Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system

Glafenine-induced intestinal injury in zebrafish is ameliorated by -opioid signaling via enhancement of Atf6-dependent cellular stress responses

Beside their analgesic properties, opiates exert beneficial effects on the intestinal wound healing response. In this study, we investigated the role of μ-opioid receptor (MOR) signaling on the unfolded protein response (UPR) using a novel zebrafish model of NSAID-induced intestinal injury. The NSAID glafenine was administered to zebrafish larvae at 5 days post-fertilization (dpf) for up to 24 hours in the presence or absence of the MOR-specific agonist DALDA. By analysis with histology, transmission electron microscopy and vital dye staining, glafenine-treated zebrafish showed evidence of endoplasmic reticulum and mitochondrial stress, with disrupted intestinal architecture and halted cell stress responses, alongside accumulation of apoptotic intestinal epithelial cells in the lumen. Although the early UPR marker BiP was induced with glafenine-induced injury, downstream atf6 and s-xbp1 expression were paradoxically not increased, explaining the halted cell stress responses. The μ-opioid agonist DALDA protected against glafenine-induced injury through induction of atf6-dependent UPR. Our findings show that DALDA prevents glafenine-induced epithelial damage through induction of effective UPR

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology

Background & Aims The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development(1-5), physiology(6-11), disease(12-16), and host-microbe interactions(17-25). Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae(26). Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system

Glafenine-induced intestinal injury in zebrafish is ameliorated by μ-opioid signaling via enhancement of Atf6-dependent cellular stress responses

SUMMARY Beside their analgesic properties, opiates exert beneficial effects on the intestinal wound healing response. In this study, we investigated the role of μ-opioid receptor (MOR) signaling on the unfolded protein response (UPR) using a novel zebrafish model of NSAID-induced intestinal injury. The NSAID glafenine was administered to zebrafish larvae at 5 days post-fertilization (dpf) for up to 24 hours in the presence or absence of the MOR-specific agonist DALDA. By analysis with histology, transmission electron microscopy and vital dye staining, glafenine-treated zebrafish showed evidence of endoplasmic reticulum and mitochondrial stress, with disrupted intestinal architecture and halted cell stress responses, alongside accumulation of apoptotic intestinal epithelial cells in the lumen. Although the early UPR marker BiP was induced with glafenine-induced injury, downstream atf6 and s-xbp1 expression were paradoxically not increased, explaining the halted cell stress responses. The μ-opioid agonist DALDA protected against glafenine-induced injury through induction of atf6-dependent UPR. Our findings show that DALDA prevents glafenine-induced epithelial damage through induction of effective UPR

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells

<div><p>The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from <i>her6/HES1</i> that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.</p></div

Identification of genes with conserved regional transcriptional specification along the length of the intestine in zebrafish and mouse.

<p><b>(A)</b> A heat map comparing gene median-centered z-scores of 1-to-1 orthologs from previously published datasets profiling expression levels along the length of the zebrafish [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref015" target="_blank">15</a>] and mouse intestine (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#sec020" target="_blank">Materials and methods</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.s003" target="_blank">S3 Fig</a>) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref047" target="_blank">47</a>]. Similarly expressed genes are ordered by expression values of mouse <b>(A)</b> duodenum, <b>(B)</b> jejunum, <b>(C)</b> ileum, and <b>(D)</b> colon. Gene names with asterisks are also intestinal epithelial cell (IEC) signature genes as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.g001" target="_blank">Fig 1</a>. Genes can exist in multiple subfigures.</p

Accessible chromatin maps in intestinal epithelial cells (IECs) from multiple species reveals common regulatory information without substantial sequence conservation.

<p><b>(A)</b> Accessible chromatin and RNA sequencing (RNA-seq) data from representative replicates for the IEC signature gene <i>ELF3</i> for each organism. For each organism, gene models are represented with thick bars (exons) and thin bars (introns and untranslated regions). <b>(B)</b> Accessible chromatin signal at the 1,000 bp surrounding the transcription start site (TSS) of 1-to-1-to-1-to-1 orthologs ordered by zebrafish IEC Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) signal at the base pair coordinate of the gene’s TSS for zebrafish, stickleback, mouse ileum, mouse colon, and human colon (Right). Moving medians (Left) are shown for PC1 correlation value used to identify IEC signature genes (500 gene window, 1 gene step; black), Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of associated genes (250 gene window, 1 gene step; color scheme based on data sets presented in A and throughout), and IEC signature genes are marked by black horizontal bars. <b>(C)</b> Moving median of accessible chromatin signal at TSS of stickleback, mouse ileum, mouse colon, and human ordered by signal at zebrafish TSS (250 gene window, 1 step) highlight the relationship between IEC accessible chromatin data in multiple species. Numerical values can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.s011" target="_blank">S1 Table</a>. <b>(D)</b> A heatmap of cluster analysis of overlap between accessible chromatin peak calls for IEC samples and additional published accessible chromatin datasets with the region 1,000 bp upstream of the TSS (TSS-1,000 bp) for IEC signature genes (red bars). Overlap between TSS-1,000 bp and conservation metrics are represented with blue bars. The total number of overlaps is represented in parentheses for each conservation metric column, and overlaps are defined as having at least 1 shared base pair. Initials are used to specify IEC data sets: Z; Zebrafish, S; Stickleback, MI; Mouse Ileum, MC; Mouse Colon, HC; Human Colon. <b>(E)</b> Closeup of the heatmap of clustered genes from panel 3D that frequently showed putative conserved accessible chromatin at TSS in IEC samples (light blue and green). Color scheme is shared with 3D. <b>(F)</b> A heatmap highlighting common transcription factor binding sites (TFBS) motif enrichment within the accessible chromatin peaks that fall between the region 10,000 bp upstream of the TSS and 10,000 bp downstream of the transcription termination site (TTS) of IEC signature genes. Motif enrichment includes known transcription factors (TFs) involved in IEC biology which are also often expressed highly in our IEC samples. E26 transformation specific (ETS) factors include motifs for ELF5, EHF, ELF1, GABPA, ETS, Elk1, Fli1, Elk4, ETS1, ERG, SPDEF, and EWS:FLI. <b>(G)</b> The percentage of DNA bases that are either adenosine or thymine (AT%) (20 bp smooth: 20 bp window, 1 bp step) surrounding TSS as ordered by IEC zebrafish FAIRE signal from (B) and IEC mouse Ileum DNase signal (Right) shows nonrandom patterns at TSS that <b>(H)</b> vary on average from species to species. <b>(I)</b> Comparison of AT% (20 bp smooth) at conserved nonexonic elements (CNEs) that by their nature show some similar sequence composition.</p

Transcriptional profiling of intestinal epithelial cells (IECs) from multiple species show conserved expression after 420 million years since a common ancestor.

<p><b>(A)</b> (Left) A phylogenetic tree showing time since a common ancestor for human (<i>Homo sapiens</i>), mouse (<i>Mus musculus</i>), zebrafish (<i>Danio rerio</i>), and stickleback (<i>Gasterosteus aculeatus</i>) species. (Right) Simplified schematics showing the intestinal tract of all 4 organisms in gray with the region of collected IEC sample colored: green (human colon IECs), orange (mouse ileum IECs), red (mouse colon IECs), blue (zebrafish whole intestine IECs), and purple (stickleback whole intestine IECs). <b>(B)</b> Scatterplot of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values for 1-to-1 orthologs from mouse ileum IEC and zebrafish IEC samples shows a positive correlation coefficient (Pearson <i>r</i>² = .273; Spearman <i>r</i>² = .416). <b>(C)</b> Same as (B) for mouse colon IECs and zebrafish IECs (Pearson <i>r</i>² = .341; Spearman <i>r</i>² = .379). <b>(D)</b> Complete linkage cluster analysis using FPKM values for 1-to-1 orthologs show similarity between mRNA levels for IECs in comparison to other mouse tissues. Scale represents values of Pearson distance. Black data sets marked by asterisks are RNA sequencing (RNA-seq) experiments from non-IEC tissues [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref029" target="_blank">29</a>]. <b>(E)</b> Scatterplot of principal component 1 and 2 (PC1 and PC2) using principal component analysis (PCA) of FPKM values for all IEC data sets and other mouse tissues. <b>(F)</b> Top reported Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from Database for Annotation Visualization and Integrated Discovery (DAVID) using IEC signature genes. The enrichment of these gene groups do not clear Bonferroni correction thresholds (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.s012" target="_blank">S2 Table</a>). <b>(G)</b> Top tissues showing overlap with IEC signature genes from human and mouse Gene Atlas using Enrichr. <b>(H)</b> The University of California Santa Cruz (UCSC) screenshot of RNA-seq levels at cadherin 17 (<i>CDH17</i>) in 4 species’ IECs (top) and other tissues (bottom) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002054#pbio.2002054.ref029" target="_blank">29</a>] shows expression largely restricted to IECs.</p