ECB-ART-44548
PLoS Biol
2016 Mar 01;143:e1002391. doi: 10.1371/journal.pbio.1002391.
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Comparative Developmental Transcriptomics Reveals Rewiring of a Highly Conserved Gene Regulatory Network during a Major Life History Switch in the Sea Urchin Genus Heliocidaris.
Israel JW
,
Martik ML
,
Byrne M
,
Raff EC
,
Raff RA
,
McClay DR
,
Wray GA
.
Abstract
The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results indicate that distinct evolutionary processes operate on gene expression during periods of life history conservation and periods of life history divergence, and that this contrast is even more pronounced within the GRN than across the transcriptome as a whole.
PubMed ID: 26943850
PMC ID: PMC4778923
Article link: PLoS Biol
Grant support: [+]
P01 HD037105 NICHD NIH HHS , R01 HD014483 NICHD NIH HHS , T32 HD040372 NICHD NIH HHS , R01 HD14483 NICHD NIH HHS
Species referenced: Echinodermata
Genes referenced: ago1b gscl hbn LOC100887844 LOC100893907 LOC115919910 LOC115922368 LOC575170 LOC578257 pcsk2 pdx1l
Article Images: [+] show captions
Fig 1. Gene expression during sea urchin development reflects known phylogenetic relationships between species.(A) Our study includes three sea urchin species: the sister species H. tuberculata (planktotroph) and H. erythrogramma (lecithotroph), which diverged approximately 5 Myr, and an outgroup species L. variegatus (planktotroph), which diverged approximately 35–45 Myr ago [44, 50]. (B) Our developmental time course includes seven stages across each species, from unfertilized eggs to early larvae. (C) Principal component (PC) analysis of gene expression (S1 Data). PC1 explains 36% of the overall variation and clearly separated early (egg through 32-cell stage) from later developmental stages (blastula through early larva), whereas PC2 separated L. variegatus from the two Heliocidaris species, corresponding to their phylogenetic relationships. | |
Fig 2. Comparative clustering strategy reveals cases of expression profile conservation and change within a phylogenetic framework. (A) Cluster centroids identified in the outgroup L. variegatus are shown (S2 Data), along with the number of genes in each species that were assigned to a given cluster. Green = L. variegatus, blue = H. tuberculata, and purple = H. erythrogramma. The x-axis represents developmental time, and the y-axis represents expression change. (B) Comparative clustering strategy identifies genes that exhibit the same expression profile among all three species (conservation), differ among all three species (divergence), differ between L. variegatus and the Heliocidaris species (genus-level change), or change specifically along a particular branch of the phylogeny (cluster jump in H. tuberculata or H. erythrogramma). (C) Using a comparative clustering strategy (see 2B and Methods), we characterized expression profile divergence (white, 29.8%), conservation (dark grey, 19.1%), and genus-level change (light grey, 19.8%) across the transcriptome. We also detected branch-specific change in H. tuberculata (blue, 13.5%) and H. erythrogramma (purple, 17.8%) (S1 Table). | |
Fig 3. Subtle shifts are more common than dramatic changes in gene expression between species. (A) Along the H. erythrogramma branch, Gsc displays a subtle change in expression and is slightly accelerated compared to the planktotrophs. Accordingly, Gsc is assigned to cluster 6 in the planktotrophs and cluster 5 in H. erythrogramma. In contrast, Elov6.3 exhibits a dramatic change in expression and is assigned to cluster 7 in the planktotrophs and cluster 3 in the lecithotroph. Biological replicates are represented as circles, and average expression profiles across replicates are represented as lines. Expression values below the horizontal line are less than 5 counts per million (cpm) and are designated as VLE (S1 Data). (B) In the PCA plot, each ortholog is a light grey circle and each cluster centroid is a dark grey circle. The PCA was performed on S3 Data (see Methods). To calculate a jump score from the L. variegatus ortholog of Elov6.3 (green circle) to the H. erythrogramma ortholog of Elov6.3 (purple circle), we calculated a scaled distance, or “jump score,” from the first two PC loadings (see Methods). This calculation was repeated to obtain the distance between the L. variegatus and the H. tuberculata ortholog (blue circle). The position of Gsc orthologs for each species is also shown. All profiles designated as VLE have the coordinates (0,0). (C) Distribution of jump scores calculated from L. variegatus to H. tuberculata (x-axis) and from L. variegatus to H. erythrogramma (y-axis) (S4 Data). Jump scores are colored according to comparative clustering classification: H. erythrogramma branch jumps are purple, H. tuberculata branch jumps are blue, and genus level changes are grey. For clarity, jump score distributions of conserved and diverged genes are plotted separately in S3 Fig. (D) Kernel density curves of jump scores for genes that exhibited a cluster jump in either H. tuberculata (blue) or H. erythrogramma (purple) (S4 Data). | |
Fig 4. Life history strategy is a major component of gene expression divergence across the GRN. (A) Compared to the transcriptome, the GRN exhibits increased expression profile conservation among species (dark grey, 41.5%) and reduced divergence (white, 11.3%). Genus level and H. tuberculata-specific cluster jumps are also reduced (light grey, 8.5% and blue, 5.7%, respectively), while cluster jumps specifically along the H. erythrogramma branch increased at the GRN level compared to the transcriptome (purple, 33%) (S4 Table). (B) H. erythrogramma-specific branch jumps (purple) occur in each GRN territory, though they are concentrated in the skeletogenic lineage (Binomial test, p < 0.01). Names of genes measured in this study are shown in black, other genes in grey. The associated jump score for each H. erythrogramma-specific branch jump is represented by box color. Jumps into the VLE group are represented by open boxes. Examples of GRN change in H. erythrogramma are highlighted, including EM subcircuit compression (green boxes, S4A Fig) and altered regulatory interactions between VegF and VEGFR and between Not and Gsc (red lines, S4D–S4F Fig). Generalized categories of GRN change are depicted in S5 Fig. | |
Fig 5. Genes coexpressed during planktotrophic development significantly overlap with those coexpressed during lecithotrophic development. (A) Overlap matrix of H. tuberculata and L. variegatus cluster assignments (S1 Table). The x-axis represents H. tuberculata clusters, and the y-axis represents L. variegatus clusters. Each cell contains the number of intersecting genes, and the cell color represents the significance of the intersection. Cells along the matrix diagonal are “consensus clusters” between the planktotrophic species (e.g., consensus cluster 1 contains 186 genes that are coexpressed in both H. tuberculata cluster 1 and L. variegatus cluster 1). Consensus cluster cells are outlined in black. In general, genes coexpressed below the matrix diagonal represent genes that have accelerated expression profiles in H. tuberculata compared to L. variegatus, whereas genes coexpressed above the matrix diagonal represent genes that have delayed expression in H. tuberculata compared to L. variegatus. (B) Overlap matrix of planktotrophic consensus cluster and H. erythrogramma cluster assignments (S1 Table). The x-axis represents planktotrophic consensus clusters (detailed in A), except for the grey cluster, which represents genes that did not exhibit consensus expression profiles between planktotrophs (i.e., genes that were off-diagonal in A). The y-axis represents H. erythrogramma clusters. Each cell contains the number of intersecting genes, and the cell color represents the significance of the intersection. Cells along the matrix diagonal are coexpressed between all three species and are outlined in black. In general, genes coexpressed below the matrix diagonal represent genes that have accelerated expression profiles in the planktotrophs compared to H. erythrogramma, whereas genes coexpressed above the matrix diagonal represent genes that have delayed expression in the planktotrophs compared to H. erythrogramma. | |
Fig 6. Cross-species transcriptome comparison reveals novel candidates for planktotrophic development. (A) Whole mount in situ hybridization reveals expression of Nkx6.1 is restricted to the endoderm in the early larva and to the hindgut in the larva in L. variegatus. (A’–A”‘) Double fluorescent in situ hybridization shows that Nkx6.1 and hindgut marker Lox colocalize in the hindgut of L. variegatus larvae. (B) Whole mount in situ hybridization reveals expression of Mab21l2 is first restricted to the apical plate and later to two lateral patches in the anterior neurogenic ectoderm in L. variegatus larvae. (B’–B”‘) Double fluorescent in situ hybridization reveals that Mab21l2 and anterior neurogenic ectoderm marker Hbn colocalize in the anterior neurogenic ectoderm of L. variegatus larvae. |
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