Exploring the Mechanisms of Regeneration in Ptychodera flava

Thesis (Ph.D.)--University of Washington, 2017-08Hemichordates are marine acorn worms and a sister group to the echinoderms. Acorn worms share numerous developmental features with the chordates that the echinoderms do not share, suggesting that the common ancestor of the deuterostome clade was likely more similar to hemichordates than to echinoderms. Hemichordates possess pharyngeal gill slits that are homologous to chordate gill slits, a Hox specified anterior-posterior body plan, an endostyle, and a post-anal tail in some species. The hemichordate, Ptychodera flava, progresses from a pelagic, feeding tornaria larva to a tripartite, benthic worm with an anterior head or proboscis, a middle collar region, and a long posterior trunk. The collar houses a hollow, dorsal neural tube in ptychoderid hemichordates and numerous chordate genes involved in brain and spinal cord development are expressed in a similar anterior–posterior spatial arrangement along the hemichordate body axis. Remarkably, P. flava regenerates all structures, including their dorsal neural tube. This work investigates the morphological and genetic mechanisms underlying this fascinating trait I examined anterior regeneration in the hemichordate Ptychodera flava and detailed the spatial and temporal morphological changes that occur. Additionally, we sequenced, assembled, and analyzed the transcriptome for eight stages of regenerating P. flava, and revealed significant differential gene expression between regenerating and control animals. Importantly, we uncovered developmental steps that are regeneration-specific and do not strictly follow the embryonic program. We also showed that P. flava larvae are capable of robust regeneration after bisection through the sagittal, coronal, and axial planes. We used antibody staining to show that the anterior apical region, including the eyespots and larval brain, regenerates a rich, serotonin positive complex of cells within two weeks after amputation. Cells labeled with EdU (5-ethynyl-2'-deoxyuridine) confirmed that regeneration is occurring through epimorphic processes as new cells are added at the cut site and throughout the regenerating tissue. This study verified that P. flava larvae can be used for future functional studies aimed at identifying the genetic and morphological mechanisms controlling CNS regeneration in a stem deuterostome