Author: Nathalie Feiner

Readers of this blog will be well aware of the conspicuous variation in dorsal color patterns of Anolis lizards – think of the spots of A. sabanus, or the bands of A. transversalis (both worthy a google search if you happen to be unfamiliar with them). Such patterns are particularly interesting when several patterns exist within a single population – a polymorphism. This is the case in A. sagrei where females have either a chevron-like pattern, same as all males of this species, or a diamond-like pattern on their backs.

In 2016, I visited some A. sagrei populations in Florida and became interested in this female-limited color pattern polymorphism. Being a developmental biologist, what puzzled me most was not if selection can maintain multiple patterns, but how the two patterns actually develop.

On the one hand, the difference between the two patterns ought to have a simple genetic basis. Diamond- and chevron-females mix within the same population and the two morphs are discrete (although there is a continuous variation within both morphs, and the diamonds sometimes look more like a band [1]). On the other hand, the developmental biology of pattern formation is far from trivial. Here is a little teaser of the complexity: in vertebrates, pigment cells are derived from neural crest cells that originate along the dorsal midline of the early embryo. Once detached from the crest of the neural tube, pigment cell precursors migrate towards their final destination in the epidermis, where they differentiate into pigment cells – xanthophores, melanophores and iridophores. What these cells do and how they organize themselves are what gives rise to patterns. And this does not seem to be very simple at all. So how can we reconcile a simple genetic basis with the complex developmental biology of pattern formation of diamonds and chevrons?

Simply out of curiosity, we set out to solve this puzzle. Thanks to our anole breeding group at Lund University, we already had three generations of A. sagrei of both morphs that we could use to address the pattern of inheritance of diamonds and chevrons, and to identify the underlying gene(s). Our intuition about the simple genetic architecture of the polymorphism held true, and we identified a single Mendelian locus that perfectly segregated with the morph patterns.

Surprisingly though, the locus was not on a sex chromosome, so this could not explain why only females are polymorphic. Instead, the identity of one of the two genes located at the Mendelian locus pointed to a solution: the estrogen receptor 1 gene is crucial for the development of female traits and therefore expressed at higher levels in females compared to males. Because of the close physical proximity of the two genes at the Mendelian locus, also the second gene shows a female-biased expression pattern. So while the estrogen receptor 1 gene does not explain the pattern differences, it explains why the polymorphism is only present in females.

But what is this second gene at the Mendelian locus, and how does it explain the difference in patterning? It is a gene encoding the coiled-coil domain-containing protein 170, or CCDC170. Not exactly a usual suspect of color pattern formation. In fact, not much is known about this gene, but the cancer literature had demonstrated that it codes for a structural protein that regulates the migratory capacity of cells (mutations in CCDC170 can make cancer cells migratory and are associated with an aggressive type of breast cancer). The proteins encoded by the diamond and chevron alleles were predicted to form CCDC170 proteins with structural differences. This is likely to affect the function of the protein – perhaps by influencing migratory behaviors of pigment cell precursors.

Testing this hypothesis is easier said than done. As a first proof-of-principle, we used an in silico modelling approach to test if tinkering with the migratory capacity of cells can switch a system from generating chevron-like to diamond-like patterns. Miguel Brun-Usan is a magician with cell-based computer modelling and his trick is to create computer models that are complex enough to capture real biological phenomena, yet simple enough to allow us to trace and understand what is going on. To get to the bottom of the diamond/chevron morph differences, he implemented cells on a growing epithelium with a Turing-type mechanism consisting of a gene regulatory network (GRN) with up to five genes. By running a number of experiments (yes, computer scientists also run experiments, I learned), Miguel could demonstrate that some GRNs generated back patterns that very much resembled those of real lizards. Moreover, he found that modifying a single gene in the GRN that regulates the migratory capacity of cells is sufficient to switch the system from generating chevrons (if migration is switched ON) to diamonds (if migration is switched OFF).

So it seems that what reconciles the simple genetic basis of the polymorphism with the complex process of pattern formation is that the underlying developmental system exhibits a high degree of controllability. Simply modifying migratory capacities of cells by tinkering with one gene in the core GRN leads to changes in collective cell migration, visible as a distinct and new dorsal color pattern. Such high controllability could explain the evolvability of dorsal color patterns and result in high turn-over rates between patterns, as beautifully demonstrated by geckos [2]. I am excited to continue this research to see if the model can be substantiated mechanistically. I would also like to test if convergent evolution of diamond patterning in different Anolis species is underpinned by convergent developmental and genetic mechanisms. If you are interested in joining our research group, please get in touch (nathalie.feiner@biol.lu.se; www.feiner-uller-group.se).

You can read the full article here:

Feiner, N., M. Brun-Usan, P. Andrade, R. Pranter, S. Park, D. B. Menke, A. J. Geneva, and T. Uller. 2022. A single locus regulates a female-limited color pattern polymorphism in a reptile.
Science Advances 8:10 DOI: 10.1126/sciadv.abm2387

Other references:

• Moon, R.M., and Kamath, A. (2019). Re-examining escape behaviour and habitat use as correlates of dorsal pattern variation in female brown anole lizards, Anolis sagrei (Squamata: Dactyloidae). Biol J Linn Soc 126, 783-795.
• Allen, W.L., Moreno, N., Gamble, T., and Chiari, Y. (2020). Ecological, behavioral, and phylogenetic influences on the evolution of dorsal color pattern in geckos. Evolution 74, 1033-1047.

Transposable elements are DNA sequences that move around in the genome. Do they also play any roles in evolution and development? I answered this question by looking at our favourite group of animals – lizards – and found some surprising answers. My most recent paper in Evolution Letters is the last of a trilogy of papers – one, two, and three – that reveal that Anolis lizards, by breaking the rules, allow us to link TEs to speciation and evolvability.

Mobile DNA sequences – transposable elements or TEs for short – are found in the genome of virtually all organisms. As their name implies, TEs can cut or copy themselves from one location in the genome to another. This can wreak havoc as insertion of TEs may interfere with gene regulation or in fact knock out entire genes. Cells therefore have mechanisms that prevent TEs from jumping, including DNA methylation and other epigenetic tools. Thus, TEs are not roaming freely through the genome, but are restricted from entering functionally important parts. Preventing TE invasion is particularly important when genes are regulated through spatial proximity to each other. The textbook example of this situation are the Hox genes, which are the key players in embryonic development with an ingenious mode of action: Hox genes are arranged in tight clusters and their position in the cluster defines their time and space of expression, and thus their effect on the patterning of the early embryo. It is therefore fitting that Hox gene clusters of mammals and other well-studied vertebrates have been found to be almost completely free of TEs. My new study reveals that Anolis lizards have broken this paradigm. Moreover, the invasion of TEs into Hox clusters of Anolis lizards can be linked to aberrant gene expression and increased rates of speciation.

Ever since the discovery of TEs, people have speculated about their evolutionary implications. One possible consequence of high TE activity is structural genomic variation. This may accelerate genomic incompatibility between populations, effectively making TEs engines of speciation.

Occasionally, TE insertions may also generate phenotypic novelty. As noted above, some genes are regulated through their proximity to other genes, which means that invasion of TEs can change expression of a number of genes simultaneously. Furthermore, since jumping TEs often drag along neighbouring genomic regions, they can translocate regulatory sequences that cause genes to be expressed in new cell types or at different stages in development. 

While these are good reasons to expect TEs to promote evolution, examples are few and their role often appears idiosyncratic. An excellent group for a more systematic survey of TE-driven diversification is squamate reptiles, a group that includes lizards and snakes. Squamate genomes do not only appear particularly rich and variable in TEs, but their body plan is also highly malleable. Illustrative examples include the adaptive radiation of Anolis lizards and the repeated evolution of limbless and elongated bodies.

I decided to study how TEs have shaped the genomes, and in particular, the Hox clusters, of squamates. My first surprise was to discover that lizards possess more Hox genes than all other tetrapods since they retained some genes that other lineages have ditched. The second surprise came when I looked at the TE content of Hox clusters. Despite the high TE content in their genomes, squamates follow other vertebrates in generally protecting their Hox clusters from TEs. But there was one exception: I found massive invasion of TEs in the Hox clusters of two out of three Anolis species, with TE contents almost as high as the average place in the genome.

Why in Anolis? Anolis lizards are famous in evolutionary biology due to their adaptive morphological radiation involving high rates of speciation – amassing close to 400 species. In a previous study (explained in a previous Anole Annals post) I showed that Anolis lineages with more speciation events in the past have more TEs in their Hox clusters. My new genome-wide study reveals that this signature of speciation is indeed pronounced in Hox clusters: only the two Anolis species from amply speciating lineages exhibit unusually TE-rich Hox clusters, while a third species (Anolis auratus, black circle in figure above) follows the norm and keeps its Hox clusters relatively free from TEs. Looking in detail at genome-wide TE landscapes of these three Anolis species, I discovered that the two species with TE-rich Hox clusters had a larger population of young, more active TEs in their genomes. In addition, the inferred timing of peak activity of these TEs broadly coincided with past speciation events.

These results suggest that – during speciation events – TEs are unusually active and proliferate throughout the genome. As a result, even crucial regions such as Hox clusters become invaded. Subsequently, TEs are removed from Hox clusters by selection until a ‘healthy equilibrium’ of TE content relative to the genome-wide TE content is reached. This equilibrium appears highly conserved as the Hox clusters of almost all lizards and snakes contain close to 50% of the global TE content. This proposed model generates a number of predictions that can be tested with genomic data from lineages with variable rates of speciation.

How then do some Anolis species cope with having their Hox clusters invaded by TEs? Clearly, the inflation of Hox clusters – increasing the distance between genes – does not disrupt the patterning of the early embryo. Genes located at one end of the cluster remain expressed early in the head of the embryo, while genes located at the other end are expressed late in the tail. However, the successive activation of Hox genes predicts that disruption, if occurring at all, should be most pronounced towards the end of the Hox clusters. I found that this indeed is the case: one out of four Hox13 genes showed aberrant expression in the two Anolis species with TE invaded Hox clusters, but this gene was expressed as ‘normal’ in other Anolis and more distantly related lizards.

My study reveals that, despite being THE textbook example of our conserved developmental toolkit, Hox genes can be tinkered with. What is more, the TE invasions of Hox clusters appear to be intimately linked to diversification. Now that Anolis lizards have shown us that it can happen, perhaps they can also show us why it happens and how.

The original version of this blog post was published on the Evolution Letters Editors’ Blog.

References:

Feiner N. 2019 Evolutionary lability in Hox cluster structure and gene expression in Anolis lizards. Evol Letters. https://doi.org/10.1002/evl3.131

Feiner N. & Wood N.J. 2019 Lizards possess the most complete tetrapod Hox gene repertoire despite  pervasive structural changes in Hox clusters. Evolution & Development. 2019;21:218–228 https://doi.org/10.1111/ede.12300

Feiner N. 2016 Accumulation of transposable elements in Hox gene clusters during adaptive radiation of Anolis lizards. Proc. R. Soc. B 283: 20161555. http://dx.doi.org/10.1098/rspb.2016.1555

Studying Caribbean lizards when you are based in Northern Europe is maybe not the most obvious thing to do. But I couldn’t resist the charm of Anolis and embarked on a postdoc project with the aim of unlocking some of their mysteries. Since I have a background in comparative genomics, I was particularly excited about one odd feature of the green anole genome: unlike other vertebrates, it is remarkably cluttered with transposable elements.

Transposable elements (or TEs for short) are popularly referred to as jumping genes because they can copy and paste themselves within a genome. Traditionally TEs have been considered to be a ‘junk’ part of the genome, selfishly proliferating in an arms race with the host genome that is trying to keep TEs in check. As a defense, the host genome is usually restricting TEs from entering functionally important regions. But in the green anole even the Hox gene clusters, developmental control regions of the genome that are usually kept neat and tidy, got invaded by these TEs.

Even junk can become valuable in a different context. Indeed, there is circumstantial evidence that TEs can contribute to diversification and adaptation. For example, genomic incompatibilities arising from TE insertions have therefore been suggested to promote reproductive isolation. In other words, proliferation of TEs should be positively associated with speciation. Furthermore, some evolutionary innovations, like the mammalian placenta, appear to involve co-option of TEs for gene regulation.

Does the odd feature of the green anole genome indicate that something interesting is going on with TEs also in the evolutionary history of Anolis lizards? My study published in the Proceedings of the Royal Society of London B is a first attempt to take a closer look.

To this end, I compared the DNA sequences of Hox gene clusters of 30 lizard and snake species, including 20 Anolis species. I reconstructed the history of TE invasions of Anolis lizards and linked this to patterns of diversification across the phylogeny. The results revealed that there was a burst of TE activity in the lineage leading to extant Anolis. It did not stop there – TEs have continued to accumulate during speciation events, such that extant Anolis whose evolutionary history is characterized by many speciation events also have accumulated more TEs than lineages with relatively fewer speciation events. This finding supports the hypothesis that proliferation of TEs contributes to reproductive isolation, but what is cause and what is consequence remains to be seen.

Could TE activity also have contributed to the morphological differences that characterize Anolis ecomorphs? Well, I did not find evidence for this as yet, but this hypothesis is much more difficult to test since we need to learn more about developmental genetics to know where in the genome we should look. Nevertheless, I think this study shows that we can begin to unravel the genomics of adaptive radiation of these wonderful lizards!

 

Nathalie Feiner. 2016. Accumulation of transposable elements in Hox gene clusters during adaptive radiation of Anolis lizards. Proc. R. Soc. B. DOI: 10.1098/rspb.2016.1555.