Skip to main content
. 2017 Jan 10;6:e21052. doi: 10.7554/eLife.21052

Figure 5. Early-embryo-enriched transcripts are downregulated as organogenesis begins.

(A) Hierarchical clustering of S2–S4-enriched transcripts (n = 1,756) using scaled RPKM data. Left: Heat map. Y, yolk. Colored bars (left) denote Clusters 5, 6 and 8 containing early-embryo-enriched (EEE) transcripts. Cluster f5 sequences (blue, n = 413) were expressed at roughly equivalent levels during S2 and S3, with 66% (n = 275) transcripts showing five-fold or greater declines in average expression values between S3 and S5. Cluster 6 sequences (red, n = 523) exhibited maximal expression during S2, and average expression levels declined more than five-fold between S2 and S4 for 81% (n = 426) of these transcripts. Cluster 8 sequences (green, n = 112) showed peak expression during S4, with 52% (n = 60) of the transcripts showing five-fold or greater declines in average expression values by S5. Right: Normalized expression trends for EEE transcripts in Clusters 5 (blue), 6 (red) and 8 (green) plotted as a function of developmental time. Median 50% of transcripts based on expression maxima are plotted. (B–E) Colorimetric WISH depicting expression of the EEE transcripts tct-like (B), BTF3-like (C), DDX5-like (D) and eIF4a-like (E) (blue) in S2–S8 embryos and C4 asexual adults. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. O, oral; V, ventral. Scale bars: 100 µm. (F–I) EEE transcripts were expressed throughout the piwi-1+ compartment in S3–S4 embryos. Fluorescent double WISH with riboprobes against piwi-1 (red) and the EEE transcripts tct-like (F), BTF3-like (G), DDX5-like (H) and eIF4a-like (I) (green) in S4 embryos. Percentage piwi-1+ cells coexpressing the indicated EEE marker (red) and percentage EEE+ cells coexpressing piwi-1 (green) appear in the lower left corner of merged images. (F) n = 895 piwi-1+ cells, n = 905 tct-like+ cells, n = 7 S3–S4 embryos. (G) n = 692 piwi-1+ cells, n = 728 BTF3+ cells, n = 6 S3–S4 embryos. (H) n = 676 piwi-1+ cells, n = 681 DDX5-like+ cells, n = 5 S3–S4 embryos. (I) n = 312 piwi1+ cells, n = 332 eIF4a+ cells, n = 4 S3–S4 embryos.

DOI: http://dx.doi.org/10.7554/eLife.21052.047

Figure 5—source data 1. Hierarchical clustering of S2–S4-enriched transcripts across embryogenesis.
S2–S4 transcripts (n = 1,756) were clustered using scaled RPKM values across embryogenesis (Y, S2–S8). Cluster membership and average RPKM values (Y–S8, C4 and SX) are provided. Early-embryo-enriched (EEE) transcripts (n = 1,048), which were downregulated by S5 and were lowly expressed through S8, comprise Clusters 5, 6 and 8. Separate tabs for EEE transcript Clusters 5, 6 and 8 include annotation based on best BLASTx hits (E < 0.001) versus the NR, Swiss-Prot, C. elegans, D. melanogaster, D. rerio, X. tropicalis, M. musculus and H. sapiens RefSeq databases.
DOI: http://dx.doi.org/10.7554/eLife.21052.048
elife-21052-fig5-data1.xlsx (796KB, xlsx)
DOI: 10.7554/eLife.21052.048
Figure 5—source data 2. Validation of transcript expression trends using the Nanostring nCounter platform.
Normalized Nanostring data tab: Normalized expression counts for positive ERCC spike-in controls (POS A–F), negative controls (NEG A–H), housekeeping genes, the blastomere and mitotic cell markers H2B and piwi-1, the differentiating progenitor marker prog-1, and 107 early-embryo-enriched (EEE) transcripts. Clusters tab: hierarchical clustering results for 107 EEE transcripts using normalized count data. Raw data tab: raw expression count data for positive ERCC spike-in controls (POS A–F), negative controls (NEG A–H), housekeeping genes, the blastomere and mitotic cell markers H2B and piwi-1, the differentiating progenitor marker prog-1, and 107 EEE transcripts. Nanostring probe sequences tab: target sequence region for capture and reporter probes.
DOI: http://dx.doi.org/10.7554/eLife.21052.049
elife-21052-fig5-data2.xlsx (141.6KB, xlsx)
DOI: 10.7554/eLife.21052.049
Figure 5—source data 3. EEE transcript expression patterns detected by colorimetric WISH.
Colorimetric whole mount in situ hybridization screen results detail expression patterns across embryos and C4 intact adults for select EEE transcripts. Numbers in parentheses indicate the number of embryos scored with expression in a given tissue (numerator) versus the total number of embryos scored (denominator). pT4P-EEE transcript plasmid insert and cloning primer sequences are also provided.
DOI: http://dx.doi.org/10.7554/eLife.21052.050
elife-21052-fig5-data3.xlsx (56.3KB, xlsx)
DOI: 10.7554/eLife.21052.050

Figure 5.

Figure 5—figure supplement 1. Validation of early-embryo-enriched transcript expression trends using the Nanostring nCounter platform.

Figure 5—figure supplement 1.

(A) Count sums for positive controls(left), housekeeping genes (middle) and experimental probes (right) for four biological replicates per sample: yolk (Y), S2–S8 embryos, C4 asexual adults (C4) and virgin sexually mature adults (SX). Note the low read counts for the experimental S2 samples. (B) Log-scale plot of count sums for positive controls (POSITIVE A–F) and negative controls (NEGATIVE A–H) across all 40 samples assayed. Positive control probes detect exogenous ERCC control sequences added to the reactions at known concentrations. Negative control probes are not homologous to any known Smed sequence and measure background signal. (C) Raw counts for the housekeeping genes ACT-B, RPL-27, G6PD, LUC7L3, clathrin-1, hgp-1, rpl13a, ubiquilin-1 and zfp207-1 across all 40 experimental samples. ACT-B showed significant variation in expression across samples. (D) Normalization factors calculated by taking the geometric mean of the positive control sequences POSITIVE A–F (left) or the suite of housekeeping genes (right) across sample replicates. (E) Normalized expression trends for piwi-1, H2B, and prog-1 during embryogenesis and adulthood (Y, S2–S8, SX, C4) mirror those observed in the single embryo RNA-Seq time course. (F) Heat map (left) generated by hierarchical clustering using normalized count data for 108 EEE transcripts across developmental time (Y, S2–S8, SX, C4). Line graphs display expression trends for Clusters 1 (blue), 2 (red), and 4 (green), which corroborate early-embryo-enriched expression observed in the RNA-Seq time course. Transcripts in Clusters 5–8 (n = 9) exhibited low expression throughout the time course and/or exhibited expression trends different from those observed using RNA-Seq.
DOI: http://dx.doi.org/10.7554/eLife.21052.051