The XenCart Protocol: A method for Alcian blue labeling and quantitative analysis of craniofacial cartilage in...

Craniofacial birth defects, such as cleft lip and palate, are among the most common congenital anomalies and often arise from disruptions in early facial patterning. Many of these defects are linked to environmental teratogens, yet such exposures cannot be directly evaluated in humans, making animal models essential for evaluating developmental risks. Xenopus laevis offers a powerful solution: its tadpoles develop externally, share deeply conserved craniofacial patterning mechanisms with humans, and provide an accessible platform for uncovering how environmental exposures reshape facial structures during development. Here, we present the XenCart Protocol, a reproducible workflow for Alcian blue staining and quantitative morphometric analysis of Xenopus craniofacial cartilage. This method provides clear visualization of individual cartilage elements and can be readily applied to investigate genetic or environmental perturbations. The Xenopus craniofacial skeleton contains distinct cartilaginous structures that perform key biomechanical functions and share strong homology with regions of the human craniofacial skeleton. These similarities allow direct comparison of developmental outcomes across vertebrates. As part of a Course-based Undergraduate Research Experience (CURE), the XenCart Protocol was used to measure jaw cartilage dimensions in tadpoles exposed to an emerging teratogen, e-liquids were used in vaping. X. laevis embryos exposed to e-liquid showed reductions across the major craniofacial cartilages, along with altered cartilage proportions. Together, these changes are consistent with an overall reduction in facial size and altered craniofacial shape. These findings also pinpoint the precise regions of the jaw most affected by e-liquid exposure, providing a foundation for uncovering the developmental mechanisms driving these craniofacial changes. The ability to detect clear differences in specific structures shows that the protocol is sensitive and well suited for student-led research. In summary, the XenCart Protocol provides a standardized, scalable method for quantifying craniofacial cartilage development and offers a powerful platform for both mechanistic research and undergraduate training in developmental biology and toxicology.