Baby Anoles – Cute, Cuddly, and Easily Staged!

Check out this piece in the New Scientist, which picked up on our images of Anolis embryos and Thom’s awesome research!

Embryo of Anolis longitibialis, a trunk-ground anole from the Dominican Republic.

The readers of this blog do not need to be convinced that anoles are an amazing model system in evolutionary biology. New and exciting research often finds its way to the Anole Annals. Here we’ve learned about emerging trends in Anolis genomics, speciation, and comparative phylogenetics, to list just a few. In recent years, Anolis has also become a model system for developmental biology. For example, a recent study by Dr. Thom Sanger demonstrated that the diversity of limb dimensions among ecomorphs have evolved from similar developmental mechanisms.

This summer I worked a bit with Thom to learn how to stage Anolis embryos using his handy staging series as a guide. The goal of the project was to determine the stage at which female anoles laid eggs under two treatment conditions – a hot treatment (32°C) and a cold treatment (20°C). I had females from three populations of A. cybotes (55, 700, and 1400 meters in elevation), one population of A. shrevei (2450 m), and one population of A. longitibialis (100m). Unfortunately, I was unable to collect very many eggs despite letting the experiment run for six weeks. I did, however, manage to get several beautiful embryos, which I have imaged and staged. Here I’ll provide some pictures and give a few shorthand methods for staging Anolis embryos.

Figure 1. Stage 2 Anolis longitibialis embryo.

What first struck me from this experiment was the variation in embryonic stage at laying. Consider, for example, this Stage 2  Anolis longitibialis embryo that was laid in the hot room (Fig. 1). The two edges of the eye cup have not yet met, and so a choroid fissure is still visible, although it is fairly narrow. The tail bud, from which the tail will grow, still remains unsegmented and there are few somites, which are the tissue that will later become skin, skeletal muscle, and vertebrae. The mandibular processes, which contain the tissue that will later form the lower jaw, abut although they remain unfused.

Figure 2. Stage 5 Anolis longitibialis embryo.

You might also notice that the embryo in Figure 1 lacks limb buds. Those appear a little later in development. Limb bud condensations appear in Stage 3. Once limb buds appear, so do a whole suite of other characters, as well. For example, see this Stage 5 Anolis lonigitibalis embryo (Fig. 2). The maxillary process is visible in the embryo. The telencephalon and mesencephalon, which constitute the fore- and midbrain, respectively, are much more developed and the lobes are distinct. Moreover, the otic cup is fused. You’ll notice that the hindlimb bud is larger than the forelimb bud. In stage 4 embryos the size disparity between the fore- and hindlimb buds are less noticeable.

Figure 3. A. Stage 6 Anolis cybotes embryo. B. Stage 7 Anolis cybotes embryo.

An interesting transition happens between Stages 5 and 7. In Stage 6, in addition to lengthening the limb, the distal portion of bud widens to form a more paddle-like structure than in earlier stages (Fig. 3a). In Stage 7, the paddle widens further and medial digit condensations are now visible (Fig. 3b). Both the Stage 6 and the Stage 7 embryos are from A. cybotes eggs laid in the cold room.

Figure 4. A. Stage 8 Anolis cybotes embryo. B. Stage 9 Anolis cybotes embryo.

Between Stage 8 and Stage 9 the digits become much more developed (Fig. 4). During Stage 8 the digital condensations are much more clearly visible. Although the picture is slightly out of focus, one can clearly see that all the digits are much more developed with respect to Stage 7. Moreover, the interdigital webbing becomes thinner, although it does not yet regress. In Stage 9 (Fig. 4B), the digital webbing begins to regress, a process that continues through Stage 11. The two embryos above are from A. cybotes eggs laid in the cold room.

Because I was interested at developmental stage at laying, I did not see any embryos beyond Stage 9. However, development continues for several weeks after laying, often up to almost a month before anoles hatch. Thom mapped out a total of 19 stages, all the way from the earliest pre-limb bud stages to hatching, which you can read about in his paper. While I’ve listed a few characters that I found useful for staging, there are many more, such as tail development, somite number, development of the maxillary process, and others. If Anolis embryology and developmental biology interests you, then I suggest reading some of Thom’s papers to orient you with this fascinating field.

About marthamunoz

Martha is working on her PhD on the thermal ecology and evolution of anoles in the Losos Lab at Harvard University.
This entry was posted in Research Methods and tagged , , , . Bookmark the permalink.

9 Responses to Baby Anoles – Cute, Cuddly, and Easily Staged!

  1. polychrotid says:

    Wow – great images

  2. thsanger says:

    What an endorsement! Thanks. Sometime in the near future I will write a brief post about how to capture nice, high resolution photos of embryos. There are a few tricks that might be helpful for the community to be aware of.

  3. Pablo says:

    Please Thom write that post. I have a few embryos and i need to take some good photos. They are not anoles but i guess the procedure would be similar, no?

  4. marthamunoz says:

    Thanks so much! Thom taught me how to do it. It’s not difficult with the right microscope and software.

  5. thsanger says:

    Pablo, I learned these methods from someone working on bird development. While their use may be limited to relatively early to mid-stage embryos these simple methods can be easily applied to many species.

  6. Griff says:

    Looking at those cute little five-fingered “hands” in B. Stage 9, one could be forgiven for thinking it was a human embryo.

  7. Pingback: Not All Embryos are Created Equal |

  8. Dale Hoyt says:

    I hate to nit-pick about such beautiful pictures, but shouldn’t the label be “optic cup” and not “otic cup”? An otic cup would be located more posteriorly.

  9. marthamunoz says:

    Hi Dale,
    Yikes! Thanks for catching that. I’ll try to fix that promptly.

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s