Biological Implications of the Coelacanth Genome an Ancient Mariner

Molecular Evolution Features

Biological implications of the coelacanth genome An Ancient Mariner

Chris T. Amemiya (Benaroya Research Institute and the University of Washington, USA)

The coelacanth is an iconic species that has captured the public’s imagination owing to its unusual appearance and its special history. The fish possesses paired fins resembling limb-like structures and other features that have placed it in an outgroup position to the terrestrial vertebrates. The recent sequencing of the coelacanth genome has enabled insights into the fin–limb transition and the origin of terrestriality. Here, I discuss some of the more salient findings and interpret them in an evolutionary context.

Charles Darwin was a brilliant naturalist who Mariner4 who was the sole survivor of his ship’s crew, recognized seemingly disparate connections in the the living coelacanth is the sole survivor of an ancient natural world; he saw evolutionary relationships lineage of fishes that had literally gone in absentia for among forms living and extinct, over wide geographical 70 million years. We now know that there are two barriers, and he used powerful deductive reasoning closely related species of coelacanths (Latimeria), both and logic to establish the now obvious parallels from the Indian Ocean: one from the south-east coast of natural selection and the principles underlying of Africa and the other from Indonesia, and that both domestication of plants and animals. Well before are rare and endangered. As a child growing up in rural the publication of On the Origin of Species1, Darwin Hawaii, I read Old Four Legs5, Smith’s account of the speculated on the mechanism by which evolutionary living coelacanth’s discovery and his efforts to procure a change occurred: “With belief of transmutation & second specimen; immediately I became smitten by this geographical grouping we are led to endeavour to large prehistoric-looking fish with lobed-fins (Figure discover causes of change, the manner of adaptation... 1), an obsession that would continue throughout my instinct & structure become full of speculation & life. We published our first coelacanth paper 20 years line of observation”2. Darwin today would no doubt ago6, although I could never have fathomed at the time revel in our ability to draw biological inferences from that its genome would eventually be sequenced and that genome sequences, especially for an organism such as we would have a chance to peer into its evolutionary the coelacanth, which holds a special place as a living history or to be able to do biology with this creature, ‘transitional’ form*. albeit from the inside-out. A few of the more salient The story of the living coelacanth is a remarkable biological implications of the coelacanth genome one, filled with intrigue, disbelief, pathos and even are discussed below, particularly with reference to geopolitical posturing3. Much like Coleridge’s Ancient evolutionary transitions.

*Darwin discusses the nagging problem of ‘Transitional Varieties’ in Chapter VI of On the Origin of Species2. Although the term is usually referred to in a paleontological context, the living coelacanths and the six extant species of lungfish represent long evolutionary lineages that are unique in their departure points in-between the ray-finned fishes (like zebrafish) and the tetrapods (like us).

Abbreviations: Key words: actinodin gene, CNE, cis-non-coding element; hpf, hours post-fertilization; SINE, short interspersed repetitive element; TE, transposable element cis-non-coding element, sarcopterygian, teleost, tetrapod, transposable element

February 2014 © Biochemical Society 23 Features Molecular Evolution

elements) that have been lost or gained during evolutionary divergence in the respective lineages. Two different scenarios are given in Figure 2 for genomic components lost or gained among three different vertebrate lineages (teleost fishes, coelacanths and tetrapods). As depicted by the Venn diagrams, the patterns of losses and gains can implicate putative elements that may have been of importance in evolutionary processes. In Figure 2(a), a scenario is given in which specific sequences have been lost in the lineage leading to tetrapods, but are retained among teleosts and coelacanths. Our analysis detected at least 50 genes that satisfied this criterion9, and included genes involved in development of the fins, eye, ear, kidney, brain and axial structures (trunk, somites and tail). How could loss of such genes facilitate or contribute to the evolution of terrestriality? One idea is that certain fish-specific structures are maladaptive in a terrestrial environment and that their losses led to evolution of structures better suited to life on land. Teleological arguments could be made to this end for several of the 50 genes lost among Figure 1. African coelacanth photographs. (a) An African coelacanth and diver in a cavern off tetrapods; however, without empirical evidence, these the coast of South Africa. Note the lobed fins and prominent scales. (b) A facial shot of Sydney, remain ad hoc hypotheses. One particularly intriguing one of the members of the Sodwana Bay population of coelacanths. Sydney is a coelacanth example for which there is some supporting evidence is named in honour of Dr Sydney Brenner, who is originally from South Africa and an early provided by the actinodin genes. proponent of sequencing of the coelacanth genome. (c and d) Close-up views of Sydney’s The coelacanth has a single actinodin (And) gene, pectoral and pelvic fins respectively. The paddle-like appearance of the fins was the impetus whereas teleost fishes have multiple copies due to for Smith’s term, ‘Old Four Legs.’ ancestral gen(om)e duplications; there are no And genes in tetrapods. The And genes are involved in the formation Why sequence the coelacanth genome? of fibre-like collagenous structures in developing fins of fish and are necessary, ultimately, for the development of The overarching reason for sequencing the genome of the the soft bony fin rays, the lepidotrichia. Certainly, the fins coelacanth is that it represents a key evolutionary departure of fish are extremely well-adapted to life in the aquatic point in-between the ray-finned fishes (such as carp and environment and are used in many ways in addition salmon) and the tetrapods (amphibians, reptiles, birds to locomotion. A compelling study by Akimenko and mammals), a position occupied by only two extant and colleagues showed that knockdown of two of the lineages, one of which (lungfish) is particularly difficult to And genes in zebrafish results in structural disarray study due to the enormity of its genome. As a prelude to in the developing pectoral fin bud and subsequent genome sequencing, our studies had shown, importantly, misexpression of developmental genes long implicated that selected coelacanth gene families were highly in the fin–limb transition10. The overarching inference conserved in terms of their genomic organization and from these studies is that loss of actinotrichia and fin that their rates of molecular evolution were comparatively rays on the paired appendages during the evolution of lower than the same genes in tetrapods7,8. These findings tetrapods may have enabled the development of the augured well for subsequent genome sequencing of the endochondral skeletal structures that are necessary for coelacanth, and supported the contention that it would terrestrial locomotion and land dwelling. be well suited to ‘informing’ aspects of tetrapod biology, In contrast with the loss of genes during evolution, namely the molecular changes that may have occurred a scenario is given in Figure 2(b) in which those during adoption of a terrestrial environment. sequences that are newly arisen in the lineage that led to the lobe-finned vertebrates or sarcopterygians, are Evolutionary transitions: genomic present in coelacanth and tetrapods, yet absent from losses and gains teleosts. Although a definitive analysis has yet to be carried out for protein-coding genes, such an analysis Comparisons of genome sequences from different groups has been carried out for the so-called cis-non-coding of species can provide candidate genes (or regulatory elements, or CNEs. These are stretches of DNA that

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are found by virtue of both their conserved sequence and proximity relative to genes in multiple sequence alignments. A substantial fraction of the vertebrate CNEs detected in this manner is enhancer elements that activate transcription11,12. Moreover, it is thought that cis-regulation by transcription factors has played an integral role in effecting evolutionary change13–15. Thousands of CNEs were detected that arose in the sarcopterygian lineage, i.e. after the divergence of coelacanths and tetrapods from the teleost fishes. Subjecting these datasets to gene ontology statistical analysis allows rough determination of the biological nature of the genes that these CNEs regulate. Among the highest scoring categories were those associated with embryonic morphogenesis, differentiation, regulation of transcription, sensory organ development, brain and endocrine system development, and limb development. One of these newly arisen CNEs was found in a region upstream of the HOX-D homeobox gene cluster (Figure 3). This region contained six CNEs that were shown previously to consist of enhancer elements Figure 2. Inclusion of the coelacanth genome in comparative genome analysis can identify (blue ovals in Figure 3a). Previous analyses of these six genes (and conserved non-coding sequences) that have been lost (red) or gained (green) enhancers using a mouse reporter transgene assay gave in vertebrate evolution. (a) Loss of genes (and CNEs) from the tetrapod lineages, i.e. these distinct patterns of expression in the developing forelimb sequences were present in teleost fishes and coelacanth, but absent (lost) in tetrapods. (autopod) as shown in the illustration below the map. (b) Gain of genes (and CNEs) in the lobe-finned vertebrates (coelacanths plus tetrapods). One of the enhancers (I1) was found in coelacanths and tetrapods, but not in teleost fish and thus represents a facilitators of developmental neuronal plasticity17–19. new sarcopterygian enhancer. Validation experiments Well before the coelacanth genome project, Bejerano using the coelacanth enhancer in the transgenic mouse et al.20 computationally analysed the small amount of reporter assay gave an expression pattern similar to that Latimeria genomic data that had been deposited in seen previously with the mouse version (Figure 3b). GenBank®, and showed that the origin of a particular The evolutionary implication from this analysis is that, ‘ultraconserved’ element in the human genome could be whereas the autopod is considered a tetrapod invention, traced to the lobe-finned fishes. This short interspersed an enhancer element that drives autopodial expression repetitive element (SINE), importantly, was still is already present in the genome of a lobe-finned fish. conserved in the coelacanth and found in high copy From these preliminary analyses, it is impossible to number; whereas in humans and other tetrapods, this know to what extent such an element has played in family had degenerated beyond recognition except in the fin–limb transition as the observations are largely those instances where it had become ‘exapted’ to new correlative. However, these kinds of experiments set the functions in both coding and non-coding sequences. The stage for further more hypothesis-driven research into authors concluded that the coelacanth, by preserving what the rudiments of tetrapod appendages. in other species would be transient TE families, might serve as a ‘living molecular fossil’ and that the coelacanth Repository of transcriptionally active genome could very well hold relicts of additional events transposable elements that helped shape our own evolution. Indeed, additional studies showed similar patterns for two other coelacanth Transposable elements (TEs) are curious genetic TEs: a SINE21,22 and a Harbinger-type transposon23. Thus sequences that have the ability to undergo mobilization it was with great anticipation when the entire Latimeria within the genome, either by self-contained genetic genome sequence became available, as it was assumed that machinery or via assistance from other intact TEs. It it would provide a veritable treasure trove of evolutionary is estimated that the human genome comprises ~44% information on TEs. TEs, many of which have degenerated and are no longer The analysis of TEs in the coelacanth genome mobile16. TEs are thought to influence myriad biological has since been extensively carried out and the initial processes, including the regulation and diversification prediction that the genome would consist of very high of the genome, and can act as mutagenic agents and levels of TEs proved incorrect. It is now known that

TEs comprise approximately 25% of the coelacanth genome, lower than that for humans. However, more importantly, the number of different TE families in the coelacanth was shown to be markedly higher than in humans and that surprisingly, 23% of all coelacanth TEs were transcriptionally expressed24,25. Taken together, these observations suggest that the coelacanth genome is transpositionally dynamic, an inference that seems to be at odds with the slower relative rate of coding sequence evolution. The underlying evolutionary biology of TEs is still largely a black box as many of the assumed biological effects of transposition have been difficult to assess directly. One feature of TEs is that they endow their neighbouring milieu with better chromatin accessibility. This would have effects on gene regulation of neighbouring genes and would certainly have Figure 3. Identification of a coelacanth enhancer element that drives expression in a limb- evolutionary implications. One TE in the coelacanth, specific manner. Long-range multiple alignments were computationally derived for several the LatiHarb1 transposon23, contains a transposase gene vertebrate genomes using the multi-Z tool and filtered extensively to identify both genes and a second gene, whose function is still unknown, but and CNEs, allowing delineation of lineage-restricted sequence patterns as per Figure 2(b). has been exapted into coding sequences of a few other The region in (a) represents 1 Mb of the mouse HOX-D locus and surrounding area. Genes vertebrate genes. This TE alone is found in >5% of the are labelled and depicted as grey boxes, and previously identified enhancers are shown as Latimeria genome and is highly conserved within the blue ovals. Enhancer-driven expression patterns of mouse transgenic reporter constructs coelacanth genome. The use of a fluorescent transgene are depicted in the illustrations below the map for the mouse limb bud (forelimb). One of reporter system in zebrafish confirmed that indeed this these enhancers, I1, is notable in that it is shared by both tetrapods and coelacanths, but not transposon facilitates transcription in a very general by teleost fishes. (b) The coelacanth version of this enhancer element drives expression in sense and in many tissues (Figure 4), although its the mouse limb bud in a similar manner to the mouse enhancer, and suggests that this is a exaptation into the regulatory landscape of tetrapods has sarcopterygian innovation. Modified from Figure 2 of the study in 2013 by Amemiya et al.9 yet to be assessed. Two of the SINEs in the coelacanths have been shown to be exapted into transcriptional regulatory positions for neuronal genes in the mouse20,21. Clearly, we have just touched the surface insofar as our understanding of the evolutionary influence that TEs have had on tetrapod evolution.

Caveats and future prospects

As with all genome reports, there is the possibility of over-interpretation of the sequence data26. The genome of an extant species represents but one snapshot in time and this snapshot may not necessarily reveal anything tangible about morphological and physiological changes that have occurred over time. In addition, each species has also undergone its own evolution since divergence from a common ancestor, further complicating the analysis. As far as the lobe-finned fishes are concerned, there is a rich history of other fossilized forms (including Figure 4. Expression of a transposon-reporter construct in zebrafish. An intact ‘fishapods’) whose genomes, if they could be determined, LatiHarb1 transposon was placed upstream of a basal promoter and GFP reporter gene would certainly be better suited than Latimeria in the p339hsp70GFPrc vector32 and injected into single cell embryos of zebrafish. for understanding the mechanisms involved in the (a) Embryos at 9 hpf (hours post-fertilization) (late gastrula stage) showing early, although evolutionary transition to a tetrapod bauplan and life largely unspecific, expression of GFP. (b) Side view of a head of a 32 hpf embryo showing history27–29. Genome reports are not meant to be a be-all expression primarily in the integument (skin). (c) Side view of an embryo at 32 hpf showing and end-all evolutionary analysis, but rather a starting GFP expression in the notochord. point for further biological inquiry and hypotheses.

Although we will never be able to replay evolution, 8. Noonan, J.P., Grimwood J., Danke J. et al. (2004) Genome creative experimentation can help us to unravel the Res. 14, 2397–2405 rudiments of key steps that undoubtedly were necessary 9. Amemiya, C.T., Alfoldi J., Lee, A.P. et al. (2013) Nature 496, for evolutionary transitions10,30,31. This is our grand 311–316 challenge in the field of evolutionary developmental 10. Zhang, J., Wagh P., Guay D. et al. (2010) Nature 466, biology: to incorporate and reconcile morphology and 234–237 natural history (including the fossil record) with modern 11. Pennacchio, L.A. and Rubin, E.M. (2001) Nat. Rev. Genet. 2, genetics and developmental biology. ■ 100–109 12. Visel, A., Bristow, J. and Pennacchio, L.A. (2007) Semin. Cell I thank Dr Jessica Alföldi, Dr Axel Meyer and Dr Kerstin Dev. Biol. 18, 140–152 Lindblah-Toh, and the other members of the Coelacanth 13. Carroll, S.B., Grenier, J.K. and Weatherbee, S.D. (2001) From Genome Sequencing Consortium for working together DNA to Diversity: Molecular Genetics and The Evolution of to sequence and analyse the coelacanth genome. All Animal Design, Blackwell Science, Malden photographs of the African coelacanth were taken by 14. Carroll, S.B. (2005) PLoS Biol. 3, e245 Laurent Ballesta during mixed-gas deepwater dives 15. Shapiro, M.D., Marks, M.E., Peichel, C.L. et al. (2004) Nature in Jesser Canyon, Sodwana Bay, South Africa; these 428, 717–723 photographs are used with permission and are not to 16. Lander, E.S., Linton, L.M., Birren, B. et al. (2001) Nature 409, be duplicated and/or distributed without the express 860–921 consent of Laurent Ballesta (www.coelacanthe-projet- 17. Thomas, C.A. and Muotri, A.R. (2012) Front. Biosci. (Elite gombessa.com). Dr Nicolas Rohner contributed the Ed.) 4, 1663–1668 picture of the mouse forelimb expression with the 18. Bundo, M., Toyoshima, M. and Okada, Y. (2014) Neuron, coelacanth enhancer-reporter construct. Additional doi:10.1016/j.neuron.2013.10.053 data discussed here were generated by Dr Igor Schneider, 19. Kazazian, Jr, H.H. (2004) Science 303, 1626–1632 Dr Byrappa Venkatesh and Dr Jeramiah Smith. 20. Bejerano, G., Lowe, C.B., Ahituv, N. et al. (2006) Nature 441, 87–90 Chris Amemiya is a Full Member at the 21. Nishihara, H., Smit A.F. and Okada, N. (2006) Genome Res. Benaroya Research Institute and Professor 16, 864–874 of Biology at the University of Washington, 22. Xie, X., Kamal, M. and Lander, E.S. (2006) Proc. Natl. Acad. Seattle, Washington USA. His lifelong Sci. U.S.A. 103, 11659–11664 fascination with natural history was 23. Smith, J.J., Sumiyama, K. and Amemiya, C.T. (2012) Mol. fostered by being raised in Hawaii, a Biol. Evol. 29, 985–993 natural laboratory for evolution, and by a father who was an avid 24. Forconi, M., Chalopin, D. and Barucca, M. et al. (2013) J. fisherman. His laboratory studies developmental mechanisms Exp. Zool. B Mol. Dev. Evol., doi:10.1002/jez.b.22527 and the evolution of vertebrates, with an eye for novel strategies 25. Chalopin, D., Fan, S., Simakov, O., Meyer, A., Schartl, M. and that organisms have acquired to deal with their internal and Volff, J.N. (2013) J. Exp. Zool. B Mol. Dev. Evol., doi:10.1002/ external environments. email: [email protected] jez.b.22521 26. Zimmer, C. (2010) Yet-another-genome-syndrome. References Discover Magazine, The Loom Science Blog. Also available 1. Darwin, C. (1859) On the Origin of Species by Means of at: http://blogs.discovermagazine.com/loom/2010/04/02/ Natural Selection, Random House, New York yet-another-genome-syndrome/#.UtZelrQbGg8 2. Darwin, C. (1838) Notebook B: Transmutation (1837–1838), 27. Daeschler, E.B., Shubin, N.H. and Jenkins, Jr, F.A. (2006) Cambridge University Press, Cambridge. Also available at: Nature 440, 757–763 http://darwin-online.org.uk 28. Shubin, N.H., Daeschler, E.B. and Jenkins, Jr, F.A. (2006) 3. Weinberg, S. (2000) A Fish Caught In Time: The Search For Nature 440 764–771 The Coelacanth, HarperCollins, New York 29. Clack, J.A. (2012) Gaining Ground: The Origin and 4. Coleridge, S.T. (1883) The Rime of The Ancient Mariner, Evolution of Tetrapods (2nd ed.), Indiana University Press, University Press, Cambridge Bloomington 5. Smith, J.L.B. (1956) Old Fourlegs: The Story of the 30. Abitua, P.B., Wagner, E., Navarrete, I.A. and Levine, M. Coelacanth, Longman Green, London (2012) Nature 492, 104–107 6. Amemiya, C.T., Ohta, Y., Litman, R.T., Rast, J.P., Haire, R.N. 31. Carroll, S.B. (2005) Endless Forms Most Beautiful: The and Litman, G.W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, New Science of Evo Devo and the Making of the Animal 6661–6665 Kingdom. W.W.Norton, New York 7. Amemiya, C.T., Powers, T.P., Prohaska, S.J. et al. (2010) Proc. 32. Mongin, E., Auer, T.O., Bourrat, F. et al. (2011) PLoS One 6, Natl. Acad. Sci. U.S.A. 107, 3622–3627 e19747