Single Cell RNA Sequencing Provides Clues for the Developmental Genetic Basis of Syngnathid Fish Evolutionary Adaptations

Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genomes revealed suggestive gene content differences and provide the opportunity for detailed genetic analyses. We created a single cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how their novelties evolved. Overall design: We created scRNAseq atlases from embryos of 2 wild caught Gulf pipefish (Syngnathus scovelli). 20 embryos per pouch were isolated for libraries. Embryos were at the 'frontal jaws' stage whereby they possessed cartilaginous craniofacial skeletons but it has not elongated yet. Embryos were dissociated in 460ul of .25% trypsin and 40ul 100mg/ml collagenase I. Cell concentratoins were quantified with the TC20 Automated Cell Counter then diluted to 800 cells/ul in .04%. The University of Oregon Genomics and Cell Characterization Core prepared single cell libraries for the two samples using the 10X Genomics Single Cell 3' Genome Expression mRNAseq kit with NextGEM v3.1 chemistry. Grants: William Cresko, OPP-2015301, National Science Foundation, the evo-devo of male pregnancy and its effects on the brood pouch microbiome Hope Healey, F31DE032559, National Institute of Health NIDCR, Discovery of craniofacial genes capable of compensation through evolutionary mutant model