Several weeks ago, Anole Annals highlighted a recent paper that uncovered the molecular bases of craniofacial dimorphism in the carolinensis clade of Anolis lizards (for full disclosure, I am the lead author of that paper). Hidden deep within that research is a relatively new technique for precisely measuring rates of skeletal growth that may be of interest to the community. I briefly introduced this technique several years ago in a post about methods of skeletal preparation, but with the details of this method now available it is worth highlighting once more.

Double labeled facial skeleton of A. carolinensis. Green label (calcein) and red label (alizarin complexone) separated by 30 days.

Because some images shouldn’t be lost in the supplementary materials. Double labeled facial skeleton of A. carolinensis. Green label (calcein) and red label (alizarin complexone) separated by 30 days.

Growth in body size can often be measured using calipers or a ruler. But in some situations a finer-scale analysis may be necessary, such as when differences in growth rate may be subtle, within the range of error associated with those manual methods. Fluorescent calcium chelators provide the precision needed to measure differences on the order of microns per day. In the recent paper, this technique was used to measure facial elongation in sexually mature green anoles, which was only ~8um per day in males and ~4um per day in females. These compounds are stable, are not highly toxic to animals, are relatively inexpensive, and can be easily used in the field or the lab. They can also be applied to adults or hatchlings with little modification to the protocol as injection volumes are typically 10-20ul depending on size. Ultimately, there is a lot of versatility to the way in which this method can be applied.

Dimorphism in facial growth rates between male and female A. carolinensis. Modified from Sanger et al. 2014.

Dimorphism in facial growth rates between male and female A. carolinensis. Modified from Sanger et al. 2014.

While new to herpetology, this technique was adopted from the biomedical literature on fracture repair where precise spatiotemporal measure of bone deposition is required. The general experimental framework is that pulses of chelators with different fluorescent properties are delivered at distinct intervals, the skeleton prepared, and the distance between the labels recorded from digital photographs. Calcium chelators are available that fluoresce under many of the standard filters used in modern microscopy – including green (calcein), red (alizarin complexone), orange (xylenol orange), and blue (calcein blue and oxytetracycline) – offering great experimental flexibility. Once incorporated into the bone, their signature remains strong for at least 30-45 days, until it is remodeled away as the living skeleton continues to grow and reshape itself. In the recent paper on craniofacial dimorphism, fluorescence in the facial skeleton could be observed following simple removal of the skin because the face has little to no overlying connective tissue. Measuring growth of the vertebrae or limbs is also possible, but may require careful sectioning of the bone using either plastic or paraffin protocols. Ultimately I think that there is a lot of potential with this method that has yet to be explored in the context of organismal biology. I hope that by highlighting this method here more people become aware of its utility and give it a try.

Thomas Sanger
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