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Read all about it in the latest post from Daffodil’s Photo Blog.

After a bit of a hiatus, Daffodil’s Photo Blog is back with its splendid anole photos and natural history tidbits. Check out the recent offering.

From the pages of Nova.

BY KATHERINE J. WU TUESDAY, AUGUST 27, 2019 

Compared to mammals, reptiles have a weird way of reproducing—and in the spring of 2017, that put Ashley Rasys in something of a pickle.

For months, the University of Georgia biologist was struggling to come up with a way to tinker with the genes of the brown anole (Anolis sagrei), a petite, pointy-faced lizard native to Cuba and the Bahamas.

The reptile had initially caught Rasys’ eye because of, well, its eyes. People with albinism often have poor vision due to problems with their foveae, the dense pits of cells at the back of the eyes that confer visual acuity. While foveae are lacking in most mammals, they’re present in lizards—making them intriguing candidates for studying the genes that impact foveal function.

There was just one problem: Reptiles aren’t easy to genetically manipulate. In other common laboratory animals, like mice and zebrafish, a tool called CRISPR has made DNA editing a breeze. The procedure typically involves injecting freshly fertilized eggs with gene-editing machinery, creating a change that would propagate when the cell divided.

But a few quirks ruled out that particular strategy in these lizards. Female anoles can store sperm for many months before fertilizing their eggs internally, making it difficult to time the introduction of the CRISPR cocktail. Anole fertilization also cues the formation of a soft, delicate eggshell that’s hard to penetrate without damaging the embryo.

That meant Rasys and her advisor, Doug Menke, had to get creative. So they decided to shift the injection back a developmental step, targeting eggs still maturing in the females’ ovaries. “At this point, they’re just hanging out in the lizard, waiting to be fertilized,” Rasys says.

The procedure took more than a year to perfect. But in the fall of 2018, Rasys, Menke, and the rest of their team hatched the world’s first gene-edited non-avian reptile: a red-eyed albino anole with near-transparent skin. According to the team’s study, published today in the journal Cell Reports, its birth marks a breakthrough for the field of developmental genetics, and hints that similar experiments may be possible in some of the other 10,000-plus species of non-avian reptiles that scuttle the Earth.

“This technology is really important and exciting,” says Martha Muñoz, an evolutionary biologist and anole researcher at Yale University who was not involved in the study. “This really opens up the door for other groups to think outside of traditional model organisms [like mice and zebrafish]…the sky’s the limit.”

With albinism in mind, Rasys and her colleagues set out to mutate the anoles’ tyrosinase gene, which governs pigmentation and has been linked to foveal function in humans. Manipulating this gene, Rasys explains, also made for an easy marker of success: If the procedure ended up generating albino anoles down the line, they’d be pretty tough to miss.

After rounding up 21 female brown anoles from the wilds of Orlando, Florida, the researchers gently anesthetized the lizards and opened them up. In anoles, the ovaries are transparent, making it easy to eyeball their contents “like a train of developing eggs,” Menke says.

The team selected 146 of these growing eggs and injected them with the classic CRISPR recipe: a pair of molecular scissors and a series of DNA-binding “guides” that would show them where to cut—in this case, the tyrosinase gene.

The researchers then had to wait another three months or so for the females to fertilize and lay the eggs. And even when this generation hatched, they thought there’d likely be more work to do, Rasys says. Since the CRISPR concoction had been delivered to eggs that were later fertilized by unaltered sperm, the offspring were expected to be hybrids—half edited, half unedited. These lizards then would need to be bred further to yield albinos, which must inherit the mutation from both parents for the trait to manifest.

But as Rasys watched her first clutch of gene-edited eggs grow, she noticed something strange. About a week before they were due to hatch, most of the embryos had darkened from pink to gray—an indication that they’d started producing pigment. A handful, however, retained their initial pallor, even as they continued to swell in size.

A few days later, Rasys arrived at the lab to find a newly-hatched, inch-long albino, stretching its ghostly pink legs. “It was so exciting to see it,” she recalls. “I thought, ‘It’s so cute.’”

In total, four out of the team’s 146 CRISPR-injected embryos were obvious albinos, surprising the entire team. There’s no way to know exactly what happened, but Menke’s leading theory is that the CRISPR components remained active in some of the eggs long enough to work their magic on both the maternal and paternal copies of the tyrosinasegene.

Genetic screening revealed another five embryos to be the half-edited hybrids the team had initially expected. And when the researchers partnered one of these CRISPR mutts with an unmanipulated mate, the mutation was passed on to some of the pair’s offspring, suggesting the edited gene was heritable.

There’s still plenty of tinkering to do, Menke says. As they report in the study, the team’s gene-editing success rate was around 6 percent—a figure that pales in comparison to the near-perfect efficiency rates that have been reported in zebrafish and mice.

But just showing gene-editing is possible in this system is a big deal, says Ambika Kamath, a behavioral ecologist at the University of California, Berkeley who was not involved in the study. Albinism implications aside, anoles have long been studied by evolutionary biologists and ecologists. In their native Caribbean, the lizards have split into many lineages, but understanding this diversification “has primarily been a historical science…involving stitching together patterns that happened a long time ago,” Muñoz says. “By extending CRISPR to Anolis, we can now mechanistically test some [evolutionary] hypotheses.”

As more applications surface, however, “we don’t want to be releasing CRISPRed lizards into the wild willy-nilly,” Kamath says, without a better understanding of how these sorts of introductions would affect the population at large.

And it might be more than lizard lives at stake. Menke thinks the team’s technique is likely to work in a variety of reptiles, many of which share the anole’s mode of reproduction. There’s even the possibility, he says, that the method could be adapted for birds, which are cut from the same evolutionary cloth. Scientists have hatched CRISPant chicks in the past, but as in lizards, bird embryos are hard to pinpoint at the single-cell stage, making current editing procedures complex and laborious.

Carolyn Neuhaus, a bioethicist at the Hastings Center who was not involved in the study, cautions that as CRISPR continues to be debuted in more and more organisms, the how, when, and in whom of gene editing will need to remain transparent. Though many experiments—including the ones in this study—have the potential to advance science and human health, she says, technology like this shouldn’t be used in a new species “just because it’s there.”

“We rely on scientists to create accurate and reliable knowledge, and that’s a huge responsibility,” she says. “With the CRISPR craze…I just hope it happens as mindfully and carefully as possible.”

We’ve had previous posts on parasites of anoles (for example, here), but now a new paper in Herpetology Notes adds to the literature on this topic, reporting blowfly parasitism of Anolis parvauritus from northwest Ecuador.

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

When you hear about a spa, you visualize a relaxing place where soft skin peeling is commonly practiced. Even if the place is human-specific, skin peeling itself has been recorded in the “animal world,” and in a way that goes beyond your imagination.  

Indeed, some species of scincid and gekkonid lizards are known to lose a part of their skin during drastic regional integumentary loss (Bauer et al., 1989, 1992a, 1993; Bauer & Russell, 1992). Such loss is dependent on the bilayering of the dermis and the inherent weakness of the outer layers of the skin and is used as an antipredator strategy (such as the tail autotomy), particularly during the early stages of subjugation by the predator (Bauer et al., 1989, 1992, 1993; Bauer & Russell, 1992). Nonetheless, this practice has severe costs related to radiation exposure (particularly in diurnal species), osmoregulation and immunological integrity (Bauer & Russell, 1992).    

Back to the Caribbean island of Dominica the past month, I sampled populations of Anolis cristatellus. During the routine measurement process on a big male, I was surprised to see that the lizard lost his entire skin as soon as I touched him, the muscle being nearly apparent (Fig. 1). I measured more than 2.000 anoles, and this guy was the first one to lose his skin in my hands. Did I sample a very special individual? The response is probably “no.” Within the same day, this regional integumentary loss occurred in two adult males and one adult female A. cristatellus in total. To my knowledge, such a skin loss was not observed in anoles before. Could it be an underestimated antipredator strategy in Anolis?  If yes, could it be different on island vs mainland species as suggested Bauer & Russell (1992) in gekkonid lizards? The anole world has not finished to surprise us! 

References:

Bauer, A.M.; Russell, A.P.; Shadwick, R.E. 1989. Mechanical properties and morphological correlates of fragile skin in gekkonid lizards. J. Exp. Biol. 145(79-102)

Bauer, A.M.; Russell, A.P.; Shadwick, R.E. 1992. Skin mechanics and morphology in Sphaerodactylus roosevelti (Reptilia: Gekkonidae). Herpetologica. 48(124-133)

Bauer, A.M.; Russell, A.P. 1992. The evolutionary significance of regional integumentary loss in island geckos: a complement to caudal autotomy. 4(343-358).

Bauer, A.M.; Russell, A.P.; Shadwick, R.E. 1993. Skin mechanics and morphology of two species of Pachydactylus (Reptilia: Gekkonidae). S. Afr. Tydskr Dierk. 28(192-197)