RiboTRAP-seq identifies spatially distinct functions for the anterior and posterior intestine in immune and metabolic regulation in Caenorhabditis elegans
- Department of Neuroscience and Dorris Neuroscience Center, The Scripps Research Institute, United States
Figures
Figure 1
Conditions and workflow for INT1- and INT2-9-specific translatomics.
(A) Representative images of wild-type animals bearing integrated INT1::GFP and INT2-9::mCherry transgenes. Upper panel, GFP expression in INT1 cells and mCherry expression in INT2-9 cells; lower panel, merge with DIC. Scale bar, 100 μm. (B) Representative images of wild-type animals in fed, 30 min fasted, and 30 min refed states fixed and stained with Oil Red O. (C) Fat content was quantified for each condition and expressed as a percentage of wild-type animals in fed state ± SEM (n=20). nsp>0.05 by one-way ANOVA (Tukey). (D) Representative images of wild-type animals in fed, 180 min fasted, 30 min refed, 90 min refed, and 180 min refed states fixed and stained with Oil Red O. (E) Fat content was quantified for each condition and expressed as a percentage of wild-type animals in fed state ± SEM (n=18–20). ***p<0.001, nsp>0.05 by one-way ANOVA (Sidak). (F) 100000 C. elegans expressing INT1::RPL-22::3xHA or INT2-9::RPL-22::3xHA were grown to day 1 adults. Worms were fasted and refed for the time indicated and harvested for TRAP-Seq. Panel F created with BioRender.com.
Figure 2 with 2 supplements
Identification of the fundamental differences between INT1 and INT2-9 translatomes.
(A) Principal component analysis showing translatomic variability across the first two principal components of TRAP-Seq data from INT1 and INT2-9 promoters under both acute and chronic conditions (fed, fasted, and refed). Ellipse was computed using the mean and covariance of PC1 and PC2 for each line. (B) Cluster dendrogram produced by average linkage hierarchical clustering in WGCNA. The color row under the dendrogram indicates the module assignment. (C) Pearson’s correlations of TRAP-Seq conditions with WGCNA modules. Each row represents a WGCNA module and its mean correlation to the indicated trait across replicates. The color of each cell indicates the mean Pearson’s correlation coefficient between the module and the condition; red represents a positive correlation and blue represents a negative correlation. (D) Average expression of red module genes in the TRAP-Seq conditions. The y-axis shows mean variance stabilized expression across all genes and replicates per condition with standard deviation indicated by the error bars. (E) WormCat visualization of categories enriched in genes of module red. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (F) Volcano plot of differentially expressed genes for INT1 fed versus INT2-9 fed. High-confidence differentially expressed genes are labeled. The horizontal dashed line indicates an adjusted p-value cutoff of 0.05. (G) WormCat visualization of categories enriched in genes upregulated in INT1 cells versus INT2-9 cells in the fed state. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (H) A hierarchical clustering tree of significantly enriched GO terms (Biological Process, BP) in the upregulated genes for INT1 fed versus INT2-9 fed by ShinyGO. GO terms with many shared genes are clustered together. Larger dots indicate more significant adjusted p-values. (I) Fluorescent images of transgenic animals bearing PB0024.4::mNeonGreen, Pclec-86::mNeonGreen, Pclec-160::mNeonGreen, PF01D5.5::mNeonGreen or PF01D5.1::mNeonGreen transgenes. The white arrowhead indicates mNeonGreen expression in INT1 cells. Scale bar, 100 μm. (J) Fluorescent image of a transgenic animal bearing Pnpr-28::mNeonGreen transgene. Scale bar, 100 μm.
Figure 2—figure supplement 1
The genes differentially expressed in INT1 cells.
(A) Module-trait relationships for selected conditions in WGCNA. Rows indicate the WGCNA module and the columns comparative traits. The color indicates the strength of the Pearson’s correlation and bracketed number the significance. The color red represents a positive correlation; the color green represents a negative correlation. (B) GO enrichment of selected modules in the WGCNA analysis. Bubble size indicates the gene ratio; color indicates the adjusted p-value. (C) Dotplot showing increased expression in the anterior intestine clusters from Ghaddar et al. for genes upregulated in the INT1 versus INT2-9 fed datasets. We selected the top 10 significant (FDR <0.05) genes ranked by effect size that were expressed in at least 5% of anterior intestinal cells to show. Size indicates the percent of cells expressing the gene, and color indicates the scaled effect size. (D–H) Expression profiles of B0024.4, clec-86, clec-160, F01D5.5, and F01D5.1 under all conditions in INT1 and INT2-9 cells.
Figure 2—figure supplement 2
InterPro annotations of differentially expressed genes for INT1 fed versus INT2-9 fed.
(A, B) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 fed versus INT2-9 fed.
Figure 3 with 2 supplements
Identification of the differences between INT1 and INT2-9 translatomes under acute and chronic conditions.
(A, D, G, J) Volcano plot of differentially expressed genes for INT1 versus INT2-9 in acute fasted, acute refed, chronic fasted, and chronic refed states. High-confidence differentially expressed genes are labeled. The horizontal dashed line indicates an adjusted p-value cutoff of 0.05. (B, E, H, K) WormCat visualization of categories enriched in genes upregulated in INT1 cells versus INT2-9 cells in acute fasted, acute refed, chronic fasted, and chronic refed states. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (C, F, I, L) A hierarchical clustering tree of significantly enriched GO terms (Biological Process, BP) in the upregulated genes for INT1 versus INT2-9 in acute fasted, acute refed, chronic fasted, and chronic refed states by ShinyGO. GO terms with many shared genes are clustered together. Larger dots indicate more significant adjusted p-values.
Figure 3—figure supplement 1
InterPro annotations of differentially expressed genes for INT1 versus INT2-9 under acute conditions.
(A, B) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 30 min fasted versus INT2-9 30 min fasted. (C, D) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 30 min refed versus INT2-9 30 min refed.
Figure 3—figure supplement 2
InterPro annotations of differentially expressed genes for INT1 versus INT2-9 under chronic conditions.
(A, B) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 180 min fasted versus INT2-9 180 min fasted. (C, D) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 90 min refed versus INT2-9 90 min refed.
Figure 4
Comparisons of InterPro categories for INT1 versus INT2-9 under acute and chronic conditions.
(A–H) Venn diagrams showing the number of upregulated or downregulated genes in specific categories for INT1 versus INT2-9 under the three acute conditions (fed, fasted, and refed). (I–Q) Venn diagrams showing the number of genes in specific categories for INT1 versus INT2-9 under the three chronic conditions (fed, fasted, and refed). (R) Venn diagram showing the overlap between overlapped upregulated ShKT proteins for INT1 versus INT2-9 under acute and chronic conditions.
Figure 5 with 3 supplements
Comparison of fasting and refeeding responses between INT1 and INT2-9 cells under acute and chronic conditions.
(A, B) Scatterplots comparing the acute and chronic fasting response genes in INT1 versus INT2-9 differential expression. The top 1000 DEGs by significance were selected for each line and fasting response to be compared, with a regression line fitted to compare INT1 and INT2-9 responses. (C) Table of differentially expressed genes (FDR <0.1) under acute and chronic conditions for INT1 and INT2-9 datasets. (D) Venn diagrams showing the number of differentially expressed genes (FDR <0.1) in fasting and refeeding conditions for INT1 and INT2-9 datasets. (E, F) WormCat visualization of categories enriched in upregulated genes in INT1 cells and INT2-9 cells under chronic fasting or refeeding conditions. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (E) is part of Figure 5—figure supplement 2A, and (F) is part of Figure 5—figure supplement 3A.
Figure 5—figure supplement 1
WormCat annotations of differentially expressed genes under acute fasting and refeeding conditions for INT1 and INT2-9 cells.
(A, B) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under acute fasting condition. (C, D) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under acute refeeding condition. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.
Figure 5—figure supplement 2
WormCat annotations of differentially expressed genes under chronic fasting condition for INT1 and INT2-9 cells.
(A, B) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under chronic fasting condition. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.
Figure 5—figure supplement 3
WormCat annotations of differentially expressed genes under chronic refeeding condition for INT1 and INT2-9 cells.
(A, B) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under chronic refeeding condition. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.
Figure 6 with 1 supplement
INT1-specific genes modulate behavior and metabolism.
(A) The vector, clec-86, and clec-160 RNAi-treated sid-1;INT1::sid-1 animals after 8 hr exposure to PA14 lawns are shown. (B) The distribution of indicated RNAi-treated sid-1;INT1::sid-1 animals at hour 8 after being transferred to PA14 plates. At hour 0, a total of 30 animals were placed near the PA14 lawn. Data are expressed as a percentage of the animals staying inside PA14 lawn at hour 8± SEM (n=3, 3 plates per RNAi treatment, 30 animals on each plate). **p<0.01 by one-way ANOVA (Dunnett). (C) The lifespan of each indicated RNAi-treated animal after being transferred to PA14 lawn was determined by counting the number of live and dead animals every 6 hr until all animals had died. Data are plotted as the percentage of animals that survived on any given hour relative to the number of live animals at hour 0. The number of animals for each RNAi treatment is indicated in the figure panels. ***p<0.001 by log-rank test. The median survival is 44 hr for all the RNAi treatments. (D–F) Average expression of brown, magenta, and turquoise module genes in the TRAP-Seq conditions. The y-axis shows mean variance stabilized expression across all genes and replicates per condition and module. (G) Representative images of sid-1;INT1::sid-1 animals bearing integrated INT1::INS-7mCherry and CLM::GFP transgenes treated with vector RNAi at the indicated time points. Scale bar, 5 μm. (H, J, L) sid-1;INT1::sid-1 animals bearing integrated INT1::INS-7mCherry and CLM::GFP transgenes were treated with vector, aakg-4 or fmo-2 RNAi. INS-7 secretion levels were measured after 30 min fasting and after 30 min refeeding. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each RNAi treatment. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of fed animals treated with vector RNAi ± SEM (n=21–27). *p<0.05, **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (I, K) Normalized expression plots of aakg-4 and fmo-2 under all conditions in INT1 and INT2-9 cells by replicate.
Figure 6—figure supplement 1
WormCat annotations of undulating genes in INT1 and INT2-9 cells.
(A, B) WormCat visualization of categories enriched in genes of module brown and turquoise. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.
Figure 7 with 1 supplement
Pyruvate is critical for maintaining INS-7 secretion dynamics in INT1 cells.
(A) INS-7 secretion dynamics during fasting and re-feeding with OP50, latex beads, day 1 OP50 supernatant (D1SN), day 1 OP50 supernatant with latex beads (D1SN + Beads), and UV-killed OP50 were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=20–26). **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (B) INS-7 secretion dynamics during fasting and re-feeding with OP50, 2% glucose, and 100 mM pyruvate were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=14–26). **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (C–F) INS-7 secretion dynamics in INT1-specific vector RNAi, mpc-1 and mpc-2 RNAi, pyc-1 RNAi, pdha-1 and pdhb-1 RNAi-treated animals during fasting and re-feeding with 100 mM pyruvate were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of vector RNAi fed animals ± SEM (n=13–24). *p<0.05, **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (G) INS-7 secretion dynamics during fasting and fasting with the presence of 100 mM pyruvate were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of fed animals ± SEM (n=16–19). ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3).
Figure 7—figure supplement 1
The role of pyruvate in refeeding responses of INS-7 secretion from INT1 cells.
(A) INS-7 secretion dynamics during fasting and re-feeding with different concentrations of pyruvate (0.01–100 mM) were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=15–23). ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (B) INS-7 secretion dynamics during fasting and re-feeding with 10 mM pyruvate, 10 mM 3-FP, and 10 mM pyruvate and 3-FP were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=17–20). *p<0.05, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3).
Additional files
-
Supplementary file 1
C. elegans strains used in this study.
Genotypes, sources, and identifiers of the C. elegans strains used in this study. Integration and backcrossing information is provided where applicable.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp1-v1.xlsx
-
Supplementary file 2
Plasmids used in this study.
Construct descriptions, sources, and identifiers of plasmids used for translating ribosome affinity purification (TRAP), fluorescent marking of intestinal cells, and generation of mNeonGreen reporters. mNG, mNeonGreen; 3′ UTR, 3′ untranslated region.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp2-v1.xlsx
-
Supplementary file 3
Cloning primers used in this study.
Oligonucleotide sequences, primer functions, and supplier information for Gibson assembly of the clec-160, clec-86, B0024.4, F01D5.5, F01D5.1, and npr-28 reporter constructs. Primer pairs were used to amplify the corresponding promoters and 3′ untranslated regions, the mNeonGreen coding sequence, and the pUC19 vector backbone.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp3-v1.xlsx
-
Supplementary file 4
Quality control, differential expression, and co-expression network analysis of intestinal TRAP-Seq datasets.
Quality-control measurements and analysis results for TRAP-Seq profiling of the four anterior intestinal cells (INT1) and the remaining 16 intestinal cells (INT2-9). The acute paradigm comprised 30 min of fasting followed by 30 min of refeeding; the chronic paradigm comprised 180 min of fasting followed by 90 min of refeeding, with separate fed controls for each paradigm. Worksheets provide RNA concentrations, immunoprecipitation recovery relative to input (%IP), mean RNA integrity numbers (RIN), sample-exclusion notes, DESeq2 differential-expression results, weighted gene co-expression network analysis (WGCNA) module assignments, Gene Ontology enrichment analysis results, and module eigengenes. Differential-expression tables include gene identifiers, mean normalized counts, log₂ fold changes, standard errors, test statistics, and unadjusted and Benjamini–Hochberg-adjusted p-values, including nonsignificant results. In comparison codes, G and P denote INT1 and INT2-9; F, S, and R denote fed, fasted, and refed states; and 30 and 180 identify the acute and chronic paradigms. Positive log₂ fold changes indicate higher expression in the first member of each comparison. The ‘DE’ labels indicate fold-change direction; statistical significance is reported by the adjusted p-values.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp4-v1.xlsx
-
Supplementary file 5
Differentially expressed genes and functional annotations for INT1 versus INT2-9.
Gene lists comparing INT1 with INT2-9 in fed, fasted, and refed states within the acute and chronic paradigms. Acute experiments comprised 30 min of fasting followed by 30 min of refeeding, and chronic experiments comprised 180 min of fasting followed by 90 min of refeeding, with separate fed controls for each paradigm. The summary worksheet contains genes selected at Benjamini–Hochberg-adjusted p<0.05. Additional worksheets separate genes with higher (‘Up’) or lower (‘Down’) expression in INT1 relative to INT2-9 for each condition. Annotations include WormBase identifiers, gene names, log₂ fold changes, adjusted p-values, WormCat functional categories and descriptions, UniProt identifiers, and InterPro protein-family and domain annotations.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp5-v1.xlsx
-
Supplementary file 6
Differential expression and functional annotations for fasting and refeeding responses in INT1.
TRAP-Seq differential-expression results for INT1 during acute fasting (30 min) and subsequent refeeding (30 min), and chronic fasting (180 min) and subsequent refeeding (90 min). The merged worksheet contains DESeq2 results for fasted versus fed, refed versus fasted, and refed versus fed comparisons within each paradigm, including nonsignificant results. Additional worksheets provide gene lists selected at Benjamini–Hochberg-adjusted p<0.1, with WormBase identifiers, gene names, log₂ fold changes, adjusted p-values, and WormCat functional annotations. ‘Fasted’ worksheets compare fasted with fed animals; ‘Refed’ worksheets compare refed with fasted animals. ‘Up’ and ‘Down’ indicate positive and negative log₂ fold changes, respectively.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp6-v1.xlsx
-
Supplementary file 7
Differential expression and functional annotations for fasting and refeeding responses in INT2-9.
TRAP-Seq differential-expression results for INT2-9 during acute fasting (30 min) and subsequent refeeding (30 min), and chronic fasting (180 min) and subsequent refeeding (90 min). The merged worksheet contains DESeq2 results for fasted versus fed, refed versus fasted, and refed versus fed comparisons within each paradigm, including nonsignificant results. Additional worksheets provide gene lists selected at Benjamini–Hochberg-adjusted p<0.1, with WormBase identifiers, gene names, log₂ fold changes, adjusted p-values, and WormCat functional annotations. ‘Fasted’ worksheets compare fasted with fed animals; ‘Refed’ worksheets compare refed with fasted animals. ‘Up’ and ‘Down’ indicate positive and negative log₂ fold changes, respectively.
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp7-v1.xlsx
-
Supplementary file 8
Protocol for translating ribosome affinity purification from C. elegans.
Detailed protocol used to isolate intestinal ribosome-associated RNA from C. elegans expressing HA-tagged RPL-22 in INT1 or INT2-9. Procedures cover animal synchronization and culture, harvesting in the presence of cycloheximide, flash-freezing and cryogenic grinding, lysate preparation and pre-clearing, anti-HA immunoprecipitation, washing, and RNA elution. Adapted from McLachlan and Flavell, 2019, Bio-protocol 9 (15) (doi:10.21769/BioProtoc.3328).
- https://cdn.elifesciences.org/articles/109237/elife-109237-supp8-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/109237/elife-109237-mdarchecklist1-v1.pdf
-
Source data 1
Numerical data underlying the figures and figure supplements.
Numerical source data are provided in a single Excel file, with separate worksheets labeled according to the corresponding figure and panel. Each worksheet contains the associated data and experimental group labels.
- https://cdn.elifesciences.org/articles/109237/elife-109237-data1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
RiboTRAP-seq identifies spatially distinct functions for the anterior and posterior intestine in immune and metabolic regulation in Caenorhabditis elegans
eLife 15:RP109237.
https://doi.org/10.7554/eLife.109237.3
