Early cranial patterning in the direct-developing frog Eleutherodactylus coqui revealed through gene expression.

Genetic and developmental alterations associated with the evolution of amphibian direct development remain largely unexplored. Specifically, little is known of the underlying expression of skeletal regulatory genes, which may reveal early modifications to cranial ontogeny in direct-developing species. We describe expression patterns of three key skeletal regulators (runx2, sox9, and bmp4) along with the cartilage-dominant collagen 2alpha1 gene (col2a1) during cranial development in the direct-developing anuran, Eleutherodactylus coqui. Expression patterns of these regulators reveal transient skeletogenic anlagen that correspond to larval cartilages, but which never fully form in E. coqui. Suprarostral anlagen in the frontonasal processes are detected through runx2, sox9, and bmp4 expression. Previous studies have described these cartilages as missing from Eleutherodactylus cranial ontogeny. These transcriptionally active suprarostral anlagen fuse to the more posterior cranial trabeculae before they are detectable with col2a1 staining or with the staining techniques used in earlier studies. Additionally, expression of sox9 fails to reveal an early anterior connection between the palatoquadrate and the neurocranium, which is detectable through sox9 staining in Xenopus laevis embryos (a metamorphosing species). Absence of this connection validates an instance of developmental repatterning, where the larval quadratocranial commissure cartilage is lost in E. coqui. Expression of runx2 reveals dermal-bone precursors several developmental stages before their detection with alizarin red. This early expression of runx2 correlates with the accelerated embryonic onset of bone formation characteristic of E. coqui and other direct-developing anurans, but which differs from the postembryonic bone formation of most metamorphosing species. Together these results provide an earlier depiction of cranial patterning in E. coqui by using earlier markers of skeletogenic cell differentiation. These data both validate and modify previously reported instances of larval recapitulation and developmental repatterning associated with the evolution of anuran direct development.