Expression Of Fluorescent Reporters Research Articles

BCOR Loss Confers Increased Stemness and Partially Rescues RUNX1-Deficient Phenotypes in Human Hematopoietic Stem and Progenitor Cells

Introduction: Loss of function mutations in BCL-6 corepressor (BCOR) commonly occur in de novo acute myeloid leukemia (AML) and germline hematopoietic malignancies, most notably in those arising from RUNX1 familial platelet disorder (RUNX1-FPD). Second only to loss of heterozygosity of RUNX1, mutations in BCOR are the most commonly acquired mutations upon progression to myeloid malignancy in RUNX1-FPD. Additionally, RUNX1 and BCOR mutations commonly co-occur in de novo AML, and both are classified as “adverse risk” by the European LeukemiaNet if found at AML diagnosis. However, the role of BCOR in leukemogenesis is poorly understood, and there are limited studies investigating co-loss of BCOR and RUNX1 in hematopoietic stem and progenitor cells (HSPCs). Methods: We developed a model of BCOR loss in primary human CD34+ HSPCs using CRISPR/Cas9 and AAV6-mediated homology directed repair to knock-in a fluorescent reporter expression cassette into the BCOR locus. We used this system to investigate how BCOR knockout (KO) perturbs normal HSPC function. We combined this approach with a previously developed analogous system to disrupt the RUNX1 locus to investigatehow BCOR loss in the setting of RUNX1 KO modulates the effects of RUNX1 loss on HSPC function. Results: Here, we found that BCOR loss increased capacity for serial replating and conferred a competitive advantage over control HSPCs. In serial replating assays, BCOR-deficient HSPCs had a 5-fold increase in colony formation after one passage with a striking majority of colonies being erythroid. In liquid differentiation assays, BCOR-deficient HSPCs consistently had a 2-fold increase in CD34+ cells over a 21 day time course. When co-cultured with control HSPCs in a 1:1 ratio in stem expansion media (base media + SCF, FLT3-L, TPO, IL-6, UM-171), BCOR-deficient HSPCs expanded to comprise 70% of the culture at 14 days. This competitive advantage was recapitulated in vivo. At the 12-week timepoint of a competitive transplantation assay in NSG mice using HSPCs from 3 different donors, BCOR KO cells (marked by BFP) on average made up 75% of the engrafted human CD45+ population, while control cells (expressing GFP from the AAVS1 safe harbor locus) made up only 21%. Additionally, BCOR loss partially rescued RUNX1-induced stem cell dysfunction in vitro. Compared to RUNX1 deficiency alone, HSPCs deficient in both RUNX1 and BCOR had a 4-fold increase in colony formation after two passages in serial replating assays. Our lab has previously shown that RUNX1 loss ablates erythroid colony formation and skews towards monocytic colonies. Despite BCOR loss alone having a strong skew towards erythroid colonies, BCOR loss in RUNX1-deficient HSPCs did not induce erythroid colony formation. This suggests that in double mutant HSPCs, RUNX1 loss drives lineage decisions while BCOR loss drives the increase in serial replating potential. Ongoing efforts are aimed at characterizing combinatorial effects of BCOR and RUNX1 loss on hematopoiesis and stem cell function through in vitro liquid differentiation assays and in vivo transplantations, as well as investigating the molecular underpinnings of these phenotypes through RNA-seq. Conclusion: In summary, we have established a model of BCOR loss in primary human HSPCs to evaluate not only the role of BCOR in hematopoiesis, but also to characterize how BCOR is involved in leukemic transformation when combined with RUNX1. We show that BCOR deficiency enhances several attributes associated with stem cell function, including increased potential for serial replating, increased CD34 positive population, and increased engraftment compared to control HSPCs. When combined with RUNX1 loss, BCOR loss increases the serial replating potential of RUNX1-deficient cells, suggesting some rescue of RUNX1 KO-induced stem cell dysfunction. Ultimately, this model will enable us to elucidate the molecular mechanisms underlying increased stemness and competitive advantage in BCOR-deficient HSPCs, interrogate RUNX1-FPD disease progression associated with BCOR mutation, and validate potential therapeutics.

Analysis of Xylem Cells by Nucleus-Based Transcriptomics and Chromatin Profiling.

Nuclei contain essential information for cell states, including chromatin and RNA profiles - features which are nowadays accessible using high-throughput sequencing applications. Here, we describe analytical pipelines including nucleus isolation from differentiated xylem tissues by fluorescence-activated nucleus sorting (FANS), as well as subsequent SMART-seq2-based transcriptome profiling and assay for transposase-accessible chromatin (ATAC)-seq-based chromatin analysis. Combined with tissue-specific expression of nuclear fluorescent reporters, these pipelines allow obtaining tissue-specific data on gene expression and on chromatin structure and are applicable for a large spectrum of cell types, tissues, and organs. Considering, however, the extreme degree of differentiation found in xylem cells with programmed cell death happening during vessel element formation and their role as a long-term depository for atmospheric CO2 in the form of wood, xylem cells represent intriguing and relevant objects for large-scale profilings of their cellular signatures.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Isolation of Tissue Macrophages in Adult Zebrafish.

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

A robotic system for automated genetic manipulation and analysis of Caenorhabditis elegans.

The nematode Caenorhabditis elegans is one of the most widely studied organisms in biology due to its small size, rapid life cycle, and manipulable genetics. Research with C. elegans depends on labor-intensive and time-consuming manual procedures, imposing a major bottleneck for many studies, especially for those involving large numbers of animals. Here, we describe a general-purpose tool, WormPicker, a robotic system capable of performing complex genetic manipulations and other tasks by imaging, phenotyping, and transferring C. elegans on standard agar media. Our system uses a motorized stage to move an imaging system and a robotic arm over an array of agar plates. Machine vision tools identify animals and assay developmental stage, morphology, sex, expression of fluorescent reporters, and other phenotypes. Based on the results of these assays, the robotic arm selectively transfers individual animals using an electrically self-sterilized wire loop, with the aid of machine vision and electrical capacitance sensing. Automated C. elegans manipulation shows reliability and throughput comparable with standard manual methods. We developed software to enable the system to autonomously carry out complex protocols. To validate the effectiveness and versatility of our methods, we used the system to perform a collection of common C. elegans procedures, including genetic crossing, genetic mapping, and genomic integration of a transgene. Our robotic system will accelerate C. elegans research and open possibilities for performing genetic and pharmacological screens that would be impractical using manual methods.

Optical opening of the blood-brain barrier for targeted and ultra-sparse viral infection of cells in mouse cortex

Adeno-associated viruses (AAVs) are used in a wide array of experimental situations for driving expression of biosensors, recombinases, and opto-/chemo-genetic actuators in the brain. However, conventional approaches for minimally invasive, spatially precise, and ultra-sparse AAV-mediated transduction of cells during imaging experiments have remained a significant challenge. Here, we show that intravenous injection of commercially available AAVs at different doses, combined with laser-based perforation of cortical capillaries through a cranial widow, allows for ultra-sparse, titratable, and micron-level precision for delivery of viral vectors with relatively little inflammation or tissue damage. Further, we show the utility of this approach for eliciting sparse expression of GCaMP6, channelrhodopsin, or fluorescent reporters in neurons and astrocytes within specific functional domains in normal and stroke-damaged cortex. This technique represents a facile approach for targeted delivery of viral vectors that should assist in the study of cell types and circuits in the cortex.

Robustness and innovation in synthetic genotype networks

Genotype networks are sets of genotypes connected by small mutational changes that share the same phenotype. They facilitate evolutionary innovation by enabling the exploration of different neighborhoods in genotype space. Genotype networks, first suggested by theoretical models, have been empirically confirmed for proteins and RNAs. Comparative studies also support their existence for gene regulatory networks (GRNs), but direct experimental evidence is lacking. Here, we report the construction of three interconnected genotype networks of synthetic GRNs producing three distinct phenotypes in Escherichia coli. Our synthetic GRNs contain three nodes regulating each other by CRISPR interference and governing the expression of fluorescent reporters. The genotype networks, composed of over twenty different synthetic GRNs, provide robustness in face of mutations while enabling transitions to innovative phenotypes. Through realistic mathematical modeling, we quantify robustness and evolvability for the complete genotype-phenotype map and link these features mechanistically to GRN motifs. Our work thereby exemplifies how GRN evolution along genotype networks might be driving evolutionary innovation.

Evaluation of drug-free methods for the detection of gene silencing in Saccharomyces cerevisiae.

Multiple studies in Saccharomyces cerevisiae have measured the levels of gene silencing by inserting the URA3 gene at various loci and selecting against URA3-expressing cells by 5-flouroorotic acid (5-FOA). However, 5-FOA affects the cellular pools of dNTPs and can produce side effects. To circumvent this issue, we and others have introduced drug-free techniques to detect silent and active gene states. In this study, we compared three drug-free methods based on the expression of fluorescent reporters in the VIIL telomere of S. cerevisiae. Our results point out that only one of these methods is suitable for large-scale drug-free analyses of gene silencing.

In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates.

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

Fluorescent Reporters Distinguish Stem Cell Colony Subtypes During Somatic Cell Reprogramming.

Somatic cell reprogramming was first developed to create induced pluripotent stem (iPS) cells. Since that time, the highly dynamic and heterogeneous nature of the reprogramming process has come to be appreciated. Remarkably, a distinct type of stem cell, called induced extraembryonic endoderm (iXEN) stem cell, is also formed during reprogramming of mouse somatic cells by ectopic expression of the transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). The mechanisms leading somatic cells to adopt differing stem cell fates are challenging to resolve given that formation of either stem cell type is slow, stochastic, and rare. For these reasons, fluorescent gene expression reporters have provided an invaluable tool for revealing the path from the somatic state to pluripotency. However, no such reporters have been established for comparable studies of iXEN cell formation. In this study, we examined the expression of multiple fluorescent reporters, including Nanog, Oct4, and the endodermal genes, Gata4 and Gata6-alone and in combination, during reprogramming. We show that only simultaneous evaluation of Nanog and Gata4 reliably distinguishes iPS and iXEN cell colonies during reprogramming.

Modulating myoblast differentiation with RNA-based controllers.

Tunable genetic controllers play a critical role in the engineering of biological systems that respond to environmental and cellular signals. RNA devices, a class of engineered RNA-based controllers, enable tunable gene expression control of target genes in response to molecular effectors. RNA devices have been demonstrated in a number of systems showing proof-of-concept of applying ligand-responsive control over therapeutic activities, including regulation of cell fate decisions such as T cell proliferation and apoptosis. Here, we describe the application of a theophylline-responsive RNA device in a muscle progenitor cell system to control myogenic differentiation. Ribozyme-based RNA switches responsive to theophylline control fluorescent reporter expression in C2C12 myoblasts in a ligand dependent manner. HRAS and JAK1, both anti-differentiation proteins, were incorporated into RNA devices. Finally, we demonstrate that the regulation of HRAS expression via theophylline-responsive RNA devices results in the modulation of myoblast differentiation in a theophylline-dependent manner. Our work highlights the potential for RNA devices to exert drug-responsive, tunable control over cell fate decisions with applications in stem cell therapy and basic stem cell biology research.

Comparative Evaluation of Inducible Cre Mouse Models for Fibroblast Targeting in the Healthy and Infarcted Myocardium.

Several Cre recombinase transgenic mouse models have been generated for cardiac fibroblast (CF) tracking and heart regulation. However, there is still no consensus on the ideal mouse model to optimally identify and/or regulate these cells. Here, a comparative evaluation of the efficiency and specificity of the indirect reporter Cre-loxP system was carried out in three of the most commonly used fibroblast reporter transgenic mice (Pdgfra-CreERT2, Col1a1-CreERT2 and PostnMCM) under healthy and ischemic conditions, to determine their suitability in in vivo studies of cardiac fibrosis. We demonstrate optimal Cre recombinase activity in CF (but also, although moderate, in endothelial cells (ECs)) derived from healthy and infarcted hearts in the PDGFRa-creERT2 mouse strain. In contrast, no positive reporter signal was found in CF derived from the Col1a1-CreERT2 mice. Finally, in the PostnMCM line, fluorescent reporter expression was specifically detected in activated CF but not in EC, which leads us to conclude that it may be the most reliable model for future studies on cardiovascular disease. Importantly, no lethality or cardiac fibrosis were induced after tamoxifen administration at the established doses, either in healthy or infarcted mice of the three fibroblast reporter lineages. This study lays the groundwork for future efficient in vivo CF tracking and functional analyses.

Application of Transgenic Zebrafish Models for Studying the Effects of Estrogenic Endocrine Disrupting Chemicals on Embryonic Brain Development.

Endocrine disrupting chemicals (EDCs) are environmental pollutants that mimic hormones and/or disrupt their function. Estrogenic EDCs (eEDCs) interfere with endogenous estrogen signalling pathway(s) and laboratory animal and human epidemiological studies have provided evidence for a causal link between exposure to them during embryonic/early life and neurological impairments. However, our understanding of the molecular and cellular mechanism(s) underlying eEDCs exposure effects on brain development, tissue architecture and function and behaviour are limited. Transgenic (TG) zebrafish models offer new approach methodologies (NAMs) to help identify the modes of action (MoAs) of EDCs and their associated impacts on tissue development and function. Estrogen biosensor TG zebrafish models have been applied to study eEDC interactions and resulting transcriptional activation (via a fluorescent reporter expression) across the entire body of the developing zebrafish embryo, including in real time. These estrogen biosensor TG zebrafish models are starting to deepen our understanding of the spatiotemporal actions of eEDCs and their resulting impacts on neurological development, brain function and behaviour. In this review, we first investigate the links between early life exposure to eEDCs and neurodevelopmental alterations in model organisms (rodents and zebrafish) and humans. We then present examples of the application of estrogen biosensor and other TG zebrafish models for elucidating the mechanism(s) underlying neurodevelopmental toxicities of eEDCs. In particular we illustrate the utility of combining estrogen biosensor zebrafish models with other TG zebrafish models for understanding the effects of eEDCs on the brain, spanning cellular processes, brain circuitry, neurophysiology and behaviour. Finally, we discuss the future prospects of TG zebrafish models as experimental models for studying more complex scenarios for exposure to contaminant mixtures on neurological development and function.

The Long and Winding Road-Vestibular Efferent Anatomy in Mice.

The precise functional role of the Efferent Vestibular System (EVS) is still unclear, but the auditory olivocochlear efferent system has served as a reasonable model on the effects of a cholinergic and peptidergic input on inner ear organs. However, it is important to appreciate the similarities and differences in the structure of the two efferent systems, especially within the same animal model. Here, we examine the anatomy of the mouse EVS, from its central origin in the Efferent Vestibular Nucleus (EVN) of the brainstem, to its peripheral terminations in the vestibular organs, and we compare these findings to known mouse olivocochlear anatomy. Using transgenic mouse lines and two different tracing strategies, we examine central and peripheral anatomical patterning, as well as the anatomical pathway of EVS axons as they leave the mouse brainstem. We separately tag the left and right efferent vestibular nuclei (EVN) using Cre-dependent, adeno-associated virus (AAV)-mediated expression of fluorescent reporters to map their central trajectory and their peripheral terminal fields. We couple this with Fluro-Gold retrograde labeling to quantify the proportion of ipsi- and contralaterally projecting cholinergic efferent neurons. As in some other mammals, the mouse EVN comprises one group of neurons located dorsal to the facial genu, close to the vestibular nuclei complex (VNC). There is an average of just 53 EVN neurons with rich dendritic arborizations towards the VNC. The majority of EVN neurons, 55%, project to the contralateral eighth nerve, crossing the midline rostral to the EVN, and 32% project to the ipsilateral eighth nerve. The vestibular organs, therefore, receive bilateral EVN innervation, but without the distinctive zonal innervation patterns suggested in gerbil. Similar to gerbil, however, our data also suggest that individual EVN neurons do not project bilaterally in mice. Taken together, these data provide a detailed map of EVN neurons from the brainstem to the periphery and strong anatomical support for a dominant contralateral efferent innervation in mammals.

A Transgenic Rat for Noninvasive Assessment of Chondrogenesis in Vivo.

To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.

ExBoX - a simple Boolean exclusion strategy to drive expression in neurons.

ABSTRACTThe advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here, we build on previous knowledge to develop a simple adeno-associated virus (AAV)-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This expression by Boolean exclusion (ExBoX) system encodes for a gene of interest that is turned on by a particular recombinase (Cre or FlpO) and turned off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems.

InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes

SummaryThe mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Key messageBiolistic delivery into pollen.In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.

A Dietary Cholesterol-Based Intestinal Inflammation Assay for Improving Drug-Discovery on Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) with chronic infiltration of immune cells in the gastrointestinal tract are common and largely incurable. The therapeutic targeting of IBD has been hampered by the complex causality of the disease, with environmental insults like cholesterol-enriched Western diets playing a critical role. To address this drug development challenge, we report an easy-to-handle dietary cholesterol-based in vivo assay that allows the screening of immune-modulatory therapeutics in transgenic zebrafish models. An improvement in the feeding strategy with high cholesterol diet (HCD) selectively induces a robust and consistent infiltration of myeloid cells in larvae intestines that is highly suitable for compound discovery efforts. Using transgenics with fluorescent reporter expression in neutrophils, we take advantage of the unique zebrafish larvae clarity to monitor an acute inflammatory response in a whole organism context with a fully functional innate immune system. The use of semi-automated image acquisition and processing combined with quantitative image analysis allows categorizing anti- or pro-inflammatory compounds based on a leukocytic inflammation index. Our HCD gut inflammation (HCD-GI) assay is simple, cost- and time-effective as well as highly physiological which makes it unique when compared to chemical-based zebrafish models of IBD. Besides, diet is a highly controlled, selective and targeted trigger of intestinal inflammation that avoids extra-intestinal outcomes and reduces the chances of chemical-induced toxicity during screenings. We show the validity of this assay for a screening platform by testing two dietary phenolic acids, namely gallic acid (GA; 3,4,5-trihydroxybenzoic acid) and ferulic acid (FA; 4-hydroxy-3-methoxycinnamic acid), with well described anti-inflammatory actions in animal models of IBD. Analysis of common IBD therapeutics (Prednisolone and Mesalamine) proved the fidelity of our IBD-like intestinal inflammation model. In conclusion, the HCD-GI assay can facilitate and accelerate drug discovery efforts on IBD, by identification of novel lead molecules with immune modulatory action on intestinal neutrophilic inflammation. This will serve as a jumping-off point for more profound analyses of drug mechanisms and pathways involved in early IBD immune responses.

A ClearSee-Based Clearing Protocol for 3D Visualization of Arabidopsis thaliana Embryos.

Tissue clearing methods combined with confocal microscopy have been widely used for studying developmental biology. In plants, ClearSee is a reliable clearing method that is applicable to a wide range of tissues and is suitable for gene expression analysis using fluorescent reporters, but its application to the Arabidopsis thaliana embryo, a model system to study morphogenesis and pattern formation, has not been described in the original literature. Here, we describe a ClearSee-based clearing protocol which is suitable for obtaining 3D images of Arabidopsis thaliana embryos. The method consists of embryo dissection, fixation, washing, clearing, and cell wall staining and enables high-quality 3D imaging of embryo morphology and expression of fluorescent reporters with the cellular resolution. Our protocol provides a reliable method that is applicable to the analysis of morphogenesis and gene expression patterns in Arabidopsis thaliana embryos.