Barsi Lab Internship
Results
The exactTest (edgeR) on gene expression data of 20,973 genes at 30hpf in S. purpuratus Archenteron cells and control cells (the entire embryo), in addition to thresholds of p-value 0.05 and fold change (FC) of 0.5 and 2.0, resulted in 137 statistically upregulated (active) differentially expressed genes, 123 statistically downregulated (inactive) differentially expressed genes for the Archenteron and 20173 not statistically significant genes (figure 1.A & 1.B). Of the 137 differentially upregulated genes, two were signalling genes: Wnt6 (WHL22.596784, p = 0.04, log2FC = 3.50) and Hgf (WHL22.461412, p = 0.007, log2FC = 2.88) (figure 1.C).
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The gene expression data of the 20173 not significant genes were visually analysing seperately for each of the two biological replicates. From this 23 signalling genes were identified that could potentially be significantly upregulated in the Archenteron at 30hpf despite not being statistically significant according to our analysis. Of these, 12 genes peak or rise in gene expression at 30hpf when looking at the entire embryo: F5L_1 Hgf, HgfL, Deltex4, Irsp53/58, Pcsk7, Pdgfr/vegfrL, and Ron peak (figure 3.B), and Pdgfr/vegfrL_1, Delta, Notchl1, Omi/HtrA2, and Tgif rise in expression (figure 3.C).
Deltex4 and Tgif may be involved in the signalling processes of the Wnt6 gene identified as statistically significant (table 3).
Of the remaining potentially interesting genes, only Irsp53/58, Pdgfr/vegfrL, and Omi/HtrA2 appear - according to the literature - may be significantly involved in signalling processes in the Archenteron (table 1).
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Three of the genes identified as potentially interesting signalling genes (Ron, HgfL, and ‘F5L_1, Hgf’) may be involved in Hgf signalling processes according to the literature (table 1). Notably, ‘F5L_1, Hgf’ is statistically close to being significant with a p-value of 0.08 and a log2FC of 3.03 (table 2).
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A further literature search on Wnt6 resulted in the identification of 16 genes present in our dataset that may be involved in Wnt6 signalling during gastrulation: Runt2, Lef1 Tcf, Runt1, Runx1l1, Frizz1/2/7, Frizz5/8, Frizz9/10, Dsh (three WHL IDs), Porcupine, Ets1-2, Ets1/2, FoxA, Wnt8, and Hox11/13b (table 2). Of these Runt1, Lef1 Tcf, Porcn, Frizz9/10, Ets1-2, Ets1/2, Dsh (WHL22.256138) and FoxA peak, and Runx1l1, Dsh (WHL22.256123) rise in expression at 30hpf in S. purpuratus embryos (figure 4).
Isolating Statistically Significant Genes
Figure 1 Differential gene expression analysis on 20.973 genes in S. purpuratus Archenteron cells at 30hpf, where the control is the transcriptome of the entire embryo at 30hpf. A Volcano plot log(p value) against log2(fold change) of the statistical analysis conducted using the exactTest in R (edgeR) using two biological replicates. Statistically significant differentially expressed genes were selected as follows: p value<0.05 & log 2 FC< 1 for downregulated genes (green) and p value 0.05 & log 2 FC 1 for upregulated genes (purple). We found 137 statistically upregulated (active) differentially expressed genes, 123 statistically significant downregulated (inactive) differentially expressed genes, and 20713 not statistically significant genes. B and C Gene expression for all genes in archenteron cells against cells of the entire embryo , showing statistically significant upregulated (purple) and downregulated (green) genes in figure B, and all the statistically significant upregulated genes for both replicates (red and blue) in figure C. C highlights the two statistically significant signalling genes: Wnt6 and 'Hgf, Hypp_1329, Hypp_137, Hypp_950.
Other Potentially Interesting Signalling Genes
Figure 2 Differential gene expression analysis on 20.973 genes in S. purpuratus Archenteron cells at 30hpf, where the control is the transcriptome of the entire embryo at 30hpf. The figures show gene expression for all genes in archenteron cells against cells of the entire embryo for both replicates (red and blue). A All signalling genes in the dataset, highlighting the two found to be statistically significantly upregulated: Wnt6 and 'Hgf, Hypp_1329, Hypp_137, Hypp_950'. Statistical analysis was conducted with the exactTest in R (edge R) using threshold s of p 0.05, and fold change 0.5 and 2.0 . B All signalling genes, where the statistically significant ones are circled and those visually identified as potentially interesting highlighted in colour. Potentially interesting genes were selected when they had a high expression in at least one replicate .
Figure 3 Gene expression profiles (percent age of a genes own maximum expression) of various genes between 0 and 72 hours post fertilisation , retrieved from Legacy.echinobase.org, 2021 These genes are found in S. purpuratus embryos at 30hpf in archenteron cells. A. signalling genes identified as statistically significantly upregulated according to differential gene expression analysis with the exactTest in R (edgeR, parameters: p <0.05, fold cha nge <0.5 & >2. 0). B, C, and D: Upregulated signalling genes identified as potentially interesting when visually analysing the two biological replicates separately. B. shows genes that peak at our sampling time (30hpf) C. shows genes that increase in expression at 30hpf. D. Shows genes whose expression decreases at 30hpf.
Table 1. Information (function , literature , and expression at 30hpf ) on genes visually identified as interesting signalling genes because they were highly upregulated in at least one the two biological replicates used in differential gene expression analysis on S. purpuratus archenteron cells at 30hpf. Information on the expression at 30hpf in S. purpuratus embryos is displayed (Legacy.echinobase.org, 2021). These signalling genes were not found to be statistically significantly upregulated in the archenteron according to the differential gene
expression analysis using the exactTest in R (edge R, thresholds p<0.05 an d fold change <0.5 or >2.0 ).
Genes Potentially Involved in Hgf Signalling
Table 2. Information (function and literature) on genes thought to be involved with the Hgf signalling gene. All these genes peak in expression at 30hpf in S. purpuratus embryos (Legacy.echinobase.org, 2021) The gene surrounded by black borders was found to be a statistically significant upregulated signalling gene in archenteron cells in S. purpuratus at 30hpf according to differential gene expression analysis using the exactTest in R (edge R, thresholds p<0.05 an d fold change <0.5 or >2.0 ). The unbordered genes with white backgrounds have been visually identified as interesting signalling genes because they were highly upregulated in at least one the two biological replicates. The gene with the grey background was identified from the literature to potentially be involved with Hgf signalling during gastrulation.
Genes Potentially Involved in Wnt Signalling
Figure 4 Gene expression profiles (percentage of a gene’s own maximum expression) of various genes between 0 and 72 hours post fertilisation, retrieved from (Legacy.echinobase.org, 2021). These genes were identified from the literature as being involved in Wnt6 signalling in early development (the Wnt6 expression profile is shown in black on each figure). They are all found in S. purpuratus embryo’s at 30hpf in archenteron cells. Wnt6 was identified as a statistically significant upregulated gene in the archenteron of S. purpuratus embryo’s at 30hpf according to differential gene expression analysis using the exactTest in R (edgeR, thresholds p<0.05, fold change <0.5 or >2.0). Each of the figures A-F shows a different set of genes involved with Wnt6 signalling.
Table 3. Information (function and literature) on genes thought to be involved with the Wnt6 signalling gene. Information on their expression at 30hpf in S. purpuratus embryos is displayed (Legacy.echinobase.org, 2021). The genes surrounded by black borders was found to be a statistically significant upregulated signalling gene in archenteron cells in S. purpuratus at 30hpf according to differential gene expression analysis using the exactTest in R (edge R, thresholds p<0.05 and fold change <0.5 or >2.0). The unbordered genes with white backgrounds have been visually identified as interesting signalling genes because they were highly upregulated in at least one the two biological replicates. The gene with the grey background was identified from the literature to potentially be involved with Wnt6 signalling during gastrulation.
