were found only significant with the EUT/SENRsb statistic. In order to illustrate the haplotype structure around the ancestral and derived alleles for each of the four SNPs at the peak position, we drew i) the decay of site-specific EHH or EHHS (weighted average of both EHH used for the computation of Rsb) within SEN, EUT and ZEB populations, ii) the EHH decay from the core SNP at both alleles within the SEN population and iii) haplotype bifurcation diagrams for both ancestral and derived allele in each region under selection within the SEN population , our results clearly challenge the official historical version since no significant AFT ancestry was found in the SEN population. In order to explain such discrepancies, two hypotheses might be proposed. First, NDA breeding individuals were actually not used to create the SEN breed. As mentioned above, some authors raised doubts about a possible NDA ancestry polled locus located on BTA01 in Bos Taurus and ii) the African horn locus in Bos taurus and especially Bos indicus, which has an epistatic effect on the polled locus Our study provides a first fine-scale genetic characterization of SEN through a comparison with other breeds representative of major breed groups of cattle distributed world-wide. The SEN individuals are representative of the whole population since they were sampled in different regions of Venezuela, and included some animals brought directly from the USA and the St. Croix Island, where the breed originated. No clear sub-structure was observed among the SEN individuals, suggesting an homogeneity of the population. In addition, SEN individuals displayed on average 89% of EUT ancestry meaning that SEN is predominantly a EUT breed. The origin of the EUT ancestry from North European breeds thus remains in agreement with historical and SEN breed association reports indicating that Red Poll individuals were initially used to create SEN. Although limited to 10.4% on average in SEN individuals, a ZEB ancestry was unexpectedly observed. More strikingly, given the very recent creation of the SEN breed  which is responsible for severe outcome of the disease in African livestock and its obligate vector (the tsetse fly) are absent in South America. SEN cattle are already present in South Africa, Botswana, Namibia and Zimbawe and some other African countries are planning to import SEN animals because they are more productive under tropical conditions than local breeds in a tsetse free context. It might be highly advisable to limit SEN importation to countries or areas outside of the tsetse belt.Importantly, the absence of AFT ancestry makes it unlikely that SEN animals have inherited trypanotolerance trait from their ancestries although such ability represents the main argument of some breeder associations for the diffusion of SEN to West-African countries. Indeed, both EUT and ZEB cattle are recognized as trypanosusceptible. Similarly, SEN might not have acquired trypanotolerance during their recent history since Caribbean islands such as St. Croix are Such new insights into SEN origins raised another interesting question. Indeed, how can a breed with almost 90% of European cattle ancestry be well adapted to tropical conditions? Due to the recent origin of the SEN breed, it is tempting to speculate that such adaptation was inherited from the ZEB ancestry. ZEB, that are widespread in African, Asian and South America tropical areas, are indeed recognized for their thermotolerance, their ability to survive during food shortage and their resistance to several tick-borne diseases iHS and Rsb statistics). iHS based tests allow to detect loci carrying favorable variants subjected to strong recent or ongoing selection and display maximal power when they have not yet reached fixation Rsb based tests show higher power to detect selective sweeps that have resulted in near or complete fixation of the favorable variant in one of the compared populations iHS and Rsb statistics, three located on BTA01  and one located on BTA20 (#4).In such a case, SEN represents an attractive model to identify those genomic regions from ZEB origin involved in its adaptation to tropical conditions. Alternatively, some favorable alleles might have segregated in EUT ancestry of the SEN breed in which theyhave subsequently been selected. To unravel loci potentially involved in adaptation of SEN to tropical conditions, we then searched for footprints of selection using previously described EHH-related tests , a developmentally regulated gene induced by all trans retinoic acid in vitro and can induce hair loss through premature hair follicule regression Interestingly, this latter region (#4) is within the previously described polled locus polled locus defined in Drogemüller et al. and Wunderlich et al. studies polled critical interval published extends from microsatellite BM6438 and RP42-218J17_MS1 on BTA01 polled allele is responsible for the observed signals, our study allows us to propose two candidate genes lying under these peaks. TIAM1 encoding the T-cell lymphoma invasion and metastasis protein and GRIK1, encoding a ionotropic glutamate receptor, which are located in regions #2 and #3, respectively. TIAM1 modulates the activity of Rho-like proteins and connects extracellular signals to cytoskeletal activities polled region, with a peak near the KRTAP8-1 gene, located 0.5 Mb from TIAM1 and 1.38 Mb from GRIK1 Among regions located on BTA01, no candidate genes could be found for region #1. However and interestingly, regions #2 and #3 mapped within (or close to in the case of region #3) the slick hair locus (BTA20), the underlying favorable variant might be associated to several haplotype backgrounds from EUT ancestry. Discovered in SEN, the slick allele has also been described in other Creole breeds such as the Carora For regions #2 and #3, examination of EHH decay and haplotype bifurcation diagrams suggested that the ancestral allele at the corresponding peak SNPs is associated to the underlying selected variant. For region #4, both ancestral and derived allele seem associated to an underlying selected variant. Due to the high EUT ancestry, ancestral alleles are thus more likely to be of EUT origin see . HoweverNo ethics statement was required for the collection of DNA samples. DNA was extracted either from commercial AI bull semen straws or from blood samples obtained from different veterinary practitioners visiting farms with the permission of the owners.A total of 153 SEN individuals from Venezuela were included in our genetic study. In Venezuela, the first importation of SEN cattle was done in 1988, in the form of 250 frozen embryos from American Senepol Limited , which were implanted in Zebuin and Holstein breeds between 1988 and 1992. In 1991, the first bull was imported and 30,000 doses of its semen were sold. In May 1993 a total of 200 animals were imported from Saint Croix to seven different farms in Venezuela. Up to 2011, 540 animals have been imported from St. Croix or USA, for a total of 1,774 fullblood and purebred Senepol animals and 4,974 crossbred animals that are participating in the upgrade program in Venezuela. The Venezuelan Association of Senepol Cattle Breeders (ASOSENEPOL) was created in 1998 and has 25 active members spread in the territory and 13 veterinarians that classify the cattle in the whole country. They have a collaborative program with the international Senepol Cattle Breeders Association.These 153 SEN animals  belong tBlood samples were collected in EDTA Vacutainer tubes and DNA was extracted using the Wizard® Genomic DNA Purification Kit . DNA quantity and quality were evaluated on Nanodrop and on agarose gels .http://www.illumina.com) The 153 animals  were genhttp://cran.r-project.org/web/packages/qvalue/index.html) Following Gautier et al (2010) an exact test for Hardy-Weinberg Equilibrium (HWE) was subsequently carried out within each breed separately on the remaining SNPs ITF, STF and ISF, and the diversity estimation for each locus and population both within and among individuals within a population were estimated using the program GENEPOP 4.0 ISF was derived from the average of these two quantities over all the SNPs.F-statistics ij where xij represents the proportion of alleles alike in state averaged over all genotyped SNPs.A neighbor-joining tree Principal Component Analysis (PCA) and unsupervised hierarchical clustering of individuals based on SNP genotyping data were performed using the smartpca software package and the program Admixture 1.04, respectively iHS and Rsb score iHS, the SEN ancestral allelic state was defined as the highest ancestral frequency estimated as EUT fEUT+wZEBfZEBfa = w, were EUTw and ZEBw represent the average proportions of EUT and ZEB ancestries in the SEN genome and EUTf and ZEBf the allele frequency of EUT and ZEB clusters iHS scores were further transformed into iHSp = −log[1–2|Φ(iHS)−0.5|] where Φ(x) represents the Gaussian cumulative function. Assuming iHS scores are normally distributed under neutrality (iHSp might thus be interpreted as 10log(1/P) where P is the two-sided p-value associated to the neutral hypothesis (no selection). All the above analyses and plots (including bifurcation diagrams) were performed using the newly developed R package rehh Haplotypes were estimated using fastphase 1.4 for SEN and each AFT, ZEB and EUT populations utrality , piHS miRsb was computed for two comparisons (EUT/SEN and ZEB/SEN) by standardizing the ratio of the corresponding ancestral cluster (EUT or ZEB) iES and the SEN iESRsbp = -log[Φ(Rsb)]. As above, assuming Rsb are normally distributed , Rsbp might be interpreted as log10(1/P) where P is the one-sided p-value associated to the neutral hypothesis (no selection).iHSp >4 (P<0.0001) and similarly the number of SNP with Rsbp >4 for each of the two comparisons (EUT/SEN and ZEB/SEN). Regions containing at least two SNPs exceeding these thresholds for at least one test were considered as candidate. When several overlapping and contiguous windows were candidates, the chosen one contained the highest proportion of significant SNPs and also the peak. The candidate regions were then annotated using the UCSC Genome browser (http://genome.ucsc.edu) and the Bos taurus UMD 3.1/bosTau6 assembly. A gene was considered a candidate if the peak position was located <25 kb from its boundaries.For each 1 Mb window over the genome (with a 0.5 Mb overlap), the candidate regions were identified by counting the number of SNP with Figure S1Neighbor-Joining tree relating the 629 individuals. Among the 623 individuals, 147 SEN from different origins and states and 476 animals from 18 other breeds were analysed. The tree was constructed using allele sharing distances averaged over 47365 SNPs. Edges are colored according to the individual breed of origin.(TIF)Click here for additional data file.Figure S2Unsupervised hierarchical clustering results with different number of clusters of 623 individuals genotyped for 47365 SNPs. The 623 animals, comprising 147 SEN individuals, were analyzed with an inferred number of clusters K = 2 (A), K = 3 (B), K = 4 (C), K = 5 (D) and K = 6 (E). For each individual, the proportion of each cluster (y) which were interpreted as representative of EUT, AFT and ZEB ancestries for K = 3.(TIF)Click here for additional data file.Figure S3Unsupervised hierarchical clustering results with different number of clusters of 506 individuals genotyped for 47365 SNPs. The 506 individuals, comprising 30 SEN individuals randomly chosen, were analyzed with an inferred number of clusters K = 2 (A), K = 3 (B) and K = 4 (C). For each individual, the proportion of each cluster (y) which were interpreted as representative of EUT, AFT and ZEB ancestries are plotted in blue, red and green, respectively.(TIF)Click here for additional data file.Figure S4Empirical distribution of SEN, EUT and ZEB iHS scores.(EPS)Click here for additional data file.Table S1List of population symbols, names and number of individuals.(XLS)Click here for additional data file.Table S2Percentage of the corresponding AFT, ZEB and EUT ancestries for each breed.(XLS)Click here for additional data file.Table S3Differentiation score (Fst) for all pairs of breeds.(XLS)Click here for additional data file.


A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ , PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as ‘anchor’ genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section  Many diseases of the kidney stem from disruptions to the transcriptional programs involved in normal kidney development Wnt9b in the initiation of MET and the subsequent requirement for Wnt4 expression to proceed through this event is well documented Notch2 for proximal tubule specification Kidney organogenesis in mammalian species is highly conserved anatomically and molecularly. Hence, the field has made considerable use of model organisms, predominantly mouse and rat, to study the process. The mammalian kidney is a complex organ containing more than 25 distinct functional cell types Six2 gene is regionally-enriched in the CM during kidney development Six2Six2 gene. The identification of additional compartment-specific genes is needed to advance our understanding of kidney development, notably during the later stages of patterning, segmentation and differentiation. The obvious solution to this would appear to be gene expression analysis of kidney development.The development of conditional transgenic strains in the mouse has particularly assisted in defining our existing understanding of ontological relationships during kidney development. For example, the While global analysis of gene expression in the entire developing kidney has been of limited value given the cellular complexity of this organ in situ hybridisation (SISH). A bioinformatic method was devised for the stringent prediction of ‘anchor’ genes, defined as a gene whose expression was restricted to one temporospatial anatomical compartment. A total of 200 genes across 11 anatomical compartments were analysed with 46% of genes (92 genes) being compartment-enriched and an additional 18.5% (37 genes) representing anchor genes for a defined temporospatial structure . As such, these anchor genes will fuel the generation of further mouse resources for lineage tracing, thereby extending our understanding of kidney organogenesis and ultimately molecular pathogenesis within the kidney. Equally, compartment-enriched genes redefined the developing kidney atlas into molecular events rather than regional or anatomical structures, identifying markers able to subdivide the processes of nephron segmentation, collecting duct functionalisation and interstitial differentiation.Microarray compartments in Brunskill et al (2008) Brunskill et al (2008) in situ hybridisation only proceeded for those genes selected according to the criteria determined for selection of potential anchor genes. The approach taken here is distinct from our previous analysis Prnp, a gene we have shown to mark a distinct subcompartment of the SSB, was not selected as it is also expressed at a significant level in RV. As this approach did not identify a number of genes commonly regarded as marking specific renal compartments, a retrospective analysis was performed to determine why such genes were excluded. Four major issues arose. 1) Insufficient differential in expression, e.g. Wnt11 and Wnt7b, regarded as specific to the ureteric tip and collecting duct respectively, were not selected due to insufficient differential expression between this and all other compartments. 2) Absence from microarray probeset, e.g. Crlf (syn: CLF-1), previously reported as specific to the UT Pf4 (Cxcl4), previously reported as a UT marker Cadherin 16 (Cdh16), although previously described as a marker of collecting duct Six2, a well known marker of the CM was also detected in the MI compartment. While this may reflect contamination of the MI compartment with CM, there was no such contamination detected in the more closely located CI.Validation via in situ hybridisation (SISH) of paraffin-embedded E15.5 murine kidney. A subset of genes was also examined in adult kidney. In all cases, expression patterns were annotated in accordance with the anatomical ontology previously generated for the mouse urogenital system specific , enriched , ubiquitous , not-detected (no detectable expression) or non-specific (expression detected in an unexpected compartment). As microarray data was not available for all kidney compartments, some genes validated with respect to the other ten compartments but were not regarded as anchor genes as they were found to also be expressed in a compartment not included in the analysis . The fully annotated expression patterns, riboprobe details and all ISH images for the complete set of validated genes are available on the GUDMAP website (http://www.gudmap.org).Of the 251 putative anchor genes selected, 200 were analysed using high resolution section Rspo3, Disp1, Cacnb4, Adcy8). Other genes  for all compartments was 64.5%, however this did vary considerably between compartments  and anches Eya1;  could beThe success of the validations depended on the ability to replicate the gene expression detected in the microarray study. While the majority of the genes that were validated produced an expected signal pattern (129/200), 71 other gene probes yielded false positive signal . Sources of these variations are mostly likely due to the technical challenges from comparing microarrays and ISH. These variations include contamination of LCM samples from spatially adjacent compartments during sampling non-specific expression, as these probes are capable of providing signal . Non-specific riboprobes showed less than 80% overlap with the Affymetrix probeset which may be a contributing factor towards the inaccuracy of the riboprobe and could be avoided during riboprobe design.Another source of variation comes from discrepancies of microarray and SISH probes representing a gene. One of the issues lies in the sequence length of the microarray probeset and SISH riboprobe. The riboprobes used in this study were typically designed within the 500–800 bp range, while the Affymetrix probes are 25 mers. The shorter probes are more likely to target specific transcripts whereas the longer probes can span across several transcripts and are therefore more likely to pick up different signal combinations picked up by each transcript http://uqgudmap.imb.uq.edu.au/riboprobe_design/). Here, each probeset entered, results in the generation by Primer3 of the corresponding riboprobe primer sequences. This then links to the complete set of primer statistics in the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/) and the In-Silico PCR program from the UCSC Genome Browser Since microarray and SISH based comparisons are common for the validation of gene targets, a riboprobe design pipeline that minimizes discrepancies in expression validation arising from transcriptional complexity was created by consolidating the Affymetrix probeset locations into the design of SISH riboprobes. Each of the eleven 25 mer oligo-probes in a given Affymetrix probeset were mapped against a non-redundant set of mouse cDNA transcripts (FANTOM3 cDNA clones) to obtain overlapping regions to serve as sequence templates for riboprobe primer design via the Primer3 program Gpsm3, Tdrd5, C230096N06) and one marking the juxtaglomerular arterioles (Vip). Gpsm3 and C230096N06 were expressed by both Stage III and IV nephrons however Vip and Tdrd5 were restricted to the more mature Stage IV RC. A single LOH anchor gene, Umod, was identified. This is a well known marker of the LOH and mutations in Umod have been associated with a range of chronic kidney diseases, such as hyperuremic nephropathy (OMIM: 162000) and medullary cystic kidney disease (OMIM: 603860). While many genes are expressed in the renal vesicle, as previously reported Npy) showed expression restricted to this earliest stage of the nephron. Anchor genes for EPT, MCD, UT are discussed later.In total, 37 anchor genes were identified representing 6 compartments within the developing kidney . Four anKcnj1 are associated with Bartter syndrome (OMIM: 241200), a life-threatening disorder including multiple developmental abnormalities.In addition, a further 77 genes showed specificity for previously undefined temporal or spatial compartments. This enabled the redefinition of initially anatomical subdivisions into molecular compartments more closely reflecting the biology of kidney development. Meis1+ GFP mice, a gene whose expression does extend into the CM (see GUDMAP data Accession ID GUDMAP:12521). However, overall the absence of markers that distinguish individual interstitial compartments also supports the notion of commonality in cellular composition within much of the interstitial space. It also highlights the need for ultimate compartmentalization of this organ down to the cellular level.Despite validation of a large number of genes, no gene was identified as an anchor gene for either the MI or CI. Instead, the pattern of gene expression observed within the interstitium reflected the fact that this compartment contains a variety of cell types that are present throughout all regions of the interstititum. Eleven interstitial genes were present in all mesenchymal interstitium including the CM, 16 genes were present in all interstitial regions other than the CM and 6 genes were present in MI and CI but not nephrogenic insterstitium, perihilar interstitium or CM. It should be noted that the CI compartment was isolated using FACS sorting of Calb1 and Hoxb7Wnt11 in the tip Ret, while enriched in the ureteric tip, is also expressed weakly in the ureteric trunk Slco4c1 as a novel UT anchor gene, five MCD anchor genes  but no CCD anchor genes. However, a cohort of seven additional genes revealed a complex set of overlapping gene expression patterns indicating a much greater level of segmentation of this epithelial compartment  and later markers of pan-nephron development , early nephron , presumptive podocyte (Stc2) and LOH/distal tubule   . One gro Tmem72)  but were Tmem72)  together Tmem72) . As with Tmem72) . This suRbpJ and Hnf1a have been previously shown to directly affect proximal tubule development and function Hnf1b and Hnf4a transcription factors are associated with kidney development Hnf4a. SISH analysis of Hnf1b and Hnf4a shows expression within the proximal tubule and other nephron segments with Hnf1b showing collecting duct expression and Hnf1a showing S-shaped body expression (see GUDMAP data Accession IDs GUDMAP:12560 and GUDMAP:12531 respectively).The large number of anchor genes identified in the proximal tubule provided the basis to perform downstream analyses to understand transcriptional control of tissue-specific expression. The proximal promoters of EPT anchor genes were used to identify over-represented transcription factor binding motifs defined at −500/+200 of RefSeq annotated transcription start sites (referred to as “Core”) using the CLOVER algorithm Hnf4a, this analysis also detected other nuclear receptor superfamily TF motifs including sites for binding of the PPARγ:RxRA heterodimer activation complex, COUP-TFI, and Nr4a2. Activation of perioxisome proliferator-activated receptor gamma (PPARγ) requires heterodimerization of RxRA and is therefore represented as PPARγ:RxRA. The presence of TF motifs may reflect a role for such proteins in proximal tubular gene expression or in the regulation of these genes in another context. Both PPARγ and RxRA expression have been detected in the proximal tubule Nr4a2 is expressed in early nephron tubules (GUDMAP:5209), suggesting a role in the regulation of later proximal tubule specification. Nr2f1 (COUP-TFI) expression has only been detected in the interstitial mesenchyme (GUDMAP:5232), however it is possible that this transcription factor plays a role in the repression of expression of these key proximal tubular markers. Alternatively, as the JASPAR motif library used does not include all COUP-TF motifs, it is possible that the predicted COUP-TFI sites actually represent regulation by another member of the family. Nr2f2 (COUP-TFII) expression was strongest in cap mesenchyme, persisted in transitioning nephron structures, but was absent in the adult proximal tubule RbpJ confirms the involvement of the Notch pathway in specification of EPT fate Hnf1a and Hnf1b act upstream of the Notch pathway to specify epithelial cell fate and differentiation in the gut In addition to Ace2, Snai proteins and Phd1 within the EPT anchor genes. Ace2 expression has been previously described within the proximal tubule Ace2 can result in defects in amino acid absorption including Hartnup disease Snai transcription factors are zinc finger transcription factors classically associated with inducing and maintaining a mesenchymal phenotype Snai1 is primarily detected in the cortical interstitium (GUDMAP:13622). While its role in development remains elusive, Snai1 has been extensively documented in renal fibrosis through the EMT pathway Snai motifs within EPT anchor gene promoters would imply that Snai suppression of these genes is critical for EMT.ECR-based promoter analysis also identified conserved motifs for Hnf4a, Hnf1a and members of the PPAR transcription factor family. Other transcription factors identified in this network included Ahr and p53, both of which are known to play a role in kidney development To identify other potential tissue-specific functional networks of proximal tubule, the 25 EPT anchor genes were used for genetic network analysis using Ingenuity Pathway Analysis. Network interactions were inferred using the Ingenuity Knowledge Base of pre-curated molecular and functional interactions mined from published literature. The networks are displayed as nodes (genes) and edges . Networks from 11 genes were merged as one network . This thIn summary, this study has defined a stringent bioinformatic approach for the selection of compartment-specific anchor genes and has applied high resolution SISH to the validation and detailed temporospatial characterisation of these genes during murine kidney organogenesis. Reiterative subdivision, profiling and high resolution validation will ultimately facilitate even deeper analysis of kidney development via the generation of tools with which to more accurately select the starting population of cells. Utilizing the anchor genes identified in this study, the generation of anchor-gene driven reporter mice will allow more defined populations to be identified. Such an ability to selectively and specifically target microanatomical compartments of this developing organ will enormously improve our capacity to dissect the genetics of mammalian kidney development by facilitating live imaging, lineage tracing, cellular subfractionation and conditional gene knockout. Ultimately, the information gained from such studies will inform both organogenesis and pathology, potentially leading to approaches that will tackle renal malformations and renal failure.All animal work contributing to this manuscript was conducted according to all state, national and international guidelines. Animal ethics approval was provided by AEEC3 of The University of Queensland ).http://www.gudmap.org) as well as via GEO, as detailed previously All microarray data discussed in this manuscript was generated and previously described by Brunskill et al (2008) http://frodo.wi.mit.edu/). In each case the reverse primer included a T7 polymerase promoter tag for the generation of an antisense riboprobe using T7RNA polymerase. Custom-made tracks of the riboprobe amplicons and primers were generated for visualization on the UCSC genome browser http://uqgudmap.imb.uq.edu.au/tools).Primers were designed to amplify 500–800 bp product primarily towards the 3′UTR region of each transcript using the Primer3 program (http://uqgudmap.imb.uq.edu.au/riboprobe_design/).For the riboprobe generation tool, all Affymetrix probes were mapped to a non-redundant set of full length mouse cDNA transcripts Outbred CD1 mouse embryos of unknown sex  were collected at 10.5, 13.5 and 15.5 days post coitum (dpc)  from pregnant adult female mice which were sacrificed by cervical dislocation. The embryonic kidneys and adult kidneys  were dissected in ice-cold Phosphate Buffered Saline (PBS) and fixed overnight in fresh 4% paraformaldehyde in PBS at 4°C.in situ hybridisation using digoxigenin (DIG)-labelled antisense riboprobes as described previously http://www.gudmap.org). Briefly, kidneys were processed and embedded in paraffin and sectioned at 7 µm. Following dewaxing and rehydration, sections were fixed with 4% paraformaldehyde in PBS, washed with PBS, assembled into slide chambers and inserted into the Tecan Freedom Evo 150 robot. Sections were permeabilised with Proteinase K (10 µg/ml) for 20–30minutes and acetylated  acetic anhydride). Sections were immersed in pre-hybridisation solution whilst the chamber racks were heated from 25°C to 68°C. Hybridisation occurred at 68°C for 10 hours with 0.5 µg/ml of probe in hybridisation buffer . After a series of SSC stringency washes, some sections were treated with 2 µg/ml RNase A and all sections were blocked  and incubated with 1∶4000 of anti-DIG-alkaline phosphatase Fab fragments for 6 hours at 4°C. Sections were washed with NTMT . Chromogenic substrate BM Purple was used to detect the in situ alkaline phosphatase activity. Once the signal had reached optimal intensity (4–100 hr), the slides were rinsed and fixed in 4% paraformaldehyde/PBS at 25°C for 10 min followed by PBS washes in order to preserve the mRNA in situ hybridisation signal. In order to keep the level of false negatives to a minimum, control riboprobes for monitoring high and low gene expression (Wnt4 and Shh respectively) were included in every hybridisation run. Resulting sections were scanned using a semi-automated. slide System from Olympus and Soft Imaging Systems  and images captured using Olyvia software  and Adobe Photoshop CS2. All analyses presented (both microarray and SISH) are for genes from 3 compartments of the 15.5dpc mouse kidney, including; early proximal tubule (EPT), renal vesicle (RV) and renal medullary interstitium (MI). It should be noted that RV genes originated from microarrays hybridised with renal vesicles obtained from 12.5dpc kidneys by LCM.Expression patterns were analysed by RNA section Transcription factor binding site (TFBS) motifs was scanned within two sets of core promoter regions from proximal tubule anchor genes; a) the entire core promoter sequence  only evolutionary conserved sequences within the core promoter set with at least 70% identity conserved across a minimum of three species from the ECR Browser using the CLOVER algorithm http://www.ingenuity.com), using default settings and gene interactions curated in the Knowledge Base database. Predicted gene nodes without direct interactions to anchor genes were excluded from the network diagram.Proximal tubule anchor genes network and GO analysis was generated by Ingenuity Pathway Analysis -specific genes was performed using SISH of 15.5dpc kidneys to validate the genes identified through microarray profiling. A) Schematic of capillary loop (Stage III) and maturing nephron (Stage IV) subdivided into renal tubular and renal corpuscle structures. The EPT of maturing nephrons was subdivided into presumptive S1, S2 & S3 segments based on histology and anatomical location within the kidney. B) Example SISH images of expression domains seen in EPT of Stage III (a) and Stage IV (b-d) nephrons at 15.5dpc. (a) Gpd1 in Stage III nephron (EPT and anlage of loop of Henle). The entire nephron can be seen from ureteric tip to renal corpuscle (RC). The outside edge of the kidney and nephron are outlined. Expression in visceral epithelium of RC is indicated (arrowhead). (b) Gpd1 in EPT (arrows), the proximal portion of immature loop of Henle  and the adjacent S3 EPT (open arrowhead). Gpd1 was absent from the distal portion of the ILH within the medulla. (c) Spp2 was specific to the EPT (S1 & S2). Enlarged region (c′) shows Spp2 in S1 adjacent to RC. (d) Gyk in EPT and ILH. Enlarged region (d′) shows Gyk in S3 EPT and ILH.(TIF)Click here for additional data file.Figure S2Spatial expression analysis of early proximal tubule-specific genes in the adult kidney. SISH of adult kidney revealed domains of expression in the renal proximal tubule of the mature Stage IV nephron. Transverse sections through the whole kidney (top) with high magnifications of two enlarged regions shown below; S3 in the outer stripe of outer medulla (a-d) and S1, S2 and a small subset of S3 in the renal cortex (e-h). Examples are shown from each of the adult expression types; broad expression in all renal proximal tubules S1, S2 and S3  and regional expression in either proximal convoluted tubule (Acaa1b and Spp2 - S2 and a subset of S1); or proximal straight tubule (Slc3a1 - S3).(TIF)Click here for additional data file.Table S1Complete list of all genes selected for SISH validation. All candidate anchor genes identified in microarray analysis from both 11 and 7 kidney-subcompartments. “Array Information” contains Affy 430.2 probeset IDs (Affy ID), probese raw signal intensity in raw florescent units (Raw), fold-change values based on microarray normalization performed in GeneSpring software, median raw signal intensity across other subcompartments . “SISH expression pattern across the entire TS23 kidney” contains gene expression pattern annotations scored against all known 15.5 kidney subcompartments. Candidate genes without probes were due to unavailable Fantom 2 amplicon/clones (Riken), or failed PCR.(XLS)Click here for additional data file.