Our project is mainly focusing on hemichordates, they are marine invertebrates, related to echinoderms. While no chordate is able to regenerate all regions of its central nervous system after deathly injury. Ptychodera flava, a hemichordate, exhibits an exceptional ability to regenerate its entire central nervous system and anterior structures in as little as two weeks. We are studying this process to understand the underlying gene networks involved in neural regeneration. I am currently investigating the expression of transcription factors involved in neural development with homeodomain-containing genes Pax6, Six3, DLX, Frizzled, Chordin, Pou, and Msx-2, which have been shown to be upregulated during P. flava regeneration through transcriptome analyses. I have previously cloned the isolated PCR products into plasmids to make RNA probes for in situ hybridization. I am generating RNA probes, performing in situ hybridization. I will use RT-PCR to examine when there is a high RNA expression at various stages of regeneration, then I will perform in situ hybridization on P. flava tissue samples at these different stages. I will also be sectioning these samples after in situ hybridization and staining to image the cellular and tissue structures to aid in seeing where these genes are expressed in specific tissues during regeneration. My research goal is to ascertain the gene networks underlying regeneration and then to compare these to the expression of the same genes during embryonic development in P. flava. Finally, we can ask whether these genes exhibit similar gene networks that have been reported during development in direct-developing hemichordates. Further experiments will be examining if these gene networks are necessary and sufficient in regeneration by knocking-out or overexpressing these specific genes at different stages of regeneration. By using P. flava as a model to study transcriptional regulation during regeneration, we are aiming to identify the genetic and morphological mechanisms to achieve sufficient central nervous system and whole-body regeneration in a stem deuterostome, which might also give us the insight into potential key regulator factors during human regeneration.