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Open Biol
2015 Nov 01;511:. doi: 10.1098/rsob.150079.
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Evolution of lineage-specific functions in ancient cis-regulatory modules.
Pauls S
,
Goode DK
,
Petrone L
,
Oliveri P
,
Elgar G
.
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Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.
Figure 1. (a) Schematic of the sox21 locus in Fugu showing the relative locations of the CNEs and coding sequences of the abcc4 and Sox21 genes (Sox21 has just one coding exon). (b) VISTA plot of MLAGAN alignment between Sox21 CNE17 from different species using Fugu as baseline. A vertical line indicates the division between LSR and core. (c) Multiple alignments across the boundary between the LSR and core regions using Fugu, amphioxus and sea urchin CNE17 sequences, highlighting the dramatic change in sequence identity.
Figure 2. GFP expression in stable transgenic zebrafish lines induced by different parts of the Fugu (a,c,e,g,i,k,m,n,p) or amphioxus (b,d,f,h,j,l,o,q) CNE at 30 hpf (a–f), 50 hpf (i–l,p,q) and 5 dpf (g,h,m,n,o). Elevated levels of GFP in different brain regions (a,c) are indicated by arrows (fb, forebrain; mb, midbrain; hb, hindbrain). Arrows indicate expression in the hypothalamus (g,h,o), ear (i,j) and spinal cord KA-neurons (k,l).
Figure 3. Quantification of GFP expression in the zebrafish lens. (a–d) Co-injection of CNE17::GFP reporter constructs (a) and an RFP lens standard (b) is used to measure relative GFP intensity in the lens at 52 hpf ((c, brightfield), (d, merged)). (e) Relative GFP intensity for full-length versions, LSR or core regions of CNE17 from different species. p-values Mann–Whitney test (electronic supplementary material, dataset S3), ‘n.s.’ = p > 0.05.
Figure 4. Conservation of 26 bp core repressor function in different sequences. GFP intensity relative to a co-injected RFP lens standard. The sea urchin 26 bp repressor does not inhibit the sea urchin LSR but represses the amphioxus LSR (LSR + Sp26). p-values Mann–Whitney test (electronic supplementary material, dataset S), ‘n.s.’ = p > 0.05.
Figure 5. Requirement of Sox consensus motifs for lens expression. Sox consensus motifs in the Fugu, amphioxus and sea urchin LSR are labelled with capital letters. These motifs were individually mutated and, if required for lens expression, are shown in black. Motifs dispensable for lens expression are shown in white and the Fugu repressor motif in red. p-values Mann–Whitney test (electronic supplementary material, dataset S3), ‘n.s.’ = p > 0.05.
Figure 6. Requirement of Sox consensus motifs in sea urchin. (a–c) GFP expression in ciliary band cells (a), ectodermal cells (b) and gut cells (c) in 6-day-old sea urchin larvae. (d) Average percentage of larvae with GFP expression after the injection of the WT sea urchin LSR or constructs lacking the same Sox consensus motifs shown in figure 4. The last value (WT + 26) refers to the sea urchin LSR plus the 26 bp core repressor. Numbers in parentheses indicate independent injections. Error bars indicate standard deviation.
Figure 7. Fugu-specific motifs required for lens expression. (a) Minimal lens enhancer in the Fugu LSR. Motifs different from human are indicated in white if dispensable or black if crucial for lens expression. (b,c) Relative GFP intensity in the zebrafish lens after the injection of either Fugu CNE17 including human motifs A–J instead of the corresponding Fugu motif (b) or insertion of Fugu motifs B or C or both into the human LSR (c). p-values Mann–Whitney test (electronic supplementary material, dataset S3), *p < 0.05, ***p < 0.005.
Figure 8. A model for lens enhancer evolution in CNE17. See main text for details. Core regions are shown in dark grey with the putative ancestral (An) LSR in yellow. Sox consensus motifs are shown as black lines in the LSRs (a cluster of these motifs is assumed to be already present in the ancestral LSR). Repressor motifs are shown as downward red arrows and lineage-specific activators as upward grey arrows. H, human; A, amphioxus; U, sea urchin; F, Fugu.
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