Rostrocaudal spinal cord patterning during the swim-to-limb transition of Xenopus metamorphosis

As tadpoles switch from undulatory to ambulatory locomotion a repeated array of axial swim units transitions to a limb-torso-limb architecture along the rostrocaudal axis. Exemplifying this, at early tadpole swim stages, motor neurons of a single type are evenly distributed along the spinal cord. While, at later frog stages, level-specific motor columns are added at both thoracic and limb levels. Here, we show rostrocaudal differences in inhibitory and excitatory interneuron populations emerge during frog metamorphosis. This raises the question of when differences in neuronal architecture develop, what mechanisms generate them, and whether these mechanisms operate at progenitor or neuron level. To address these questions, we profile neural and progenitor markers and use EdU pulse-chase labeling between tadpole and frog stages. We observe differential development by spinal cord level along the rostrocaudal axis, with a rapid increase but well-defined temporal order of limb-specific neurons and subtypes, and a gradual expansion of torso neurons. We then relate these differences to the size and proliferation of progenitor domains. To probe the driving mechanisms of rostrocaudal patterning, we created half- and whole- CRISPR mutants targeting the HoxC9 gene, a master regulator of transcription and spinal cord patterning. These animals we then submitted to gene marker profiling and EdU pulse-chase labeling. Our study aims to reveal how a limb-torso-limb circuit develops within a uniform axial swim circuit, providing a foundation to understand how spinal circuits support distinct swim and limb motor programs, and how an analogous swim-to-limb circuit transformation may have occurred across vertebrate evolution.