One of the major experimental advances in recent decades has been the battery of methods capable of functionally validating hypotheses regarding the molecular networks that regulate biological processes. For biologists, these emerging methods allow us to move beyond descriptive and correlational studies to new dimensions where we can experimentally validate our observations. Until recently these technologies were, by and large, reserved for the most well developed laboratory model systems (e.g., mouse, chicken, zebrafish, Drosophila), systems that rarely have direct utility to ecologists and evolutionary biologists. But the topography of biology is changing. These methods are rapidly becoming more easily applied to non-model systems, such as our favorite genus Anolis. In an upcoming paper from the Menke Lab, the tools of functional genomics are applied to anole limb development, taking another step towards making Anolis a truly integrative model system.

 

In situ hybridization showing expression of of early limb genes in A. sagrei.

In situ hybridization showing expression of of early limb genes in A. sagrei.

Park et al. describe a micromass culture system to explore the molecular regulation of anole limb morphogenesis. In their protocol, Park et al. collect cells from early limb buds of A. sagrei, dissociate the cells from one another, and then add them to a dish as a small (i.e., micro) bolus (i.e., mass) of cells with the appropriate growth media. Even when removed from the embryo, these cells maintain the characteristics of limb cells, developing cartilage after about two weeks and maintaining their molecular signature for at least eight days. This small mass of cells can be grown for up to 30 days and, therefore, provide a useful template for experimental manipulations. More details of this protocol are described in great detail in the paper. Compared to other technologies which require far greater investment, their protocol should be accessible to anyone with access to a tissue-culture laboratory.

Anolis is an emerging model of limb development, but previous studies have focused on describing morphometric patterns of limb growth, not the molecular regulation of limb development. In fact, there have been no studies systematically dissecting the molecular regulation of limb development in any squamate species despite broad interest in this topic in the laboratory mouse and chick systems for 40 years. To study the molecular mechanisms regulating limb morphogenesis, Park et al. forced the expression of the gene Pitx1 – a hindlimb-specific molecule in mouse and chick – in micromass cultures derived from both forelimb and hindlimbs. This experiment verified that one step of the limb regulatory network, the relationship between Pitx1 and Hoxc11, is likely conserved among amniote lineages. While at this time this  may have been a proof of principal experiment, this protocol may have future implications for both developmental and evolutionary research in Anolis. For example, multiple transgenes can be readily cloned and incorporated into the micromass cultures. In addition, micromass cultures derived from species with distinct limb morphologies may also open to door to finding pathways that are regulated in novel ways across Anolis lizards.

Park et al. Figure 4

Binding domains of Pitx1 in the intergenic region of Hoxc11. Note conservation of binding region throughout mammals (shaded arrows), but lack of conservation among amniotes (white arrows).

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