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Evolutionary divergence of a Hoxa2b hindbrain enhancer in syngnathids mimics results of functional assays

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Abstract

Hoxa2 genes provide critical patterning signals during development, and their regulation and function have been extensively studied. We report a previously uncharacterized significant sequence divergence of a highly conserved hindbrain hoxa2b enhancer element in the family syngnathidae (pipefishes, seahorses, pipehorses, seadragons). We compared the hox cis-regulatory element variation in the Gulf pipefish and two species of seahorse against eight other species of fish, as well as human and mouse. We annotated the hoxa2b enhancer element binding sites across three species of seahorse, four species of pipefish, and one species of ghost pipefish. Finally, we performed in situ hybridization analysis of hoxa2b expression in Gulf pipefish embryos. We found that all syngnathid fish examined share a modified rhombomere 4 hoxa2b enhancer element, despite the fact that this element has been found to be highly conserved across all vertebrates examined previously. Binding element sequence motifs and spacing between binding elements have been modified for the hoxa2b enhancer in several species of pipefish and seahorse, and that the loss of the Prep/Meis binding site and further space shortening happened after ghost pipefish split from the rest of the syngnathid clade. We showed that expression of this gene in rhombomere 4 is lower relative to the surrounding rhombomeres in developing Gulf pipefish embryos, reflecting previously published functional tests for this enhancer. Our findings highlight the benefits of studying highly derived, diverse taxa for understanding of gene regulatory evolution and support the hypothesis that natural mutations can occur in deeply conserved pathways in ways potentially related to phenotypic diversity.

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Data Availability

Upon publication, all raw sequencing data described in this study will be available via the NCBI Genbank. In situ images will be hosted by the Cresko Lab web server (http://creskolab.uoregon.edu) and distributed by the Cresko Laboratory GitHub account.

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Acknowledgements

We wish to thank S. Bassham, C. Small, M. Currey, A. Amores, Y.L. Yan, and J. Poslethwait for providing feedback on the study design and results, as well as other members of the Cresko and Postlethwait labs; H. Mason-Jones and E. Rose for help with obtaining Gulf pipefish embryos; A. Jones and A. Anderson for sending robust ghost pipefish, messmate pipefish, and dwarf seahorse tissue samples; and A. Bentley for sending bluestripe pipefish and sculptured pipefish tissue samples from the KU fish tissue collection.

Funding

This work would not have been possible without multiple sources of generous support: grant from the National Science Foundation Doctoral Dissertation Improvement Grant in the Directorate for Biological Sciences (DEB-1701854 to W. A. Cresko and A. M. Fuiten), fellowships to A. M. Fuiten from the National Institutes of Health for both the Developmental Biology Training Grant Fellow (T32 HD007348) and the Genetics Training Grant Fellow (T32 GM007413), and from University of Oregon UO Under-Represented Minority Fellow.

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Authors and Affiliations

Authors

Contributions

Allison Fuiten and Bill Cresko designed this study. Allison Fuiten performed experiments and data analysis. Interpretation of results was by Allison Fuiten and Bill Cresko. Allison Fuiten wrote the original draft, and Bill Cresko reviewed and edited the manuscript. Both authors read and approved the final manuscript.

Corresponding author

Correspondence to William A. Cresko.

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Ethics approval

The care and use of animals were in accordance under the IACUC protocol (assurance number A-3009-01).

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Conflict of interest

The authors declare no competing interests.

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Communicated by Mark Q. Martindale

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Supplementary information

Supplemental Figure 1

Evolution of Hox complex. a) Evolutionary timing of Hox complex duplications are denoted on the animal phylogeny based on (Carroll et al. 2013), with updates from (Pascual-Anaya et al. 2018; Ravi et al. 2009). Dashed arrow indicates current uncertainty where the second vertebrate Hox cluster duplication occurred relative to agnathans. b) A cartoon of the previously reported Hox clusters in Gulf pipefish, lined seahorse and tiger tail seahorse with boxes representing genes and circles representing microRNAs arranged along chromosome segments oriented left to right 5′ to 3′. The hollow box represents the hoxa7a pseudogene as described in Small et al. 2016. (EPS 3390 kb)

Supplemental Figure 2

VISTA plots for the HoxB clusters with threespine stickleback HoxBa set as reference sequence. Exons are highlighted in blue, CNE in pink, microRNAs are in the blue boxes. Shuffle LAGAN alignment was used with gray lines indicate stretches of continuous sequence. The reference, Gac, is the threespine stickleback; Tru, fugu; Ola, medaka; Tor, tuna; Ssc, pipefish; Hco, tiger tail seahorse; Her, lined seahorse; Bpe, mudskipper; Gmo, cod; Dre, zebrafish; Loc, spotted gar; Mmu, mouse; Hsa, human. Syngnathid specific peak losses are in red boxes. (EPS 1333 kb)

Supplemental Figure 3

VISTA plots for the HoxB clusters with threespine stickleback HoxBb set as reference sequence. Exons are highlighted in blue, CNE in pink, microRNAs are in the blue boxes. Shuffle LAGAN alignment was used with gray lines indicate stretches of continuous sequence. The reference, Gac, is the threespine stickleback; Tru, fugu; Ola, medaka; Tor, tuna; Ssc, pipefish; Hco, tiger tail seahorse; Her, lined seahorse; Bpe, mudskipper; Gmo, cod; Dre, zebrafish; Loc, spotted gar; Mmu, mouse; Hsa, human. Syngnathid specific peak losses are in red boxes. (EPS 2281 kb)

Supplemental Figure 4

VISTA plots for the HoxC clusters. Exons are highlighted in blue, CNE in pink. Gac, is the threespine stickleback; Tru, fugu; Ola, medaka; Tor, tuna; Ssc, pipefish; Hco, tiger tail seahorse; Her, lined seahorse; Bpe, mudskipper; Gmo, cod; Dre, zebrafish; Loc, spotted gar; Mmu, mouse; Hsa, human. Syngnathid specific peak losses are in red boxes. a) Gac is set as the reference with Shuffle-LAGAN alignment used. b) Ola is set as the reference with Shuffle-LAGAN alignment used. c) Tru is set as the reference with Shuffle-LAGAN alignment used. d) Gac is set as the reference with LAGAN alignment used. e) Ola is set as the reference with LAGAN alignment used. f) Tru is set as the reference with LAGAN alignment used. (EPS 5785 kb)

Supplemental Figure 5

VISTA plots for the HoxC clusters. Exons are highlighted in blue, CNE in pink. Gac, is the threespine stickleback; Tru, fugu; Ola, medaka; Tor, tuna; Ssc, pipefish; Hco, tiger tail seahorse; Her, lined seahorse; Bpe, mudskipper; Gmo, cod; Dre, zebrafish; Loc, spotted gar; Mmu, mouse; Hsa, human. Syngnathid specific peak losses are in red boxes. a) Gac is set as the reference with Shuffle-LAGAN alignment used. b) Ola is set as the reference with Shuffle-LAGAN alignment used. c) Tru is set as the reference with Shuffle-LAGAN alignment used. d) Gac is set as the reference with LAGAN alignment used. e) Ola is set as the reference with LAGAN alignment used. f) Tru is set as the reference with LAGAN alignment used. (EPS 5534 kb)

Supplemental Figure 6

A conserved non-coding element is not detectable in the pipefish HoxAb cluster with different species set as the reference and mVISTA alignment algorithms. Exons are highlighted in blue, CNE in pink. Gac, is the threespine stickleback HoxAb sequence; Tru, fugu HoxAb sequence; Ola, medaka HoxAb sequence; Tor, tuna HoxAb sequence; Ssc, pipefish HoxAb sequence; Hco, tiger tail seahorse HoxAb sequence; Her, lined seahorse HoxAb sequence; Bpe, mudskipper HoxAb sequence; Gmo, cod HoxAb sequence; Dre, zebrafish HoxAb sequence; Loc, spotted gar HoxA sequence; Mmu, mouse HoxA sequence; Hsa, human HoxA sequence. Red arrows indicate missing CNE in syngnathid fish. a) Threespine stickleback (gac) is set as the reference with LAGAN alignment used. b) Fugu (tru) is set as the reference with LAGAN alignment used. c) Medaka (ola) is set as the reference with LAGAN alignment used. d) Fugu (tru) is set as the reference with Shuffle-LAGAN alignment used. e) Medaka (ola) is set as the reference with Shuffle-LAGAN alignment used. (EPS 3593 kb)

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Fuiten, A.M., Cresko, W.A. Evolutionary divergence of a Hoxa2b hindbrain enhancer in syngnathids mimics results of functional assays. Dev Genes Evol 231, 57–71 (2021). https://doi.org/10.1007/s00427-021-00676-x

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  • DOI: https://doi.org/10.1007/s00427-021-00676-x

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