Tau accumulation and progressive loss of neurons are associated with Alzheimer’s disease (AD). Aggregation of tau has been associated with endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). While ER stress and the UPR have been linked to AD, the contribution of these pathways to tau-mediated neuronal death is still unknown. We tested the hypothesis that reducing C/EBP Homologous Protein (CHOP), a UPR induced transcription factor associated with cell death, would mitigate tau-mediated neurotoxicity through the ER stress pathway. To evaluate this, 8.5-month-old male rTg4510 tau transgenic mice were injected with a CHOP-targeting or scramble shRNA AAV9 that also expressed EGFP. Following behavioral assessment, brain tissue was collected at 12 months, when ER stress and neuronal loss is ongoing. No behavioral differences in locomotion, anxiety-like behavior, or learning and memory were found in shCHOP mice. Unexpectedly, mice expressing shCHOP had higher levels of CHOP, which did not affect neuronal count, UPR effector (ATF4), or tau tangles. Overall, this suggests that CHOP is a not a main contributor to neuronal death in rTg4510 mice. Taken together with previous studies, we conclude that ER stress, including CHOP upregulation, does not worsen outcomes in the tauopathic brain. The UPR pathway is initiated by sensing the accumulation of misfolded proteins. UPR activation leads to the induction of molecular chaperones, slowed general translation, and initiation of degradation pathways including autophagy and ER-associated degradation (ERAD)2. The end goal of this process is to reduce the load of damaged proteins and restore cell homeostasis. Failure to complete this quality control favors the buildup of damaged proteins, which increases ER stress3.Cell adaptation to stress stimuli is essential to support neuronal health through our lifespan. Neurons, like other cells, need to respond and adapt quickly to harmful stimuli to ensure cell survival and prevent development of pathologies. The endoplasmic reticulum (ER) unfolded protein response (UPR) has an essential role in the cell to maintain homeostasis6. UPR drives CHOP transcription by activating three upstream stress-sensing membrane proteins: Protein Kinase RNA-Like ER Kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring protein 1 (IRE1α). PERK is the most characterized sensor protein, and like the others, it contains a cytoplasmic and ER luminal domain to transfer stress signal across the membrane7. Upon UPR activation, PERK dissociates from BiP , homodimerizes, and autophosphorylates (active form)8. Activated PERK prevents translation of proteins by the phosphorylation (inactivation) of the translation initiation factor, eIF2α, which then induces the activating transcription factor 4 (ATF4) followed by CHOP transcription10. Simultaneously, accumulation of misfolded proteins causes BiP to dissociate from ATF6 and IRE1α, which are activated after proteolytic cleavage and mRNA splicing, respectively13. Together, these UPR pathways continually respond to stress and decide the fate of the cells under normal or pathological conditions.Prolonged ER stress conditions can shift UPR pathway from a pro-survival to a pro-apoptotic state involving the induction of cell death proteins including CCAAT-enhancer binding protein (C/EBP) homologous protein (CHOP)18. The pathological accumulation of the microtubule associated protein tau has been observed in AD and has been correlated with ER stress markers19. Together with other studies, our lab showed increased signs of UPR activation in human AD and tau transgenic mouse brain areas such as the cortex and hippocampus20. The hippocampus is one of the first areas affected during AD pathogenesis21. It is a main center for learning and memory as well as stress adaptation processes. In conditions where stressors are not properly regulated, like prolonged metabolic stress, UPR is activated in the hippocampus resulting in increased phospho-tau levels, which may accelerate AD pathology17. While not consistent across all reports24, at least three tau transgenic mouse lines  have increased ER stress-induced CHOP26. In the rTg4510 model, the line used in the current study, activation of UPR was accompanied by higher levels of ubiquitinated proteins and tau aggregation19. This activation was progressive and could be reversed by reducing tau levels. rTg4510 mice also show deficits in synaptic plasticity, learning and memory, and neuronal loss, which are common pathological hallmarks in AD27.Recent evidence from in vitro and in vivo studies have linked ER stress to variety of neurodegenerative and age-related diseases, like Alzheimer’s disease (AD)30. This suggests that, when compared to young animals, chronic activation of UPR during aging may be a route to cell death in tau-related diseases like AD. However, the extent of tau neurotoxicity mediated through the ER stress pathway is still unknown. A recent study showed that mice lacking CHOP were protected from ER stress and cell death32. Thus, we hypothesized that silencing CHOP in tau transgenic mice would block ER stress mediated neuronal death and reveal the contribution of this pathway to overall neurotoxicity. The main goal of this study was to evaluate this hypothesis by targeting CHOP in the hippocampi of rTg4510 mice using AAV delivered shRNAs. Cognitive function and histopathological changes, including neuronal loss, were evaluated in the brains of these mice.Interestingly, tau is not overexpressed in AD, nor is it found in the ER, rather it accumulates in the cytosol over time suggesting a problem with ER adaptive response and UPR mechanisms. It is known that proteins homeostasis, including ER molecular chaperones, declines with aging, which debilitates our resistance to stress and promotes cell death. For example, PERK mRNA is reduced while CHOP and caspase 12 are elevated in the brains of aged rats33, we evaluated if the knockdown of this protein could mitigate neurotoxicity in the brains of tau transgenic mice. After identifying an shRNA that reduced CHOP levels in the HT22 mouse hippocampal neuronal cell line  task Fig. a. SpatiaTo determine whether neuronal loss was affected by altered CHOP levels, neuronal density was calculated using unbiased stereological analysis in which neurons positively stained for NeuN/cresyl violet in the CA1 and dentate gyrus hippocampal regions were counted Fig. a,b. BothThe effect of CHOP levels on tau pathology was assessed by Gallyas silver staining. Here, the presence of argyrophilic tau remained unchanged when comparing hippocampal sections obtained from shScramble (control) to shCHOP injected rTg4510 mice , causing an environment with reduced ER chaperone function and poor stress response28. Similarly, UPR-related proteins like p-PERK are significantly increased in aged rTg4510 mice and AD brains29. This is interesting because higher levels of p-PERK appear to precede neurofibrillary tangles in AD and high p-PERK triggers degeneration of neurons via CHOP in mouse hippocampi52. Although our study did not assess impact of aging or exposure to different stressors, these factors will be highly relevant to understand the role of CHOP in age-related and neurodegenerative diseases.Key cellular processes responsible for supporting protein homeostasis, like the UPR pathway, are known to decline with aging55. Without the direct comparison to AAV naïve mice, we cannot conclude if this contributed to our results. However, we do expect if AAV-mediated UPR activation did have any contribution that it would be similar between the groups based on their comparable GFP levels. We limited this study to the use of rTg4510 mice but investigating CHOP contribution in other tau transgenic models will be important to conclude whether CHOP mediates tau neurotoxicity. Our study suggests that future work should include crosses with CHOP knockout mice31 since knockdown of CHOP and the use of AAV may not give the desired effect. Finally, to better understand the contribution of CHOP and ER stress in neurodegeneration, further studies are needed to evaluate the contribution of sex differences, aging, damage to non-neuronal cells, and involvement in other pathways like neuroinflammation.Here, we investigated whether modulating CHOP expression in the hippocampi of rTg4510 affected Gallyas-positive tau inclusions. Measurements of toxic soluble tau species are missing in this study. Examining changes in tau aggregates, oligomers, and hyperphosphorylated species will be important to conclude whether the observed CHOP upregulation induced tau pathological changes. We showed that ATF4 was unaffected in shCHOP injected mice, however, including other proteins involved in the UPR signaling, like BiP, would be informative about the degree of activation of this pathway in neuronal health in this tau transgenic tau model. The lack of pathological changes in an environment where levels of CHOP are increased and ATF4 is unchanged, leaves an open pathway to explore in this model regarding whether both factors are needed to induce cell death in the brain. It is also important to note that AAV transduction has been previously shown to active the UPR, including the PERK and IRE1 pathways, which can trigger CHOP activationIn conclusion, our results suggest that CHOP is not the main contributor to neuronal death in rTg4510 mice. CHOP may not be the primary player in rTg4510 neuronal death, but we cannot exclude the possibility of contributing to other processes like autophagy and protein translation. Thus, given its role in a variety of pathways and interplay with other cell regulatory factors, it may be important to examine CHOP’s function throughout AD pathological stages. Further studies could provide new insights for understanding whether CHOP plays a decisive role in protecting or driving cell death during the development of AD and other tauopathy-related neurodegenerative diseases.Male mice were housed up to five per cage or individually when aggression was observed within the cage, which were acquired from the colony we maintained at the University of South Florida vivarium. Animals were kept in standard conditions with a 12-h light cycle (lights on at 6:00 and lights off at 18:00 h) while having free access to food and water. All experimental methods were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of South Florida Institutional Animal Care and Use Committee (IACUC). All animal studies were conducted following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. The sample size was based on prior studies with this strain of mice using intracerebral injections of AAV for similar types of analysis.56. Since the tTA response element is controlled by a CaMKIIα promoter, tau is the restricted to excitatory neurons in the forebrain of the rTg4510 mice. Mice genotyping was performed after extracting DNA from ear tissue (2 mm punch) by a polymerase chain reaction (PCR) using the following primers: (1) Human Tau cd1F gene F , (2) tTA gene F [5′CGC TGT GGC ATT TTAC TTT AG 3′ and tTA gene R 5′CAT GTC CAG ATC GAA ATC GTC3’] and housekeeping gene T-cell receptor delta chain (Tcrd) . A QIAxcel Advanced system  was used to analyze the DNA amplified PCR product.rTg4510 mice were obtained by crossing a Tg4510 line (FVB/N mouse genetic background containing a human MAPT P301L cDNA) and an activator line (mixed 129S6 and FVB/N mice genetic background expressing a tetracycline response element) under the control of a CaMKIIα promoterHT22 cells, an immortalized mouse hippocampal cell line, were cultured and transfected for 72 h with non-targeting shRNA  or CHOP/Ddit3-targeting shRNA (Sigma Mission library clone ID: TRCN0000324349) plasmids. Twenty-four hours before harvesting the cells, 10 µg/mL of tunicamycin was added to induce ER stress. Then, cells were collected for Western blot analysis. Membranes were probed overnight using CHOP/GADD153  and actin  antibodies.57.The short hairpin vectors targeting CHOP (shCHOP) or a control scramble sequence (shScramble) as validated above expressed the following RNA targeting sequence for shCHOP (GAAACGAAGAGGAAGAATCAACTCGAGTTGATTCTTCCTCTTCGTTTC) and for shScramble (CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG). AAV9-shCHOP and AAV9-shScramble plasmids were designed and purchased from Vector Builder with a U6 promoter for the shRNA and a CAG promoter for an EGFP fluorescent tag. The production of AAV9 and purification was done in our lab as previously described12 viral particles/mL were delivered per injection site for each mouse using a robot stereotaxic drill and injection instrument .At 8.5 months of age male rTg4510 mice were randomized to receive bilateral injections of AAV9 shCHOP or shScramble into the hippocampus  and cortex . Two microliters of 1 × 10General locomotor activity and behavioral testing started eight weeks after viral injection. Following this, animals were anesthetized with Somnasol overdose and perfused with 0.9% saline at 11 months of age. Brains were collected and the left hemisphere was immersed overnight in 4% paraformaldehyde and transferred to gradient sucrose solutions  during the subsequent days. Using a sliding microtome, horizontal sections at 25 μm for immunofluorescence and Gallyas staining or at 50 μm for stereological analyses were obtained from these brains.56 using GADD153  antibody followed by Alexa Fluor Goat α-Rabbit IgG-594 secondary antibody. Tissue was mounted onto slides and coverslipped with Prolong Gold Antifade reagent. ATF4 staining was performed using the primary ATF4/D4B8  and anti-rabbit secondary  antibodies. To prepare tissue for counting neuronal density, sections of 50 μm thickness were incubated with biotinylated NeuN antibody , mounted, and counterstained with 0.05% cresyl violet (Nissl) followed by dehydration with Xylenes and coverslipped with DPX solution (Sigma-Aldrich 06522). The Gallyas silver staining was used to detect tau positive neurofibrillary tangles as we have done previously58. Animals with low or poor expression of the virus in the hippocampal region were excluded in the analysis.To detect CHOP expression, free floating sections including hippocampal area were stained as previously described58. CHOP analysis was completed using the ImageJ/FIJI software. The “Try-it-all” option was used to set auto thresholds (16 possibilities) in all tissues. The three-best representation of thresholds among the tissue  were averaged. We then analyzed GFP and CHOP integrated density by measuring the total sum of intensity units in each image, which was then averaged across all images for a single animal and then compared between injection groups. CHOP/GFP colocalization analysis was completed using the same program (JaCoP plugin in ImageJ/FIJI software) in the images showing CHOP upregulation. Mander’s quantification provides an estimate of the fraction of CHOP being colocalized with GFP while Pearson’s overlap coefficient indicates colocalization between GFP and CHOP intensities which ranges from 0 (null) to 1 (full). GraphPad PRISM software was used to calculate group mean differences between shCHOP and shScramble mice.Immunofluorescent staining for CHOP was imaged along with GFP expression using a 20× Plan-Apochromat 20× objective on a Zeiss LMS 880 AxioObserver confocal microscope. Slides stained for Gallyas and ATF4 were imaged using an Axio Scan.Z1  microscope slide scanner. Total Gallyas positive staining and ATF4 levels were determine using Nearcyte analysis software (Nearcyte.org). Regions of interest were outlined manually by a blinded investigator, and slides were analyzed by batch processing to determine the ratio of the positive staining area within the region of interest area (Area Ratio). The resulting Area Ratio was multiplied by 100 to generate Area Ratio (%). NeuN/cresyl violet positive neurons were counted using computer-based unbiased stereological analysis as we have done previouslywww.anymaze.com). Locomotion was evaluated in this test after calculating the total distance travelled. Anxiety-like symptoms were assessed by measuring the time spent in the center and number of entries to the center of the arena. The apparatus was clean with 10% ethanol after each trial.Mice were acclimated to the testing room 1 h before all behavioral tests. These were all conducted during the morning by a blind experimenter to the study. Individual mice were placed in the center of the open field box, allowed to explore for 10 min, and video monitored using the Any-maze video tracking software  and multiplied by 100.This behavioral task was used to evaluate spatial learning and memory performance as well cognitive flexibility in shScramble or shCHOP rTg4510 injected mice. The testing apparatus consisted of a water filled circular pool with 6-metal arms. Four different spatial cues were placed on the walls around the pool. There was an escape platform located at the end on one arm  and it was made visible using a raised flag or hidden (no flag). Animals were given 12 trials on each day and allowed to swim for up to 60 s to locate the platform. During training (day 1), the platform visibility was alternated with or without a flag between the trials. During memory test (day 2), the platform remained hidden throughout the 12 trials. To test their flexibility to learn, the platform was moved to the opposite direction of the goal arm. An entry to the incorrect arm  or passing 15 s without arm entry was counted as an error. A blind experimenter to the study manually counted errors made on each trial.For the animal studies, the sample size was based on prior studies with this strain of mice using intracerebral injections of AAV for similar types of analysis. Normal distribution was examined by Shapiro-Wilks’s test. Using the GraphPad PRISM software version 9 , differences between shScramble and shCHOP injected mice were determined by an unpaired Student t-test  and by two-way ANOVA [RAWM]. Outliers were identified using the Robust regression and Outlier removal (ROUT) method with PRISM software. Only mice lacking viral expression in the hippocampus were excluded in the study. Data is presented as the standard error of the mean (± SEM). Two-tailed p-values were calculated and considered significant when alpha level was equal or less than 0.05.Supplementary Figures.


Here, we use whole-genome sequences of 48 contemporary weedy rice accessions to investigate the genomic consequences of crop-weed hybridization and selection for herbicide resistance. We find that population dynamics have shifted such that most contemporary weeds are now crop-weed hybrid derivatives, and that their genomes have subsequently evolved to be more like their weedy ancestors. Haplotype analysis reveals extensive adaptive introgression of cultivated alleles at the resistance gene ALS, but also uncovers evidence for convergent molecular evolution in accessions with no signs of hybrid origin. The results of this study suggest a new era of weedy rice evolution in the United States.Weedy rice is a close relative of cultivated rice that devastates rice productivity worldwide. In the southern United States, two distinct strains have been historically predominant, but the 21 Whole-genome re-sequencing of weedy rice accessions reveals recent genetic introgression and adaptation that is altering crop-weed dynamics across southern USA. Recently, the evolution of agricultural weeds—which, unlike crops, evolve without intentional selection by humans—are providing additional new insights into the genomics of adaptation6. Among agricultural weeds, those that are closely related to crop species can be a particularly dynamic system because of the added potential for genetic exchange with crop cultivars as a means of weed evolution and adaptation7.Understanding the genomic basis of adaptation is among the most important questions in modern evolutionary biology. Crop domestication has long been recognized as a model for studying adaptive responses to selectionOryza spp.), a de-domesticated form of cultivated rice (O. sativa)9 that has evolved multiple times independently around the world11. As a feral crop derivative, it is highly adapted and specialized to rice fields, where it competes aggressively with the crop. Just one weedy rice plant per square meter can lead to a >200 kg ha−1 loss of yield12 and reductions in harvest quality that compromise market value15. Due to its close phenotypic and genetic similarity to the crop, weedy rice is challenging to control with herbicides and often requires additional specialized field maintenance practices. As a result, weedy rice causes annual economic losses of more than $45 million in the United States (US)16 and hundreds of millions of dollars worldwide17.The genomically best-characterized weedy crop relative is weedy rice  weedy rice is descended from indica rice varieties grown in Asia, while blackhull-awned (BHA) is derived from genetically distinct aus Asian varietal group22. Outcrossing rates between SH, BHA, and local US cultivars  have historically all been <1% despite their close physical proximity within US rice fields23.Weedy rice strains worldwide are characterized by a few key shared weed-adaptive features, including a strong seed dispersal mechanism (shattering)TM rice) were first commercialized in the US as a means of controlling weedy rice and other agricultural weeds. These HR cultivars are resistant to the imidazolinone (IMI) class of herbicides due to one of two amino acid replacements in the acetolactate synthase enzyme (ALS). The first ClearfieldTM cultivars, CL121 and CL141, carried a G654E replacement24; they were quickly replaced in 2003 by CL161 and later cultivars, which instead carry an adjacent S653N replacement conferring greater herbicide resistance. US HR rice cultivation peaked at ~65% by the mid-2010s and now constitutes ~35% of rice acreage25.In 2002, non-transgenic herbicide resistant (HR) rice cultivars TM technology reported instances of HR weedy rice24. By 2010, 80% of weedy rice plants sampled in one study were classified as resistant and carrying the S653N allele derived from HR cultivars28. In the decade of continued HR cultivar use that has followed, it is unclear how weedy rice has continued to evolve and adapt, or the extent to which crop × weed hybridization has continued to shape the genetic composition of US weedy rice populations.The combined adoption of HR and hybrid rice in US agriculture has thus created a two decades long natural experiment: two genetically distinct strains of a historically self-fertilizing weedy crop relative have now been subject to strong selection for herbicide resistance, and this selective pressure has coincided with increased opportunities for crop-weed hybridization via crop volunteers. Notably, as early as 2004, farmers utilizing the ClearfieldIn this study we used whole-genome resequencing to investigate how the genomic composition of southern US weedy rice has changed since the 21st century introduction of HR and hybrid rice cultivars. We addressed the following specific questions: 1) How do the genomes of contemporary weeds differ from the historic SH and BHA strains that predominated through the 20th century? 2) Following crop-weed hybridization (creating a weed with 50:50 crop-weed genomic composition), does selection over subsequent generations in weed populations lead to a genome-wide bias toward one ancestral genome or the other? And 3) Within the weed genome, does selection drive known weed- or crop-specific alleles to high frequency in a predictable pattern based on expected advantageous traits for contemporary weeds? Our findings reveal a genomic revolution in US weedy rice in the last 20 years that has irrevocably altered crop-weed dynamics and mechanisms of weed adaptation.29, so this sampling may be considered representative of the southern US rice production region29. Whole-genome sequences (>40× average coverage) were generated using leaf tissue from one seed per maternal plant grown to the seedling stage. Genome assemblies were analyzed with 98 previously published weedy, cultivated, and wild rice samples33 resulting in a dataset of 146 samples and ~19.34 million SNPs. Previously published genomes included 22 historic weedy (11 SH and 11 BHA), 49 cultivated  and 27 wild rice accessions. Wild rice accessions were removed from analysis after they were confirmed to play no role in US weedy rice evolution, as was expected given their absence from the US agroecosystem  separated subgroups within the indica subspecies, with aus crop varieties and aus-like BHA weeds distinguished from indica and indica-like SH weed samples. Aside from four contemporary weed accessions that cluster very closely with historic SH strains, all other contemporary weeds have intermediate distributions along PC1 between historical weedy rice  and the US cultivated rice group   and ADMIXTURE analysis. The PCA revealed relatively tight within-strain grouping of cultivated and historic (pre-2000) weedy rice, with contemporary weedy rice showing a much broader dispersion Fig. . PC1 , indica cultivated varieties, historic SH weeds, historic BHA weeds, and a genetically homogeneous subgroup within the contemporary weeds that in the PCA are grouped with other crop-BHA hybrid descendants. This genetically homogeneous subset of BHA-like weeds may represent a derivative population of BHA × tropical japonica hybrids that emerged early enough after HR cultivar introduction to have evolved into a genetically homogeneous subgroup through multiple generations of inbreeding ; it is designated the ‘beta’ group in reference to this inferred early origin.For ADMIXTURE analyses of population structure, CV scores indicated try Fig. . At K = K = 5, 35 of 48 contemporary weed accessions (72.9%) had membership assignment coefficients of >15% in two or more genetic populations. Most of these admixed weeds  appear to be derived from BHA rather than SH historic weeds, which account for 6 of the 48 admixed accessions (12.5%). A single contemporary accession appears to have complex SH-BHA admixed ancestry, with >20% membership coefficients from SH, BHA, and tropical japonica genetic populations. Nine samples (18.8%) fell into the homogeneous beta group. As in the PCA, the remaining four contemporary samples (8.3%) were genetically indistinguishable from historical SH weeds. Thus, crop-weed hybridization appears to have given rise to most contemporary US weedy rice, with most of these hybrid derivatives descended from BHA-crop hybridization.Consistent with results from the PCA, ADMIXTURE analysis suggests that most contemporary US weeds are genetic admixtures descended from hybridization between the historic weed strains and US cultivated rice. At Genetic diversity measures were calculated at every SNP across the genome in order to gain a snapshot of the contemporary weedy rice genome. These measures allowed us to quantify the relative endurance of weed and crop ancestor genomes on a genome-wide scale, and to gauge the relative timing of emergence of the homogeneous beta weed population in comparison to the more heterogeneous contemporary weed groups. Heterozygous SNP quantification indicated that contemporary weeds collectively have a high number of heterozygous sites when compared to their crop ancestors , with Rc, a locus conferring seed dormancy . As hypothesized, we found consistent evidence of a crop-like ALS region on chromosome 2 .e 2 Fig. . We alsoeds Fig. . Consistles Fig. . For Rc,ALS HR locus, a median joining network tree was constructed from manually phased consensus nucleotide sequences retrieved from assembled raw reads  ancestry and those on the left side characteristic of weedy ancestry. Most of the contemporary weeds are distributed on the right side of the network and carry the S653N mutation and surrounding haplotype sequence present in the widely grown CL161 and later HR cultivars. Two weed samples, E08 and E09, are also on the right side of the network but instead carry the G654E mutation and surrounding haplotype indicative of the oldest HR cultivars (CL121 or CL141); this suggests that these two samples are descendants of the very earliest crop × weed hybridization events.To gain a finer-scale view of haplotype variation at the ads Fig. . The hapALS haplotypes of cultivar origin. These haplotypes are represented almost entirely by SH-like plants, consistent with Loter results for the ALS genomic region. Samples A01 and A08 carry the older G654E mutation, but do not show evidence of hybrid origin and occur in a distinctly weed-like haplotype background. This allele was previously shown to have been present in the historical SH population at low frequency35, likely due to infrequent exposure to imazethapyr during IMI-resistant soybean rotations. The presence of this allele in contemporary weedy rice populations is thus most likely due to selection on standing variation. Two additional samples, A05 and A06, carry the S653N resistance allele, and also show no evidence for hybrid ancestry; this suggests a convergent mutation event conferring resistance. To our knowledge, this is the first report of the S653N resistance allele occurring in weedy rice through mutational convergence rather than crop allele introgression. Notably, two common mutations linked to IMI herbicide resistance, Ala122 and Trp57436 were not found in the populations examined in this study.The left section of the haplotype tree, conversely, does not have TM technology. Thus, we suspect they are offspring of parents segregating for resistance. Most plants showing high herbicide resistance carried the common CL161 haplotype (characterized by the S653N mutation); additional samples carry the older resistance haplotype of CL121 and CL141 cultivars (characterized by the G654E mutation)  classified as highly resistant Table . Another22 suggests low hybrid fitness. Additionally, the existence of “pre-adapted” HR weedy rice strains, even at initial low frequency, would lead one to expect those fit individuals to quickly rise to high frequency. Instead, reality suggests that those high-fitness individuals only make up ~4% of contemporary samples, while the presumably low-fitness hybrids make up 92% of the contemporary population. One possibility is that while F1 fitness is low, fitness of later stages, after selfing, is higher as alleles segregate into favorable configurations.The results of this study raise a key question: why are the majority of contemporary weedy rice plants of crop-weed hybrid origin? The lack of hybrid persistence prior to 2000Local ancestry analysis of BHA-like, “beta”, and SH-like accessions revealed genomes, regardless of ancestry, built primarily of components derived from their weedy rice ancestor. This consistency across weed strains broadly suggests either a selective maintenance of weedy genome components or a selective purge of crop alleles—though these need not be mutually exclusive. The results described here could help inform discussions on crop allele escape (transgenic or otherwise) and the genome-wide process of adaptive introgression in agroecosystems.TM cropping system has reduced average field infestations drastically, but two decades of herbicide application in the presence of hybrid rice gene-flow bridges has resulted in weedy rice that is herbicide resistant and likely more competitive than historical populations. The rapid adaptation of weedy rice to herbicide application should serve as yet another example of the dangers of relying on single methods of control for agricultural pests.The shifting landscape of rice agriculture has resulted in a new generation of weedy rice. The Clearfield8. Samples were collected from fields representing three different rice cropping histories: HR inbred cultivars ; HR hybrid cultivars , and hybrid non-HR cultivars   TABLE . Where a37. It should be noted that the DNA for this study was collected from plants grown and selfed one additional generation in the greenhouse; therefore, variation observed in genome sequence data may not correspond perfectly to field-collected genotypes, particularly for segregating variants in hybrid derivatives. Illumina libraries were generated in house using a Nextera DNA Flex library prep kit with Nextera DNA CD indexes with the i5 bases recommended for HiSeq 3000/4000 . Samples were multiplexed following recommendations from Nextera and sent to Novogene  for paired-end short-read sequencing on the HiSeq X 10 platform. Raw reads were de-multiplexed by Novogene before data return.One seed per field-sampled plant was brought to seedling stage, from which fresh leaf tissue was collected and ground in liquid nitrogen for DNA extraction using a modified CTAB protocol33 resulting in a full dataset of 146 samples representing cultivated, weedy and wild rice . Vcftools was also used to remove wild samples from.vcf files for analyses where they were not required (described below).Whole-genome sequencing reads from contemporary weedy rice samples were combined with raw reads from previously published whole-genome studies pca flag within plink43 was used in conjunction with ADMIXTURE44 to determine population structure . ADMIXTURE results were visualized in pong45. Wild rice was found to show little-to-no overlap with contemporary US weedy rice and was removed from further analysis. Principal component analysis (PCA) and ADMIXTURE analysis showed that grouping contemporary weeds by field type was uninformative for explaining population structure; they were therefore grouped and analyzed based on their predominant weedy rice ancestry in subsequent analyses. From the ADMIXTURE and PCA results combined, contemporary weeds were categorized into three groups: “SH-like”, defined as >10% SH ancestry in ADMIXTURE (without BHA contribution) or placement in the PCA output as intermediate between historic SH and cultivated tropical japonica strains; ‘BHA-like’, defined as >10% BHA ancestry in ADMIXTURE (without SH contribution); and the ‘beta’ group, defined based on placement with BHA-like weeds in the PCA , but with assignment to its own unique genetic population in the ADMIXTURE analysis at K ≥ 5. A single contemporary weed accession with complex admixed ancestry was assigned its own category (‘complex’).The -het flag in the plink software. Wright’s FST was calculated for each contemporary weed group in relation to its weed and crop ancestors using the –weir-fst-pop flag in the vcftools software, with a window size of 500 kb and a 250 kb step size. The first FST calculation measured differentiation between a given contemporary weed group  and the predominant weed ancestor of that group (SH or BHA), while the second measured differentiation between that weed group and the rice variety group representing US cultivars . These FST values were then plotted together across the 12 chromosomes of the rice genome to identify genomic regions with differential contributions of the weed or crop ancestor. Average pairwise nucleotide diversity (π) values for contemporary weed groups were calculated and visualized in the same way using the –site-pi flag in the vcftools software. As a measure of inbreeding, homozygous locus counts were performed in vcftools using the -het flag and converted to fraction of heterozygous loci using the formula ((N_sites – O(HOM))/N_sites). Lastly, a custom python script was developed to identify ancestrally-informative SNPs . This script then calculated observed heterozygous genotype counts at those sites only. This analysis allowed us to estimate the number of generations since hybridization, assuming a 50% reduction in the number of heterozygous genotypes per generation and a return to a strictly selfing mating system.Heterozygous sites among genome-wide SNPs were calculated per accession using the FST analyses, local ancestry across the genome was calculated for weed groups using the Loter software34 and visualized with matplotlib46. A custom python script was used to quantify the proportion of ancestral genomes (crop vs. weed) found in the contemporary hybrids. A second custom python script was written to convert an MSU-7.0 genomic location to the corresponding bin of the Loter output. This allowed us to pinpoint potential candidate genes for weed adaptation.To complement samtools41 software package was used to retrieve raw reads mapping to the ALS gene region from sorted.bam files. These raw reads were retrieved by using the index, view (specifying the known gene boundaries) and fasta commands. Raw reads were then exported to the Geneious 8.1.6 software (https://www.geneious.com) for assembly to a reference ALS sequence obtained from GenBank (accession MH636577). After assembly, sequences were trimmed to match the reference sequence and manually phased to remove heterozygous calls from consensus sequences. Phased and trimmed sequences were exported to the PopART software48 for haplotype network visualization.The −1 imazethapyr two times, 10 days apart. Imazethapyr was applied in 187 L ha−1 spray volume, in a spray chamber with a motorized spray boom fitted with two 800067 flat fan nozzles spaced 46 cm apart. The herbicide treatment was replicated three times and a nontreated check for each sample served as reference for evaluation of plant response. Visible injury was evaluated 3 weeks after the second application of imazethapyr on a scale of 0 to 100% where 0 indicated no injury and 100 indicated a dead plant. The level of injury reflects the level of resistance to imazethapyr.Weedy rice seeds were planted at 1.27 cm depth into pots (15.24-cm top diameter) filled with 50:50 mixture by volume of field soil and Sunshine potting mix. Up to 12 seeds were planted per pot, depending on the quantity of seeds available per sample. The pots were placed in a greenhouse with supplemental lighting to achieve a 16-h daylength. The temperature was set at a minimum of 25 °C and maximum of 35 °C. At the 3-leaf stage, the plants were treated with 70 g ai haThis study is based on the short-read whole-genome sequencing of 119 weedy and cultivated rice strains, and all population genomic analyses were performed using publicly available software described in the Methods. All statistical analyses were preformed using programs described in the Methods. Reproducibility, including parameters for the population genomic analyses, sample sizes and number of replicates, are stated in Methods and Figures.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase ( Gain-of-function mutations are responsible for neoplasms originating from these cell types. These include gastrointestinal stromal tumors (GIST) originating from ICCs5, mastocytosis, acute myeloid leukemia (AML), malignant melanoma and germ cell neoplasms8. Further, certain types of malignant breast cancer are associated with KIT overexpression, but the relevance of KIT mutations for mammary carcinoma development is unclear, since activating KIT mutations are rarely found in this context10. In humans, the KITN822K mutation potentially induces several cancer types, and is associated with resistance to tyrosine kinase inhibitor (TKI) treatment using imatinib11.The proto-oncogene KIT, a cell surface receptor tyrosine kinase, plays an important role in the development of many cell types including erythrocytes, mast cells and interstitial cells of Cajal (ICCs)Kit mutations were established and significantly contributed to the current knowledge about the role of KIT in cancer development15. One of the achievements within the Munich ENU mouse mutagenesis program was the establishment of a mouse line (preliminary laboratory name MVD013) representing microcytic polycythemia16. Here we report on its comprehensive characterization concerning hematopoietic abnormalities, development of neoplasms and unexpected side effects. Details on the identification of the causative genetic alteration, a KitN824K mutation, which is homologous to the KITN822K mutation of human patients, and a comparison to previously established models are presented. As a consequence of the high conservation of the KIT protein between human and mouse17 the obtained phenotypic data is expected to add valuable information about the role of KIT mutations as a primary cause of cancer and other disorders. A potential application is given in the accompanying paper by Kraiger et al. characterizing the disease progression in C3H KitN824K/WT mice across a life span of twelve month by in-vivo magnetic resonance imaging18. This novel disease model is expected to facilitate future studies addressing the identification of genetic and environmental factors affecting disease progression in KIT-induced cancer.During the past ten years several mouse models carrying activating 16. Heterozygous mutant animals show increased red blood cell counts with marked microcytosis  and hematocrit values . Additionally, platelet counts were mildly decreased in mutants compared to controls  evenly distributed on chromosomes 1–19 was carried out in 61 N2-hybrid mice including 23 animals showing polycythemia and 38 littermates showing a wild-type phenotype. The strongest linkage of polycythemia with C3B6 heterozygosity was observed for two SNPs on chromosome 5: rs29635956 at about 67.73 mbp and rs31585424 at about 79.10 mbp. No other chromosomal region showed remarkable linkage of the phenotype to C3B6 heterozygosity.Ifi203 on chromosome 1, a missense mutation in Kit on chromosome 5 and a missense mutation in Myh13 on chromosome 11. Only the Kit mutation, a T to A substitution in exon 17, was localized within the identified mapping region on chromosome 5. It was confirmed by sequencing of the respective region in two additional animals with mutant and control phenotype  of offspring derived from matings of heterozygous mutant mice to wild-type mates showing the mutant phenotype. Together with the almost complete genotype–phenotype match for animals classified by CBC and genotyping these results indicate, that polycythemia is fully penetrant in heterozygous mutant animals. Polycythemic progeny in litters derived from mating heterozygous male and female animals (57.5% mutant and 42.5% WT), however, did not reach the proportion of around 75% of offspring with mutant phenotype expected in case of full viability of homozygous and heterozygous mutant offspring. The proportion of mutant animals was even lower than 66% that would have been expected according to Mendel in case of full viability of heterozygous offspring and complete pre- or perinatal lethality of homozygous animals. This observation suggests homozygous lethality and reduced viability of heterozygous offspring when derived from heterozygous parents. The assumption of homozygous lethality is also supported by the finding of a decreased mean litter size with in average 4.5 living offspring per litter instead of 5.2 in C3H-WT maintenance breeding. However, since litters derived from heterozygous parents mated to wild-type partners were rather bigger than mean litter size obtained from C3H maintenance breeding , fertility of heterozygous mutants in general appeared unaffected.Macroscopic and histological evaluation of 76 mutant animals ranging from age less than 2 months up to 20 months revealed signs of abnormal cecum morphology and altered intestinal passage.Kit mutants, older than 2 months, which equates to 100% penetrance of this gastrointestinal pathology. Except two very young female mutant mice (aged 2 months) all mutants displayed diffuse ICC hyperplasia in the stomach involving the complete glandular region and the limiting ridge separating glandular region and forestomach , congestion of ingesta in the cecum was found in almost all heterozygous mutant animals older than 10 months Fig. o. Old anProtruding Peyer´s patches (PPs) were found in the cecum of control animals aged more than five months Fig. p, but weKitN824K/WT mice.No nodular lesions or tumors comparable to GISTs in humans were detected in C3H KitN824K/WT mutant mouse during necropsy. Out of 24 female mutant animals aged 8 or more months, 18 animals displayed such neoplasms  in humans. Interestingly, rare cases of PV patients carrying KIT mutations have been reported26. Additionally, in C3H KitN824K/WT mice an increased proportion of granulocytes in peripheral blood leukocytes and in the spleens of mutant mice was observed. Comparable phenotypes were described for KitV558∆;T669I/+ mice, bearing a mutation in the juxtamembrane domain of KIT and an additional “gatekeeper” second site mutation12. Interestingly, both phenotypes, microcytic erythrocytosis and an increase in granulocytes and monocyte-macrophage populations in the spleen and bone marrow were found in KitV558∆;T669I/+ mice, suggesting that proliferation of the myeloerythroid lineage is affected by this double mutation in a similar way as in the KitN824K/WT single A-loop mutation27.The most prominent phenotype found is the microcytic polycythemia with significantly increased hemoglobin and hematocrit values observed in all heterozygous mice. This was associated with increased proliferation rates of bone marrow stem cells in six months old and reduced proliferation in one year old mutant animals. KIT signaling is known to be important for survival and proliferation of erythroid progenitors and so this phenotype is in line with the importance of the A-loop region for hematopoiesisKitV558∆;T669I/+ mice was a hyperplasia of ICCs in the large intestine and stomach. KIT-activating mutations are found in 60–90% of GIST patients28. Most often these mutations are localized in the juxtamembrane domain of the receptor. Only around 2% of activating point mutations in GISTs reside in the TKII domain in exon 17, where the N822K mutation is located29. Most of the mouse models established so far, are characterized by development of GIST lesions at the cecum in heterozygous or homozygous mutant animals15. ICC hyperplasia is a generic term of microscopic KIT expressing spindle cell lesions detected during surgery of patients suffering from larger GISTs or other non-GIST lesions31. It is described as a thin layer of spindle-shaped proliferating cells between the inner circular and the outer longitudinal muscle layer32. Agaimy et al. reported on cases of diffuse Cajal cell hyperplasia with involvement of the complete layer of the muscularis propria in the distal colon, which is an uncommon region for GISTs30. KitV558Δ/+ and Kit-K641E mice also develop ICC hyperplasia and neoplastic lesions in the cecum, with KitV558Δ/+ mice showing normal cell counts in blood recapitulating the main characteristics of human GIST patients15. The C3H KitN824K/WT mouse line mimics a mixture of observations made in humans. The ICC hyperplasia is of 100% penetrance with stomach, cecum and colon being affected. It is localized either between the two muscular layers or replacing the complete muscularis propria. While in humans and also in the double mutant KitV558∆;T669I/+ mice GISTs develop from ICC hyperplasia, there was no tumor progression in the GI tract of Munich C3H KitN824K/WT mice, even in advanced age.Further observed in double mutant KitN824K/WT animals suffer from slowly progressing constipation of the cecum with a penetrance of 100% in animals older than 10 months. ICCs are known to be pacemakers for different motor activities in the gut. In patients suffering from chronic idiopathic constipation or megacolon the number of ICCs is decreased33. In the Munich KitN824K/WT mice the network seems to be disturbed leading to motility problems, although the animals show an ICC hyperplasia. The KitV558Δ/ + mice also argue for this because distended ileum and cecum were found in this mutant as well15.However, https://www.jax.org/strain/000658) and age matched control mice display no tumors this pathology is genotype-related. The tumor-type pattern was ranging from solid, sometimes with comedonecrosis, to glandular with partially large lumina. A study of Mayr et al. in 2019 investigated the correlation between GIST and additional neoplasms, and 37% of patients with GIST showed at least one additional tumor, with 20% of these being neoplasms of breast/female genital system. The reason for this association remains unknown34. KIT is highly expressed in normal mammary gland tissue and is described as a progenitor marker in the mouse mammary gland35. However, carcinogenesis is mostly associated with a loss of its expression36. In human breast cancer KIT expression is downregulated during epithelial transformation by KIT promoter hypermethylation37. Other published data suggest that KIT has a dual role in cancer, acting both as an oncogene via its kinase domain or as a tumor suppressor through induction of apoptosis38. Meanwhile an association of strong KIT expression with high-grade breast carcinomas of the basal-like subtype has been shown40. Although a majority of triple-negative mammary carcinomas shows strong expression of KIT, activating mutations of KIT in this type of cancers seem to be rare in humans41. On the other hand, Hussain et al. report on KIT mutations in more than 20% of mammary carcinoma biopsy samples, and most of these mutations have been detected before in other tumor types42. In another study Funkhouser et al. found KIT mutations in more than 11% of investigated breast tumor samples and an association with serum galactin levels and occurrence of brain metastasis43. These observations suggest a role of KIT mutations in breast tumorigenesis or disease progression. However, the N822K mutation has as yet not been reported in human breast cancer. Our immunohistochemical characterization results of the mammary tumors of this mouse model go in line with those of Eroglu et al. in human breast cancer, where KIT expression was linked to ER positivity but was found independent of ErbB244. In summary, our results indicate that activating Kit mutations are a potential primary cause of mamma carcinoma development in mice. Further studies are required to characterize these tumors in more detail and elucidate underlying pathomechanisms.An interesting phenotype we observe in our mice, which to our knowledge has not been described for any other KIT-activating mutation model, is the development of mammary tumors in female mice aged 8 months and older. Since the background strain C3HeB/FeJ does not carry the mammary tumor virus  infiltration is known in a range of tumors including breast cancer where they are common components of the tumor stroma. Huang et al. showed that MC infiltration and activation in breast tumors is mainly mediated by stem cell factor and the KIT receptor45. However, the role of increased MCs in cancer is controversial and high numbers of MCs have been associated with good prognosis as well as with poor prognosis and distant metastasis in breast cancer46. Our mice are a valuable model to study the impact of Kit mutation on MC infiltration and tumor progression in mammary tumors.While mutations in the A-loop region of KIT are known to be associated to human mastocytosis29. Although we did not observe germ cell tumors in C3H KitN824K/WT mice, the observations of apparently increased fertility of heterozygous mutant animals and the hint towards homozygous lethality could be interesting in this respect. Further studies are required to investigate effects on germ cell development and embryogenesis in detail.The homologous amino acid exchange at position 822 in humans was also been found in testicular seminoma in humansKit-mutant models carrying activating mutations. However, slowly progressing constipation of the cecum and especially development of mammary tumors were not reported for any of these models. Instead, all of the models developing ICC hyperplasia also suffered from GIST tumors at the cecum with ileo-cecal obstruction leading to early killing or spontaneous death. Therefore, mutant animals of these models might not get old enough to develop mammary carcinomas, since these occurred in our model in 8 months old or even older females.Our model shares several phenotypes described for other rodent KitN824K/WT mice were bred on a pure C3HeB/FeJ background, except for the hybrid animals produced for linkage analysis. While enteric wall thickening but no protruding GIST tumors were found in mutants on C3H background, three out of four mutant hybrid animals sacrificed and dissected with an age of eight months showed bulging tumors at the cecum. One of these tumors was histopathologically analyzed and displayed typical GIST morphology. This difference likely resulted from background-specific variants in modifying genes that influence disease progression in KitN824K/WT mutant mice, and the model could probably be used to identify these factors. However, since this observation is based on accidental findings in a small number of animals, additional studies are required to confirm the influence of a distinct genetic backgrounds on disease progression.Preliminary results from mice with mixed genetic background suggest that the genetic background significantly affects disease phenotype progression. The 18. Similarly, we cannot draw final conclusions concerning the effects of genetic background on tumor development in our model from only four mutant animals investigated in this study. Nevertheless, the observation of GIST tumor development in three out of these four mice on the background of numerous investigated mutants on C3H-background not showing this phenotype, clearly indicates a significant influence, that is worth to follow up in future studies. Using this model it might be possible to identify modifying genes, with protective or oncogenic alleles in different mouse strains.The present study has limitations. Since the majority of analyses performed for phenotypic characterization was conducted before the causative mutation was identified, animals for these studies were assigned to the mutant and wild-type groups according to their hematopoietic abnormalities. Since tissue samples were not available later from all of these animals, correct classification could not be confirmed for each individual animal by genotyping. However, genotype assessment of a part of the animals used in the phenotyping studies and of additional animals from the maintenance breeding colony indicated an almost complete match of hematological abnormalities and heterozygous genotype of the causative mutation. Single mismatches could be due to a mix-up of individual mice during sample collection. Further, only a small number of mutant mice younger than five months was examined in pathology, and the same holds true for hybrid mutant animals. Therefore, it is difficult to final conclude, whether the abnormally small cecum found in young mutants is a general feature of this model. However, we were able to confirm this observation in the follow-up disease monitoring studyKitN824K/WT mice an essential platform for studying disease modifying genes and assessing novel therapeutic strategies against cancer. It is especially expected to help shedding light on the yet still unclear role of KIT mutations and KIT signaling in mammary carcinoma.In summary, the slow, predictable and traceable age-related disease progression of the presented mice bearing an activating Kit mutation render C3H MVD013 mice were bred on a pure C3HeB/FeJ (C3H) inbred background by mating heterozygous phenotypic mutants to C3H wild-type (C3H-WT) animals for more than 10 generations before in depth genetic and phenotypic characterization was started. The animals were maintained in the German Mouse Clinic (GMC) in groups of 2–5 animals in individually ventilated cages with standard chow  and water accessible ad libitum under standard housing conditions  in a 12/12-h light–dark regime.Homozygous viability was investigated in the offspring of 20 matings of heterozygous male and female mice. Fertility assessment was done by retrospective analysis of the breeding outcome of maintenance and cohort breeding for mouse line MVD013 in the GMC in comparison to maintenance breeding of the C3HeB/FeJ wild-type colony.47, one cohort of 10 animals of each sex and genotype for the pathology of 12–14 months old animals, and one cohort of 10 six months old female and four 12 months old female animals of each genotype for bone marrow analyses.In total, five cohorts of heterozygous mutant mice and wild-type littermates were used for the genetic and phenotypic characterization of this mutant line: One cohort of 100 mice on N2-hybrid mixed genetic background for linkage analysis to map the causative mutation, and 4 cohorts on C3H background for the phenotypic characterization: two cohorts of 8–11 mutant and wild-type animals each per sex were used for the two pipeline phenotyping procedure in the German Mouse Clinic (GMC)18 and few animals bred for linkage analysis of the mutation were used for macroscopic and histopathological investigations.Most of the animals used for clinical phenotyping and additional animals from the maintenance breeding colony, from the accompanying disease monitoring studyAll experimental procedures were conducted according to the German Animal Welfare Act, they were approved by the respective authorities of the Government of Upper Bavaria “Regierung von Oberbayern, Sachgebiet 54 – Tierschutz, München, Germany”  and carried out and reported in compliance with ARRIVE guidelines.49 followed by classical linkage analysis.Heterozygous mutant animals were mated to C57BL/6JIco wild-type mice to generate F1 hybrid animals. Offspring showing the characteristic mutant phenotype of the peripheral complete blood cell count (CBC) were backcrossed to C57BL/6JIco wild-type animals to generate N2 hybrid offspring used for a classical linkage analysis. Animals were grouped according to CBC characteristics in mice showing the mutant, the wild-type or an ambiguous phenotype. Subsequently mice were sacrificed, and DNA isolated from tail tip tissue was used for genome wide SNP genotyping as previously described50. Read alignment to the mouse genome assembly mm9 was done with Burrows-Wheeler Aligner . Single nucleotide variants (SNV) and small insertions and deletions (indel) were detected with SAMtools (version 0.1.18). Subsequently results were filtered for heterozygous variants present in both MVD013 mutant mice but not in any of the other mice on C3H background analysed in parallel or any of 145 control mice with unrelated phenotypes in the in-house exome sequence database.We performed in-solution targeted enrichment of exonic sequences from spleens of two heterozygous MVD013 mice on C3H background together with six mutants and two wild-type controls from three other lines on the same genetic background using the SureSelectXT Mouse All Exon 50 Mb kit . The generated libraries were indexed, pooled and sequenced as 100 bp paired-end runs on a HiSeq2000 system , as previously described51, from PCR-products of the respective region obtained with the following primer pair: forward TTCCTGTGAATGGAAGGAAG, and reverse AAAGCACCCTGGGTAGACTC.The causative mutation found by exome sequencing in the chromosomal region linked to the mutant phenotype was subsequently confirmed by sequence analysis in two mutant animals and PCR-RFLP based genotyping of heterozygous mutant and wild-type littermates. PCR-RFLP was conducted using 5′-AAACGGGAATATCACTTGCACC-3′ and 5′-CATGTGACATTACAAGGTAGGAG-3′ as primers and the restriction enzyme “Tasl” with the cutting sequence “AATT”. Due to the mutation, the cutting sequence “AATT” in wild-type is changed to “AAAT” in mutants, resulting in differentially sized restriction fragments of 251 nt and 88 nt for the wild-type and 335 nt for the mutant allele. In addition, genotyping by heteroduplex detection was performed with a LightScanner® device originally from Idaho Technology Inc.  as described previously47. Animals were derived from heterozygous mutant to wild-type matings and assigned as polycythemic mutant and normal controls according to their hematological phenotype determined at 10–12 weeks, since the causative mutation was still unknown. Groups of eight to eleven male and female mice of each phenotype (mutant or wild-type) were analyzed in each test, starting with first tests at age 14–15 weeks and finishing at 22–23 weeks. Only tests which detected polycythemia-associated phenotypes are described in detail.A systematic GMC phenotype screen using a two-pipeline procedure as described by Fuchs et al. 2012 and additional pathological investigations on aged animals were carried out for clinical phenotype assessment of mutant miceg, 10 min, 8 °C) and plasma transferred to plain sample tubes for analysis.Blood collection: Blood samples obtained by puncture of the retrobulbar vein plexus from mice anesthetized with isoflurane were collected in Li-heparin coated tubes  for clinical chemistry analyses and EDTA-coated tubes  for the determination of hematological values. Heparinized blood samples were stored at room temperature for 1–2 h before being separated by centrifugation  as describedHematological investigations: EDTA-blood samples were agitated at room temperature on a rotary until analyzed with an abc-animal blood counter  to determine parameters of the peripheral complete blood count (CBC) including automated cell size-dependent pre-differentiation of leukocytes.53.FACS analysis of peripheral blood leukocytes: Subpopulations of peripheral blood leukocytes were analyzed using a BD LSR II flow cytometer  and fluorescence-conjugated antibodies against CD45, CD3, CD4, CD8, CD19, CD25, B220, CD5, IgD, CD11b, Gr-1, MCH-class II, Ly6C, CD44 and CD62L as described previously6 cells were stained with a cocktail for lineage markers , and lineage-negative cells (Lin-) were analyzed for c-Kit  and Sca-1  expression to discriminate Kit+Sca-1- (KL) and Kit+Sca-1+ (KSL) cells. Progenitor and stem cell subpopulations were identified by staining with the following additional markers: CD34 , Fc-gamma-II/III-R .Total bone marrow (BM) from one femur and two tibiae was isolated by flushing. After cell counting 1 × 10Femurs were fixed in 10% formalin, subsequently decalcified in 10% buffered EDTA, pH 7.2, and afterwards embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) for histological analysis.Further analyses during systemic phenotype assessment are described in supplement p < 0.05. We do not apply multiple testing correction during evaluation of primary phenotyping results, since this analysis is a screening procedure for the generation of hypotheses.Data displayed in Fig. In total we investigated 106 animals on C3H background macroscopically (76 heterozygous mutant and 30 wild-type littermates) and mutants from one litter N2-hybrid animals (n = 4). Organs of 60 mutant animals on C3H background and the cecum of one N2-hybrid mutant were subjected to histopathological analysis. During necropsy macroscopical analysis was performed previous to histological examination of H&E stained, formalin-fixed, paraffin-embedded tissues. Images were taken by the slide scanning system NanoZoomer® 2.O HT . Giemsa staining was carried out for visualization of mastocytes in 3 µm paraffin sections of 8 mutant and 4 control animals. All organs were analysed, including tumors of female mice.Immunohistochemical staining was carried out on 1 µm tissue section in automated immunostainers  using the streptavidin-peroxidase method. The following primary antibodies were used: anti-B220 , anti-KIT antibody , anti-CD3 , anti-estrogen receptor alpha  anti-ErbB2 , anti-myeloperoxidase (MPO) , anti-progesterone receptor , anti-TER-119  and anti-Ki67 . To confirm antibody specificity positive controls with known protein expression as well as negative controls without primary antibody were used. The slides were analyzed independently by two pathologists.Supplementary Information 1.Supplementary Information 2.


Malignant phyllodes tumour (MPT) is a rare breast malignancy with epithelial and mesenchymal features. Currently, there are no appropriate research models or effective targeted therapeutic approaches for MPT.We collected fresh frozen tissues from nine patients with MPT and performed whole-exome and RNA sequencing. Additionally, we established patient-derived xenograft (PDX) models from patients with MPT and tested the efficacy of targeting dysregulated pathways in MPT using the PDX model from one MPT.MPT has unique molecular characteristics when compared to breast cancers of epithelial origin and can be classified into two groups. The PDX model derived from one patient with MPT showed that the mouse epithelial component increased during tumour growth. Moreover, targeted inhibition of platelet-derived growth factor receptor (PDGFR) and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) by imatinib mesylate and PKI-587 showed in vivo tumour suppression effects.This study revealed the molecular profiles of MPT that can lead to molecular classification and potential targeted therapy, and suggested that the MPT PDX model can be a useful tool for studying the pathogenesis of fibroepithelial neoplasms and for preclinical drug screening to find new therapeutic strategies for MPT. Breast fibroepithelial tumours, including fibroadenomas and phyllodes tumours, are biphasic neoplasms characterised by proliferation of epithelial and stromal cells of the mammary glands . PhyllodThe current standard treatment for MPT is surgical excision of the tumour with sufficient resection margins. The addition of postsurgical radiation therapy did not result in substantial improvement in overall survival, and the benefit of adjuvant systemic therapy has not been tested in randomised clinical trials , 4. OwinRecent studies have improved our understanding of the molecular characteristics of MPT , 5. ScieHere, we presented the genomic and transcriptomic characteristics of MPT using various tumour tissues and suggested gene expression-based molecular subtypes of MPT. Additionally, we elucidated the driver and microenvironment relationship in this biphasic neoplasm using a patient-derived xenograft (PDX) model derived from MPT. Finally, we proposed novel therapeutic approaches for MPT by targeting the platelet-derived growth factor receptor (PDGFR) and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways.Frozen tumour tissues of MPTs were obtained from the Institutional Review Board (IRB)-approved breast cancer biospecimen repository at Seoul National University Hospital (IRB no. 1509-032-702). Tissues were obtained from surgical specimens during curative surgeries. Matched germline DNA was obtained from the peripheral blood of the patient. Formalin-fixed, paraffin-embedded (FFPE) MPT tissues were obtained from the Department of Pathology, Seoul National University Hospital (IRB no. 1402-054-555).3 after transplantation into the mouse mammary fat pad, it was excised and cut into 2–3 mm3 pieces. Subsequently, a hole was made in the mammary fat pad of the mouse to be transplanted, and a piece of PDX tumour was placed in the hole. Thus, MPT PDX models were established in several passages (P1 and P2). Tumour volumes and body weight of mice were measured once or twice a week.To develop the PDX models, surgically resected tumour tissues were minced into approximately 2 mm pieces and transplanted into the fourth mammary fat pad of 8-week-old female NOD/SCID/IL-2γ-receptor null  mice. When the MPT PDX tumour exceeded 500 mmhttp://broadinstitute.github.io/picard/). For samples using a combined reference genome of human and mouse, we removed mouse sequence reads according to additional processing procedures .–50.CDH1,p/kegg/) , and thep/kegg/) , as the MED12 were found only in the “Epithelial” subtype  of MED12, appear only in the “Epithelial” group in FFPE tissues as in FF tissues . The patients with “Epithelial” tumours showed significantly smaller tumour size compared to that of the patients with “Fibrous” tumours  during the curative surgery and develop a PDX model using NSG mice (“MX-99”). Previous studies have shown that murine cells replace the microenvironmental cells around human tumours after patient-derived tumours are engrafted into mice , 56. AccThe mouse reads obtained from MPT PDX tumours showed distinct gene expression patterns compared to those of normal mouse mammary fat pads, including upregulation of the cell cycle-related gene sets   Fig. . PDGFRB mesylate . PDGFRB al cells . Examinares Fig. .Fig. 5DiPIK3R1 (NM_181504: p.Y2fs), which is a regulatory subunit for PIK3CA with tumour-suppressor properties [PIK3R1 are often associated with increased PI3K/Akt signalling in cancer [IGF1 and IGF2, which activate PI3K signalling in tumours in an autocrine manner, were also significantly upregulated in MPT tumours compared to IDC (Fig. The “MX-99” tumour also harboured somatic mutation in operties . Somaticn cancer . Our datIDC Fig. .To test whether the genetic characteristics of the PDX model can be exploited to develop therapeutic approaches, we treated the PDX model with imatinib mesylate and PKI-587  to targeIn the present study, we characterised the genomic and transcriptomic features of a rare breast malignancy, MPT. The molecular features of MPT presented here provide novel insights into the pathogenesis of this biphasic neoplasm showing both mesenchymal and epithelial components. Furthermore, by using a PDX model derived from the MPT, we demonstrated the possibility of driver and microenvironment roles for each component. Finally, we showed that targeting the PDGFR and PI3K/mTOR pathways can be an effective treatment option for patients with MPT.CLDN3, CLDN7, or apoptosis- and angiogenesis-related genes such as VIM or PIK3CA can be key molecular characteristics of MPT. In contrast, the present study included only pathologically proven MPT to identify the molecular features that can classify malignant tumours with different clinical characteristics. Our data suggested that the expression patterns of genes involved in ECM interactions, EMT, and cell adhesion could classify MPT into two subtypes. The fact that the two subtypes of MPT are characterised by genes related to epithelial and mesenchymal differentiation indicated that the biphasic pathological features of fibroepithelial neoplasms are also reflected at the molecular level. Moreover, we showed that the molecular subtype of MPT is significantly associated with the degree of epithelial cell proportion, tumour size, and stromal overgrowth. We further inferred that the presence of mutations in MED12 may be related to the molecular subtype of MPT. Notably, the MED12 mutation, found in the stroma [Previous studies of gene expression profiles of phyllodes tumours have mostly focused on the molecular characterisation of MPT within a wide range of fibroepithelial neoplasm –8. For ee stroma , 67, appSawhney et al. previously suggested that stromal growth in fibroepithelial tumours may be affected by the epithelium, showing that stromal mitosis is more prevalent in stroma close to the epithelium . Tse et The genomic and transcriptomic findings in our study identified two druggable pathways in MPT: the PDGFR and PI3K/mTOR pathways. PDGFRα and PDGFRβ proteins are upregulated in stromal cells of human cancers  and are Supplementary figures and tablesSupplementary Data 1Supplementary Data 2


Type III interferons or IFN-λs, are composed of 4 members: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4. IFN-λs are master protectors against mucosal viral infections due to the preferential expression of their cognate receptor, IFNLR1, to cells of epithelial lineage and to a restricted pool of immune cells. IFN-λ receptor activation induces intracellular signaling that overlaps with that of type I interferons (IFN-I) and culminates with the induction of Interferon-Stimulated-Genes (ISG) with antiviral functions. However, several studies have uncovered non-redundant functions of IFN-λs, compared to IFN-I, that go beyond viral restriction at epithelial barriers. In this topic, we highlighted several emerging aspects of IFN-λ biology, including: their ability to restrict non-viral infections, the mechanisms by which they modulate immune responses, and the enigmatic role of human IFN-λ4.Wallace et al. describe how regional identity and timing of disease progression contribute to the outcomes of exposure to IFN-λs in the gastrointestinal tract. They summarize how dual regulation of epithelial and immune cells contributes to protective and pathogenic outcomes during inflammatory bowel diseases (IBD). IFN-λs can also modulate the activity of several immune cells and influence the pathogenesis of autoimmune diseases as comprehensively described by Manivasagam and Klein. Mouse models provided a robust genetic system to study the distribution of Ifnlr1 across immune cells. In humans, however, our understanding of the cells contributing to the production of IFN-λs and the capacity of immune cells to respond to IFN-λs is limited. Mallampalli et al. demonstrate that human macrophages are an important source of IFN-λs following Influenza A virus (IAV) infection. They also show that human alveolar macrophages and monocyte-derived macrophages express functional levels of IFNLR1 and can thus mount protective responses against IAV infection in response to IFN-λs.One of the defining characteristics of IFN-λs is their ability to induce a targeted response in the barrier epithelia without stimulating excessive immune cell recruitment which can lead to immunopathology. This property of IFN-λs makes them central regulators of inflammation during viral and non-viral diseases. On the one hand, in addition to inducing antiviral gene programs, IFN-λs can dampen immune cell-mediated inflammation and promote physiological intestinal epithelial cell functions. On the other hand, IFN-λs can fuel the immunopathology of inflammatory diseases by preventing cell differentiation and proliferation and thus dampening barrier repair. In this Research Topic, Ye et al. build upon their previous work that demonstrates a cell-extrinsic role for IFN-λs in boosting adaptive immune responses to IAV. IFN-λs stimulate thymic stromal lymphopoietin (TSLP) release by specialized respiratory epithelial cells to activate dendritic cells and enhance adaptive immune responses following vaccination. Here, they demonstrate that this adjuvant effect is restricted to the mucosa as other non-mucosal routes of vaccination are not affected by IFN-λs, and that the IFN-λs/TSLP axis governs vaccine-induced immunity following intra-rectal vaccination, expanding upon the therapeutic potential of IFN-λs for the management of other mucosal pathogens.The observation that IFN-λs modulate host inflammation opens new questions regarding their therapeutic applications in infectious diseases. In their original paper, Overall IFN-λs’ ability to shape immunity is increasingly appreciated. Several contributions to this topic highlight the emerging role of IFN-λs as immune regulators and raise interesting questions, such as what are the differences between the mouse and human IFN-λs biology, and what other indirect connections between IFN-λs and immune responses exist.IFNL4 gene is present in most higher mammals, excluding mice, in humans, only a subset of individuals is genetically able to produce IFN-λ4. Single nucleotide polymorphisms in the IFNL4 locus impede IFN-λ4 translation by causing a frameshift in the first exon. However, a significant proportion of humans, harbor functional IFNL4 alleles. The heterogeneity in IFN-λ4 expression is clinically relevant as IFN-λ4 expression predicts host susceptibility to chronic Hepatitis C virus (HCV) infection and decreased clearance of HCV. This characteristic of IFN-λ4 is paradoxical: IFN-λ4 stimulates ISG transcription to induce an antiviral state in target cells, yet it prevents the clearance of a chronic virus infection. Thus, understanding the biological processes uniquely affected by IFN-λ4 is of utmost interest. In this Research Topic, Guo et al. compare the antiviral functions of distinct IFN-λ4 variants by following the kinetics of IFN-λ-receptor signaling, downstream ISG expression, and the ability to establish an antiviral state. By comparing human IFN-λ4 variants with their non-human primate counterparts, the authors reveal that IFN-λ4s are less potent than the other IFN-λs and display a wide range of activities, implicating that the expression of different variants can influence the susceptibility to infectious diseases.IFN-λ4 was the latest addition to the IFN-λs family and its functions have been a matter of debate since its discovery. Although the Onabajo et al. who analyze the behavior of hepatocytes expressing IFN-λ4 and describe that IFN-λ4 is a misfolded protein, whose expression induces ER stress responses associated with cell cycle arrest, apoptosis, and decreased cell proliferation, potentially explaining the pro-HCV effects of IFN-λ4. Overall, the clinical relevance of IFN-λ4 is far from elucidated. This topic has contributed additional insights which point to a model in which a combination between ER stress responses and IFN responses may be at the base of IFN-λ4 detrimental roles.However, how IFN-λ4 drives detrimental responses during infection is still unknown. One step toward the answer comes from Alphonse et al. highlight our understanding of the central role that IFN-λs play in the control of bacterial pathogens. Their work summarizes the mechanisms by which bacterial recognition drives IFN-λs production, how IFN-λs shape the cell-intrinsic control of bacterial replication, and the ability of IFN-λs to shape the inflammatory response to bacterial challenges. Further work is needed to expand our understanding of IFN-λ and bacterial interactions.Interferons have been classically viewed as potent antiviral cytokines. The induction of ISG inhibits the replication of viral pathogens by modifying intracellular biological processes necessary for pathogen dissemination. More recent studies have demonstrated that IFNs can also modify how cells respond to intracellular bacterial infections. In this Research Topic, IFN-λs were historically believed to be a redundant addition to the interferon arsenal present in epithelial cells. Their originality resided in two critical differences 1) their receptor is specific to mucosal surfaces, and 2) they confer anti-viral activity with different kinetics compared to type I IFNs. We have now started to peel back the layers and reveal unique IFN-λs-specific functions that might be even more important than their primary anti-viral function. We now know that IFN-λs are excellent immune modulators involved in regulating the response to infectious agents, autoimmunity, and chronic inflammatory disorders. Finally, the biology of the shyest IFN-λ, IFN-λ4, is starting to be unraveled. However, our understanding of IFN-λ4 biology is in its infancy.IFN-λs need to be studied beyond their canonical anti-viral functions as several pieces of evidence suggest that these cytokines have context-specific functions and act differently in different cell types. It is critical to understand how IFN-λs act in individual cell types and to integrate this understanding to define the role of IFN-λs at the tissue level as a mediator of intrinsic local protection and coordinator of tissue-specific immune cells.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.AB is supported by the excellence initiative of Aix Marseille Universit.-A*Midex, a French “investissements d’Avenir” program: AMX-20-CE-01; the FRM amor.age de jeunes equipes grant AJE202010012468; and the ANR-JCJC grant “INTERMICI” ANR-21-CE15-0022. This work was supported by research grant starting package from the University of Florida, College of Medicine, to SB DMS was supported by the University of Manitoba, Natural Sciences and Engineering Research Council of Canada, Research Manitoba, Children's Hospital Research Institute of Manitoba and Canadian Institutes of Health Research (GA1- 177700). AF was supported by OSU funds for Advancing Research in Infection and Immunity.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.