Hoxa9 compensates for the absence of Hoxc9 in suppressing limb-type motor neurons in sharks.

backgroundThe transition from fins to limbs in vertebrates required a novel organization of spinal motor neurons to coordinate limb muscle activation. In amniotes, Hoxc9 represses lateral motor column (LMC) identity at thoracic levels, restricting limb-innervating Foxp1+ motor neurons to brachial and lumbar levels. In elasmobranchs, however, the genomic organization of HoxC genes has undergone extensive modifications, and Foxp1+ LMC-like neurons have been identified at paired-fin levels in some elasmobranch species lacking Hoxc9. These observations suggest that alternative mechanisms regulate motor neuron fate in chondrichthyans, particularly in sharks, although the responsible factors remain unclear.

resultsTo identify the mechanism underlying this suppression, we examined Foxp1 and Hox gene expression in chicken and cloudy catshark (Scyliorhinus torazame) embryos. In chickens, Foxp1+ LMC neurons initially appeared at all rostrocaudal levels but became restricted to paired-limb levels through Hoxc9-mediated repression. In contrast, in cloudy catsharks, which lack Hoxc9, Foxp1 was downregulated at inter-fin levels where Hoxa9 is expressed. Sequence analysis revealed that the Foxp1 modulatory domain (MD), associated with LMC repression, is highly conserved in Hoxa9 across all examined chondrichthyan species. Hoxc9 genes are absent in most sharks but retained in rays and holocephalans, and these retained copies preserved the Foxp1 MD. Functional analysis in chicken embryos demonstrated that cloudy catshark Hoxa9 represses LMC identity and promotes preganglionic column (PGC) fate, similar to Hoxc9 in amniotes.

conclusionsThese findings suggest that conserved Hox9-dependent mechanisms restrict Foxp1+ motor neurons at thoracic levels in sharks. In the absence of Hoxc9, cloudy catshark Hoxa9 retains the ability to repress Foxp1 and promote PGC fate, thereby contributing to the organization of motor innervation at paired-fin levels.