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Recent studies underscore a role for the differential degeneration of enhancers in the evolutionary diversification of paralogue expression. However, no one has reported evidence for the involvement of innovative cis-regulatory changes. Here we show that silencer innovation diversified expression of the vertebrate paralogues, pax2 and pax8. pax2 shows multi-tissue expression, as does the ancestral amphioxus orthologue, pax2/5/8, whereas pax8 expression localizes to a subset of pax2-expressing tissues. We reveal that both pax2 and pax8 retain ancestral enhancers capable of directing pax2-like, multi-tissue expression. However, a silencer within the pax8proximal promoter suppresses pleiotropic enhancer activity outside the pax8-expressing tissues. In contrast, the combination of the pax2proximal promoter with either the pax8 or pax2 enhancer recapitulates pax2-like expression, as in the amphioxus pax2/5/8 promoter. We propose that silencer innovation, rather than enhancer degeneration, was crucial for the divergent expression of paralogues with pleiotropic enhancers inherited from their common progenitor.
Figure 2: GFP expression driven by pax8-CNSs recapitulates the pleiotropic expression of pax2. Transgenic X. laevis embryos were generated with the following reporter constructs and their expression was analysed using in situ hybridization. (a) The β-actin basal promoter alone was linked to GFP. (b–f) The β-actin promoter-GFP cassette was flanked by either X. tropicalis pax8-CNS1 (b), pax8-CNS2 (c), pax8-CNS3 (d), pax8-CNS4 (e) or pax8-CNS5 (f). (g) The β-actin promoter-GFP cassette was flanked by all the pax8-CNSs. (h) The β-globin basal promoter alone was linked to GFP. (i) The β-globin promoter-GFP cassette was flanked by all the pax8-CNSs. The X. tropicalis pax8-CNS1 to pax8-CNS5 are shown as 1pax8 to 5pax8. Representative examples of GFP expression are shown together with the injected reporter constructs. The right-hand panels show high-magnification views of the embryonic head regions illustrated in the left-hand panels. GFP expression patterns are summarized on the extreme right with the scoring results. Numbers of embryos with the positive expression similar to the representative examples, and the total number of analysed embryos injected with each construct are indicated with percentages of the former cases. White, grey and black triangles indicate expression in the pronephros, otic vesicle and eye, respectively. White, grey and black arrows indicate expression in the pharyngeal arches, somites and MHB, respectively. White and grey arrowheads indicate expression in the hindbrain and anteriormidbrain, respectively.
Figure 3: Identification of tissue-specific silencer activity associated with the pax8 proximal promoter. Transgenic X. laevis embryos were generated with the following GFP constructs. (a) The proximal promoter region of X. tropicalis pax8 (−2038 to +130) alone was linked to GFP. (b–d) A deletion series of the X. tropicalis pax8 proximal promoter (b, −2038 to +130; c, −478 to +130; and d, −214 to +130) was flanked by the X. tropicalis pax8-CNS1 to pax8-CNS5. (e) The mouse Pax8-CNS1 (shown as 1mPax8) was linked to the β-actin basal promoter-GFP cassette. (f) The proximal promoter region of mouse Pax8 (−1607 to +494) alone was linked to GFP. (g) The mouse Pax8 proximal promoter was flanked by mouse Pax8-CNS1. White, grey and black triangles indicate expression in the pronephros, otic vesicle and eye, respectively. White, grey and black arrows indicate expression in the pharyngeal arches, somites and MHB, respectively. White and grey arrowheads indicate expression in the hindbrain and an anterior part of the midbrain, respectively.
Figure 4: Silencer innovation in pax8 is crucial for generating divergence in the expression of pax8 and pax2. Transgenic X. laevis embryos were generated with the following GFP constructs. (a) The X. tropicalis pax2-CNS1 (shown as 1pax2) was linked to the β-actin basal promoter-GFP cassette. (b,c) The X. tropicalis pax8 proximal promoter (−2038 to +130) was flanked by either the X. tropicalis pax8-CNS1 (b) or pax2-CNS1 (c). (d) The proximal promoter region of X. tropicalis pax2 (−1109 to +615) alone was linked to GFP. (e,f) The X. tropicalis pax2 proximal promoter was flanked by either the X. tropicalis pax8-CNS1 (e) or pax2-CNS1 (f). (g) The amphioxus pax2/5/8 promoter (−1330 to +589) was linked to GFP. White, grey and black triangles indicate expression in the pronephros, otic vesicle and eye, respectively. White, grey and black arrows indicate expression in the pharyngeal arches, somites and MHB, respectively. White and grey arrowheads indicate expression in the hindbrain and in an anterior part of the midbrain, respectively.
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