Anolis lizards are emerging as a new squamate model organism for studying limb development. For more general information, please see the Anolis Wikipedia page. These lizards are incredibly diverse and the Anolis genera comprises of over 400 species (Sanger, 2008), with over 150 Anolis species found in the Caribbean alone as a result of adaptive radiation. Currently, very little is known about regulatory interactions that determine limb patterning and development in squamates and have only been studied extensively in mice and chickens. In Anolis lizards, the length of the hindlimb exhibits more variation than the length of the forelimb, suggesting regulatory alterations are involved. Through application of micromass culture system to Anolis lizards, a better understanding has been gained of conserved limb-type specific squamate genes such as Tbx4, Tbx5, Pitx1, HoxC10, and Hoxc11 in embryonic development. Other than investigating these genes, this paper sought to study the effects of Pitx1, an essential transcription factor found to be involved in hind limb regulation in chicks and mice, in this novel lizard model organism.
Why use Anolis as model organism?
Surprising, there is currently very little known about the development of non-avian reptiles (Sanger, 2008). There is an enormous amount of species diversity of Anolis lizards that exhibit a substantial amount of variation when it comes to limb morphology and size. This is a result of evolution of many different ecomorphs that have adapted to the various environmental niches in the Greater Antilles. Due to the extraordinary evolutionary history and the recognition of the need and importance of a reptile model organism, Anolis carolinensis was the first squamate species to have its entire genome sequenced. This allows for new methods for studies in developmental regulation and gene expression and decreases the reliance on traditional models (Sanger, 2008).
Many experiments were performed with three main Anolis species: A. carolinensis, A. sagrei, and A. ophiolepsis. Methods used included whole-mount mRNA in situ hybridization, limb micromass cultures, alcrian blue staining, transfection of Anolis cells, quantitative RT-PCR, and comparative genomics.
Anolis Staging and Development
-In the figure shown below from the TJ Sanger et al paper, the development of Anolis sangrei can be separated in various development stages. This experiments done by Sundgae Park et al focuses on forelimb and highlimb development, which can be seen on the lizard and magnified in the inserts in certain panels.
-Based on mouse global expression studies in mice, Tbx5 and Tbx4 are known to be forelimb specific genes while Pitx1, HoxC10, and HoxC11 are expression at higher levels in the hind limb. The limb-type specificity of these five transcription factors is highly conserved in vertebrates. After performing a whole-mount in situ hybridization for tbx4, tbx5, hoxc10, and hoxc11 in A. sagrei embryos, it was found that the localization was similar to patterns observed in other tetrapods. As seen in Fig 2A, tbx5 expression is limited to forelimb while the posterior (hind limb and tail) localization of tbx4, hoxc10, and hoxc11 is shown in Figure 2B-2D.
-To make the limb micromass cultures, forelimbs and hindlimbs were taken separately from A. sangrei, A. carolinensis, and A. ophiolepsis embryos at around stage 6 and stage 7 and disaggregated into a single cell suspension. These cultures were used to study chondrogenesis, the development of cartilage, by staining with Alcian blue, a cartilage specific dye. Compared to chick embryos, which begin to exhibit chondrogenesis after 3 days, it was found that Anolis cartilage nodules did not begin to develop until about 16 days, indicating slower development. Alcian blue-stained cartilage nodules can be visualized in Fig. 3.
-To assess whether expression of these limb identity genes were maintained in the limb micromass culture, real-time qRT-PCR was used to analyze the transcript levels. It was found that tbx4 and pitx1 expression was high in the hindimb micromass while remaining low in the forelimb micromass (Fig 3A, B). Also, expression of forelimb genes, such as tbx5, was maintained in the forelimb micromass while essentially remaining off in the hindlimb micromass. The stability and maintenance of these limb-specific gene expression in their respective micromass cultures indicates that the regulatory interactions that are required to control the expression of these genes is active in these cell culture.
-In mice, it was found that the transcription factor, Pitx1, is essential for regulating hindlimb morphology. Pitx1 knockouts tend to have small hindlimbs, loss of many hindlimb-specific features, and a reduction in Tbx4 expression. Gain-of-function experiments also showed that ectopic expression of Pitx1 shifted forelimb morphology to more closely resemble the hindlimb. Pitx1 can also induce Tbx4, Hoxc10, and HoxC11 expression in chick forelimbs and Tbx4 and HoxC10 expression in mice forelimbs. In previous genomic analyses in mice, it has been there are multiple pitx1 binding regions near Tbx4 and a particularly strongly marked region between HoxC10 and HoxC11. Among mammals, these binding-sites are well conserved. However, when looking at comparative sites in the A. carolinensis genome, it is found that many of these sites are not conserved.
-Even though these Pitx1 sites are not conserved, the relationship between pitx1 and hoxc11 can still be studied by nucleofecting Anolis forelimb cells with an Anolis pitx1 expression construction before it is made into a micromass culture. Real-time PCR was then able to demonstrate that ectopic expression of pitx1 very strongly induced the expression of hoxc11. This finding indicates that pitx1 does play a regulatory role in controlling hoxc11 and may act through binding sites not conserved between mammals and reptiles.
-Among different species of Anolis lizards, specific forelimb-specifying and hindlimb-specifying genes are conserved, indicating a similar limb development regulatory pattern in tetrapods.
-Limb micromass cultures proved to be a viable technique to study gene expression levels and regulatory interactions in squamates (especially Anolis).
–Pitx1, a transcription factor, was shown to induce strong expression of hoxc11, which dictates hindlimb morphology and patterning in embryonic Anolis development.
Since Anolis is currently emerging as a new squamate model organisms, novel techniques and methods still need to be explored and adapted from other more established model organisms in order to effectively study limb patterning and functional molecular analyses. Currently, there is also no effective way to introduce transgenes and make targeted alteration in vivo in the Anolis lizard, which is something that will have to be explored in future studies. Another challenge in studying patterning in development has to do with unusual female anole biology. Females can store sperm in their bodies for several months, which can make controlling mating times challenging. Also, by the time an Anolis egg is laid, the embryonic development is already at the early limb budding stages. Even so, new techniques are being developed for studying these promising model reptiles that vastly widen our currently limited understanding of squamate developmental regulation and patterning.
 Park S, Infante CR, Rivera‐Davila LC, Menke DB. 2013. Conserved
regulation of hoxc11 by pitx1 in Anolis lizards. J. Exp. Zool. (Mol. Dev. Evol.) 9999:1–10.
 Sanger, T. J., Losos, J. B. and Gibson-Brown, J. J. (2008), A developmental staging series for the lizard genus Anolis: A new system for the integration of evolution, development, and ecology. J. Morphol., 269: 129–137. doi: 10.1002/jmor.10563
 WORDLEY, CLAIRE, JON SLATE, and JESSICA STAPLEY. “Mining Online Genomic Resources In Anolis Carolinensis Facilitates Rapid And Inexpensive Development Of Cross-Species Microsatellite Markers For The Anolis Lizard Genus.” Molecular Ecology Resources 11.1 (2011): 126-133. Academic Search Complete. Web. 3 Apr. 2014.
 “Anolis”. Wikipedia. http://en.wikipedia.org/wiki/Anolis
Image 1: Anolis Lizard, photo credit to Jonathan Losos. http://ucsdnews.ucsd.edu/pressrelease/science_detective_investigates_lizards_and_evolution_at_scripps_lecture
Image 2: A. carolinensis, photo credit to Budweezer http://www.dpchallenge.com/image.php?IMAGE_ID=18556
A. sagrei, photo credit to Neil Losin, http://www.terradaily.com/reports/Castaway_Lizards_Offer_New_Look_at_Evolutionary_Processes_999.html
A. ophiolepsis, photo credit to Reptile Database, http://reptile-database.reptarium.cz/species?genus=Anolis&species=ophiolepis
Image 3: A. carolinensis 18+ embryo, photo credit to Catherine May, http://devoasu.blogspot.com/2013/04/godzilla-our-anole-lizard-embryo.html
GIF 1: A. sangei animation. GIPHY. http://giphy.com/gifs/qvxeCZ2Fh0rrq