Genetic screen of cardinal interneuron function in swim and limb behavior over frog metamorphosis

The Xenopus laevis frog shows a wide range of motor behaviors during metamorphosis, transitioning from tail-based escape swimming, to free swimming, and finally, limb-based movement. This swim-to-limb transition allows us to study in a single organism how motor and interneurons in the spinal cord generate these behaviors. Here, utilizing advances in genetic manipulation and behavioral tracking, we dissect the molecular features and contribution of each spinal cord cardinal class to swim versus limb behavior over frog metamorphosis.We devise a high-throughput CRISPR screen, in which we knock out primary markers for twenty-five cardinal classes or subclasses of ventral and dorsal spinal neurons. We utilize the lineage map of frog embryos to generate whole and half mutants, in which only one side of the animal is mutant.First, we visualize the molecular and cellular changes in the spinal cord of each CRISPR mutant, using immunohistochemistry and multiplexed in situ hybridization to pinpoint the role of each gene in determining cell-type identity and the resulting changes in spinal cord architecture. We then apply the machine-learning-based SLEAP software as a high-throughput behavioral analysis tool​ to track tail and limbs and quantify movement of Xenopus and its body parts across metamorphosis. Wildtype behavioral profiles are then compared to whole/half mutant animals to evaluate the contribution of each neuron subtype to the range of motor repertoires.Our study uses frog metamorphosis to build a comprehensive understanding of the contribution of each spinal neuron cell-type to swim-to-limb behavior.