Staging series page 1

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

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.

Thomas Sanger
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