Author: Thomas Sanger Page 1 of 6

Examples of embryos with normal and abnormal craniofacial development

Embryos are not just little organisms encapsulated within their eggs. Embryonic development is dynamic; the embryo transitions from one to a few undifferentiated cells to a stage where the various parts like arms, legs, and faces become apparent to a form that resembles the species that will eventually emerge. A panoply of signaling events and rapid rates of cell division are all tightly choreographed to make sure that development proceeds in a predictable, species-specific fashion.

But this dynamism of development also makes the embryo susceptible to environmental perturbation. Heat, chemical exposure, and pathogens can all disrupt normal embryonic development, sending the embryo down paths that may lead to fatality or reduced fitness. In our recently published study, my colleagues, students, and I demonstrate that heat stress, paralleling what will likely be experienced during the 21st century, can induce structural malformations to the brain and face of lizard embryos.

In 2014 and 2015 I was a post-doc in the Cohn Lab at the University of Florida. At the time, I had been dissecting and observing anole embryos for approximately 14 years. Throughout those 14 years I had observed only a handful of malformed embryos, maybe on the order of 10-20 embryos after collecting thousands of embryos from numerous anole species. Yet, in the summers of 2014 and 2015, while working alongside then graduate student Bonnie Kircher, I collected more malformed Anolis sagrei embryos than I had in all of my previous years. I certainly didn’t realize it in the moment as malformed embryos were still relatively rare compared to the total number of embryos we were collecting. But, by the end of my time in Florida the number jumped out at me. Sometimes the rate of development seemed to depart from the normal sequence of development. Other times the embryo was clearly not well and would likely not survive to hatching. Even as other projects accelerated, my interest in these malformed embryos remained piqued.  When I began my faculty position Loyola University Chicago, I decided to invest the lab’s time and resources into determining whether this pattern was real or just a chance observation.

In spite of other options­–unique genetic mutation running rampant in Gainesville populations of brown anoles seems highly unlikely–I decided to investigate the effects of heat stress on embryonic development. The effects of global warming had been widely discussed as a threat to ecotherm populations and anoles have been at the center of both field and lab observations since the outset. A number of studies have also shown reduced hatching success from lizards incubated at relatively high temperatures. Relating this back to my observations in Florida, it also occurred to me that Bonnie and I used a different collection strategy for our breeding colony those years; we would regularly replenish or add to the colony from field-caught lizards throughout the summer. This raised the possibility that the embryos developing within the gravid females were exposed to the environmental heat stress before being deposited into our waiting hands. Luckily, brown anoles are prolific egg producers, providing my lab with the ability to test whether heat stress induces embryonic malformations under different incubation regimes.

Embryos incubated under conditions reflecting those observed in shady nest sites exhibit malformations in only one to two embryos out of every 100 live embryos. These nest sites tend to be relatively stable in temperature, rarely rising about 30 degrees Celsius. However, embryonic heat stress induces malformations in 10-30% of embryos exposed to incubation conditions that parallel nest sites that would be located in sunny locations. These putative nest sites reach temperatures above 36 degrees Celsius, the critical thermal temperature for the embryos, for up to eight hours per days. The malformations we observed were not evenly spread across the body. Instead, we saw the greatest concentration in the brain and face of the developing lizards. Most malformations included a change in facial proportion, from subtle changes in facial length to pronounced brachycephaly and/or clefting. In one case, the entire face and forebrain were ablated in the embryo! When it comes to the induction of structural malformations, the most sensitive period of development is around oviposition, including the time that the egg is still within the female. Although we do not yet know how many of these embryos would successfully hatch, our experiments do raise concerns about the long-term impacts of global warming on ectotherm development.

Examples of extreme, but rare malformations observed in eggs reared at elevated temperatures.

The consistent pattern of thermal-induced neural and facial anomalies made us think that there may be a common underlying cause of these changes, leading us to create and test a new model of embryonic thermal stress. Based on our understanding of amniote craniofacial development, we predicted that disruption to Hedgehog signaling, one of the earliest signaling pathways needed for facial development, could create the full spectrum of observed malformations. After measuring processes up and downstream of Hedgehog signaling (e.g., cell death and signaling within the presumptive facial cells respectively), it does, in fact, appear that Hedgehog signaling is disrupted in the face of embryos experiencing thermal stress. Depending on the degree of response by a particular embryo, everything from normal to extremely malformed embryo could be induced. At this time, it appears that our model holds for brown anoles and may be applicable in species far beyond anoles and lizards.

In our proposed model, heat stress affects Hedgehog signaling, causing a disruption to normal facial morphogenesis.

There remains much to learn about normal and abnormal facial development in lizards. We do not yet know what other signaling pathways are equally disrupted during thermal stress or whether there are endogenous buffering mechanisms that help to maintain normal development in the face of external stress. Perhaps one of the most important discussions that needs to occur is how we study rare events. These events could be uncommon, but extreme heat events that exceed the “normal” conditions typically observed in the wild. These are increasing in regularity and may have significant impacts on ectotherms later in the 21^st century. Alternatively, the rare events could be the emergence of malformed embryos which occur in only a fraction of individuals, even when the average phenotype is not dramatically altered. For species such as the brown anole, this may not be alarming. But for species with relatively few viable hatchings each season, embryonic heat stress could have dramatic impacts on their long-term viability. These developmental perspectives  are needed to fully understand the ways that global change will affect the lives and longevity of lizards and other ectotherms.

Embryos are impacted by a range of challenges associated with anthropogenic change. See more in Sanger 2021, Integrative developmental biology in the age of anthropogenic change

The adhesive toe pads of anoles (above) and geckos give them these species “super powers.” (at least when compared to other lizards)

Herps make amazing wildlife ambassadors. Many small children read about them or see them in books, but rarely have first-hand contact with them. During a recent outreach event in the northern suburbs of Chicago, I met first and second graders that had never seen a live snake or lizard! When one came out of a bag, they lit up like they had just caught Santa emerging from the chimney on Christmas morning.

Based on that introduction, it is easy to conclude that I get a lot of enjoyment out of introducing the world of herps to small kids. I enjoy engaging with kids in ways that not only introduce them to the animals, but also in ways that could motivate them to pursue science throughout their education. Several years ago, I described an exercise aimed at getting kids to think about the ways that dewlaps are used during animal communication. This past weekend I tested a new exercise as an outreach activity for GEMS, Girls Empowered in Math and Science, which was hosted at Niles West High School in Skokie, IL. GEMS is organized to encourage fifth through seventh grade girls to pursue careers in STEM. This year’s event had about 135 girls registered across that age range! This exercise is meant to teach kids about the biology of the adhesive toe pad and the bioinspired engineering that led to the development of Geckskin.

Some of the tapes the students could pick from

The first objective of this exercise is to get the students thinking about how a lizard can climb rugose tree bark using their claws. Easy right? But what about clinging to a waxy leaf, hanging upside down from a ceiling, or traveling 80mph down a highway where their claws can’t be used? Compared to other lizards, this is their “super power.” After explaining the microanatomy of the toe pads to the class–the pad, setae, and spatula–I gave them a challenge. With a collection of every type of tape available at Home Depot (Duct, Scotch, painters, masking, packing, etc.), I challenged the students to choose one that could outperform a lizard’s toe pad. The students were broken into small groups, each taking a piece of wood and a small piece of Plexiglas (tree bark could also work but might not be reusable across many groups). Each group selects a type of tape that is then run through a battery of challenges during which time I provide the biological commentary:

• A lizard runs around all forest all day. Its toe pads must be reused over and over again without fail. How many times can your tape be reused before it is no longer sticky?
• A lizard must run on different surfaces–leaves, tree bark, rocks. How does the tape perform on different surfaces?
• A lizard doesn’t leave tracks where it walks. Does your tape leave a residue?
• As the lizard walks, does its foot stick to the surface it is walking on as it tries to take a step? How easy is the toe removed compared to your tape?
• Some days it will rain. Do lizards fall out of the trees when it rains? No. Now, what happens when your tape gets wet? (a moist sponge is provided)
• Lizard toes also get dirty. What happens if the tape gets dirty? Feel free to try to brush of as much dirt as possible after putting the tape in. (a dish of coconut bark  was provided)

As the students are working through these challenges, I pull out a Lepidodactylus gecko and a knight anole from behind the table at the front of the room (they are usually clinging to the side of their cage which helps with the wow factor) and clicked through slides of lizards seeming to overcome each of these obstacles. A few smiles overcame the students as they realized that they had been bested by a tiny reptile.

Good natured volunteers demonstrating Geckskin technology.

After demonstrating that nature has come up with an amazing solution for adhesion, I pose a question to the kids, “What if we apply what we learned from these lizards to develop new products that we could use in everyday life?” Here I introduce them to the ingenuity of Geckskin (developed in part by ex -officio anole biologist Duncan Irschick). I must briefly digress to sincerely thank Phelsuma/Geckskin CEO Rana Gupta for providing Geckskin samples that I could demonstrate for these kids. As he says in the video, they “feel magical.” They are not tacky like tape, but stick to a variety of surfaces like a dream. There are some useful videos of Rana demonstrating the Geckskin products on the company’s website. The climax of my demonstration was pressing a 2X2 Geckskin Griphanger against a board as several girls held either side. Then, suspended by a piece of climbing cordellette, they hung a 5lb weight on this pad with my toes directly below. I bet the girls to use any of their tapes to do the same thing, but didn’t have any takers. (This could easily be another challenge offered to the students during a longer presentation.)

At the end of this demonstration the girls had the chance to meet Bob the red foot tortoise and Spot the ball python. (Anoles and geckos don’t make the best hands-on animals for presentations.) The presentation can readily go on without the use of live animals, but it seems to help bring the kids out of their shells and leads to a more memorable experience for them. I hope that others can use this write-up for motivation for some exercises they can employ during their next outreach activity. I am off to see some second graders in two weeks.
* I always appreciate feedback on these exercises as well. Fire away! 

Bob and Spot are always big hits.

The first plate from the Sanger et al. (2008) Anolis staging series.

Long time readers of this blog will likely remember the many posts I’ve made trumpeting the utility of anoles for integrative analysis of anatomical diversity, studies that gain perspective from disparate biological fields. The community has come a long way since we published the first staging series of anole embryology only nine years ago. To some this may be old news, but I still find this pace exciting and personally motivating. Decades of ecological and evolutionary studies have created a strong foundation upon which to build new insights about the molecular and developmental underpinnings of anatomical diversity. My lab’s questions boil down to trying to shed light on the developmental origins of adaptive anatomical variation. Otherwise stated, where did the requisite phenotypic variation arise from during the adaptive radiation of anoles. The inherently comparative nature of these studies led me to use anoles as a “model clade,” a group of species that provides the capacity to obtain evolutionary insights the way that “model species” have provided pure developmental biologists and geneticists the power to deduce insights in their areas.

One of the highest hurdles to the progression of Anolis as a model system has been long-term access to living embryos. Although comparative biology is a powerful approach for evolutionary studies, one of the hallmark lessons of modern Evo-devo is the need to experimentally validate the candidate molecular changes associated with anatomical evolution. If I hypothesize that Gene X underlies some phenotypic difference between two species, I must 1) show that it is expressed at the time when the difference arises and 2) somehow tweak the expression of Gene X at that time and in that tissue to show that the changes parallel those observed in nature. To do this you must have access to an embryo in culture, unencumbered from its opaque shell.

Over the past several years several people have been working on ways to gain access to lizard embryos. The first report of a culturing method was by Tschopp et al., who used lentivirus to trace cell migration into the genitalia and limbs. I have not personally been able to consistently replicate those conditions, especially for later embryos. Bonnie Kircher and I, however, recently published two relatively “simple” culturing protocols as part of a new book, Avian and Reptilian Developmental Biology. One of the challenges of earlier culturing attempts was bacterial and fungal growth. As a first step to combatting these invaders, we developed a protocol to sterilize the eggs, soaking the eggs in a weak bleach solution (yes, a literal bleach solution). From there we were off and running.

The first method we describe, following from advice from Raul Diaz, has worked on eggs a few days old to those that are nearly half way through their incubation period. Using a fine pair of scissors, we separate the outer opaque lays of the shell from the inner membranes that surround the embryo and yolk. This bag-of-embryo is then transferred to a small culture dish with a nutrient rich media and drugs to further combat bacterial and fungal contamination. This culturing system has worked well for up to ten days, roughly from the time the limbs are developing digits to the time that the limbs have visible scales on them. (Check out the video!) In principle, this method will allow better access to the embryo for viral injection or the application of small molecule inhibitors that disrupt particular signaling pathways.

Be warned, the second method is a little more Frankensteinian. Because the membranes cover the embryo, visualizing development remains difficult. To circumvent this problem, we developed a protocol where we explant a piece of anole tissue, such as the developing

A developing A. sagrei foot explanting onto a chicken embryo

limb, to a chicken embryo. Both anole and chicken seem to fare well at 33 degrees Celsius, below the standard incubation conditions of the chicken and above that of our anoles. Development appears to proceed normally in the explanted tissue, just as it does would in an embryo within its own shell. These experiments still have a relatively low success rate, but when the explant takes, it works well. To better visualize the tissue for imaging we also stained the tissue with a vital fluorescent dye before the transfer, giving the tissue a wonderful Halloween feel.

The work is far from over. These culturing protocols are just the first step and will not work for all applications. More technically challenging steps especially await those that want to manipulate the anole genome or target distinct patterns of gene expression. This is only the start of what’s to come. For more details about these protocols you can download the chapter here.

One of the long-faced members of the carolinensis clade, Anolis brunneus.

Several years ago, I wrote a series of papers and blog posts about the diversity of anole head shape and its developmental origins. My colleagues and I touched on disparate topics such as whether the head differences among species are similar to post cranial ecomorphology, whether the patterns of cranial modularity are conserved across anoles, and the developmental bases of sexual dimorphism in skull shape.

Since starting my own lab at Loyola University in Chicago last year, I am revisiting these projects on skull evolution. Like in much of science, I have found that my early forays into this area created more questions than answers. Understanding the diversity of skull shape among anoles and other iguanid lizards will be one of the first focal areas of my new lab. We are currently mining museum collections to understand how the variation in anole skulls compares to iguanid lizards more broadly. The ultimate goal, however, is to return to questions about the developmental origins of this variation. Just how many different ways has development been modified to generate all the variation we observe in adult anatomy? We do not yet know.

This is where my attention turns to you. To thoroughly flesh out the developmental origins of anatomical diversity, I must have robust sampling of species across the iguanid phylogeny. I am asking the community to please think of me and my students if you have extra breeding animals, eggs, or hatchlings of any species of anole or another iguanid lizard. I am happy to help offset the cost of the animals or collaborate in a mutually beneficial manner.

One of the most exciting species that have recently had the fortune to work with is Anolis hendersoni. For its body size, this species has one of the longest faces of all anoles. In this case I was contacted by the owner of Backwater Reptiles who had several A. hendersoni adults that we are hoping to get eggs from over the next year at Loyola. The folks at Backwater have been great to discuss “exotic” anoles with as they occasionally receive species like A. woodi, A. cybotes, and Chamaeleolis, all of which could be great additions to my project. This is just one example of how I am trying to broaden the sampling for this project. I ask you, the broader anole community, to help me increase my sampling further. I sincerely thank anyone that has leads for me in advance.

Anolis lizards have established their place in the annals of college textbooks. There are also a growing number of resources available for elementary and high school teachers to bring the biology of anoles into their classrooms as well. The Howard Hughes Medical Institute (in collaboration with Jonathan Losos) developed several online modules around anoles: one on the diversity of Anolis lizards, another on speciation, and a virtual lab integrating those topics. Michele Johnson also has several classroom exercises on here website, LizardsandFriends.org, some of which have been discussed on AA previously (here and here). I am writing today to share another exercise with our readers that was a recent success with a group of young scientists-to-be.

Dewlapping fifth graders at GEMS 2016

I recently introduced Anolis lizards to a group of fifth and sixth grade students at a conference aimed at getting young girls interested in the STEM professions. With around 130 girls learning about topics ranging from gemstones, programming, seeds, and urban wildlife the event was a undeniable success. My session introduced the diversity of topics that our community addresses with Anolis lizards. After explaining to students how they could figure out what lizards are anoles at the local pet stores (dewlaps and toepads), I used anoles to demonstrate how animals can communicate without talking. My exercise amounts to a game of charades where the students have a dewlap, a display-action-pattern, and a key representing four species from Puerto Rico (thanks to Travis Ingram). The display patterns are not as complex as real dewlap displays, but were made to allow the students to easily act them out and distinguish between the patterns and it worked great. The kids thought this was a lot of fun and it gave me the opportunity to pepper the discussion with additional comments about animal communication. I originally designed the exercise for fourth through seventh graders, but a curious three-year-old played along just as well during one session. I would be happy if other people used this exercise for their own outreach activities. It can be downloaded here.

In closing I will add that the students were impressed by the brown anole I brought with me. I imagine I would have left a more lasting impression if I brought a knight anole. Things to remember for next year.

Over the last decade the term “model species” has taken on new meaning. Species that were once the building blocks for distinct disciplines have taken on new importance in comparative evolutionary studies that integrate perspectives across biological disciplines. Nowhere is this better illustrated than with Anolis lizards. For decades anoles were a workhorse of ecologists and evolutionary biologists, but have, more recently, been embraced by developmental biologists, genomicists, physiologists, and neurobiologists among others. This disciplinary expansion is perhaps most evident with the rapid increase of penis/hemipenis research that has been published using anoles within the year.

For many herpetologists, including those focused on anoles, the hemipenis is ripe with taxonomic characters, easily allowing for the identification of new species. Julia Klaczko and colleagues recently demonstrated that features of the hemipenis are some of the most rapidly evolving characters among anoles, a group already well known for its rapid anatomical evolution. Independent from these taxon-specific interests, developmental biologists became interested in the anole hemipenis because of its unique anatomy compared to other amniotes. Marissa Gredler and members of the Cohn Lab used anoles as one of their reptilian models of external genital development in what is arguably the broadest embryological survey of reptilian phallus development to date. In parallel, Patrick Tschopp and colleagues probed the cellular and molecular regulation of early phallus development among anoles, snakes, chickens and mice, demonstrating that the hemiphalluses (hemipenes and hemiclitores) and hindlimbs of squamates utilize similar molecular networks at the earliest embryonic stages of morphogenesis. Now, just within the last month, two more papers have used anoles in studies of phallus evolution and development, one using cutting-edge molecular techniques to better understand the relationship between limbs and external genitalia and the other addressing the fundamental question of external genital homology using museum specimens that are more than 100 years old.

Before getting into the findings of this new research, lets lay out some of the dirty details of penis evolution. First and foremost, the penises of amniotes are extremely diverse. Squamates have paired lateral phalluses while other clades have a single midline phallus. Each of the amniote lineages uses hydrostatic pressure to achieve an erection, yet accomplish this using different bodily fluids (lymph or blood). In mammals sperm is transferred to the female through a closed urethral tube, but other groups utilize an open channel. Most birds (97%) and the tuatara, have absent or highly reduced phalluses and reproduce with the famed “cloacal kiss.” These large differences in anatomy should not overshadow the spines, bulges, corkscrews, and dramatic differences in size that give species their distinctive features. But with such striking variation, we are forced to wonder how many times the penis evolved. Perhaps the amniote ancestor possessed an intromittent phallus capable to transferring sperm to the female that later diversified in each lineage independently. Or, perhaps the last common amniote ancestor used cloacal apposition to foster internal fertilization and unique phallus morphologies evolved independently at the origin of each lineage. Because adult anatomy provides few clues to phallus homology, Thom Sanger (me), Marissa Gredler, and Marty Cohn looked towards the embryo for help.

Table 1 from Sanger et al. 2015 summarizing phallus variation in amniotes

The tuatara, a species lacking an adult phallus, has presented a problem in attempts to reconstruct the last common ancestor of amniotes because it raises the distinct possibility that reproduction through cloacal apposition was the ancestral condition.

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Vimeo user “Ectopher” posted a beautiful video of beating embryonic anole hearts. You can even see the blood flow through the branchial arches at one point. Check it out here.

Size dimorphism among Anolis habitat specialists from Butler et al. 2003

In recent years the Anolis community has shown greater interest in understanding the developmental bases of anole diversity. As these data accumulate, we can start to synthetically understand the ecological (ultimate) and developmental (proximate) factors that regulated different aspects of anatomical diversification in anoles. Differences between males and females (i.e., sexual dimorphism) is one area that has received a considerable amount of attention among anole biologists interested in obtaining this integrative understanding of anatomical diversity. Over the last decade a number of papers have been published examining the evolutionary patterns of anatomical differences, ecological correlations, and the developmental/physiological processes underlying dimorphism in anoles.

Variation in body proportion (i.e., shape dimorphism) among Anolis habitat specialists from Butler et al 2003.

Compared to other anoles, crown-giant anoles have relatively low levels of size dimorphism, but vary greatly in body proportion. Males and females tend to vary in relative limb length, head proportions, and in the dimensions of their adhesive toe pads. In a recently published paper Vanhooydonck et al. examine the timing of divergence between male and female A. baracoae. They raised 23 individuals (9 males, 14 females) for 3.5 years, repeatedly measuring three anatomical traits and bite force 11 times over this time period. The authors found that bite force and dewlap size exhibit significant differences in growth between the sexes. Their analysis further suggests that these traits diverge at different times during ontogeny – bite force diverges during juvenile growth while dewlap size does not diverge until sexual maturity – illustrating the independent regulation of dimorphic traits during development. Head length and hindlimb length did not appear to have sexual differences, although it would be interesting to also perform a formal analysis of adult size and shape dimorphism on A. baracoae to see if this species has similar dimorphic trends compared to other crown-giant anoles.

The developmental bases of size dimorphism in A. sagrei from Cox et al. 2009

These results are consistent with other studies that show a mosaic pattern of male and female phenotypic divergence. Using a longitudinal study of male and female A. sagrei, Cox et al. 2009 showed us that body size dimorphism in this species begins early in juvenile life, only three weeks after hatching. Sanger et al. showed that dimorphism in facial length can emerge through two distinct developmental strategies, one early in ontogeny and one at the time of sexual maturity that appears to be clade-specific. Additional research on that compares across traits and among species will further elucidate the number of ways that dimorphism can arise in anoles. Further work that overlays an ecological perspective onto these patterns will also allow a more thorough understanding of whether natural or sexual selection is the primary driver of these differences in timing. As this future work progresses new insights into the evolutionary processes of anatomical diversification are sure to follow.

Bonnie Kircher found this image inside the cover of Florida’s Fabulous Reptiles and Amphibians. Pay close attention to the last lines of the quotation and you will realize that this is a most well-placed anole photograph. 

We once discussed what the appropriate term would be for a group of anoles, but what about a family of anoles? Is there such a thing? If not, there is now.

Just in case stick figures aren’t an appropriate representation for your family group, look no further than the green anole window clings made personally by herp. lover Andrew. These aren’t yet available on the web, but Andrew may knock a few out for you if you write him directly. I did and now I have one of the most stylish minivans in Florida!