Introduction

Regeneration has captured the interest and imagination of people for centuries. Popularized in myths, science fiction, and even horror movies, regeneration of missing and damaged tissue is a common reality in the animal kingdom. Nearly every animal phyla contains at least some species that consistently regenerate all or certain tissues and structures (Bely and Nyberg, 2010; Somorjai et al., 2012; Giangrande and Licciano, 2014). All deuterostome groups, with the possible exception of Xenoturbella, have at least some species with the capacity to regenerate (Sánchez Alvarado, 2000). Numerous chordates, including lancelets, tunicates, frogs, fish, salamanders, and even humans are able to regenerate to some degree (Brown et al., 2009; Bely and Nyberg, 2010; Somorjai et al., 2012; Giangrande and Licciano, 2014). Every extant class of echinoderms have been reported to regenerate (Candia Carnevali et al., 2009), while some species of hemichordates, which are a sister group to the echinoderms, undergo asexual reproduction and regenerate all anterior and posterior body parts when amputated (Willey 1899; Dawydoff 1902; Rao 1954; Rychel and Swalla, 2008; Humphreys et al., 2010; Miyamoto and Saito, 2010).

When and in what animal lineage did the ability to regenerate first evolve? Was regeneration a stem metazoan trait that was subsequently lost or reduced in numerous taxa or has regeneration evolved independently several times across the metazoans? Porifera (Bely and Nyberg, 2010; Giangrande and Licciano, 2014), Cnidaria (Bosch, 2007; Dubuc et al., 2014), Ctenophora (Ryan et al., 2013; Moroz et al., 2014), and Placozoa (Bely and Nyberg, 2010) have extensive regenerative abilities and, as basal metazoans, these lineages suggest an ancient and ancestral mechanism of regeneration. Comparing gene regulatory networks used during regeneration across various animal phyla will help to elucidate common molecular mechanisms of regeneration.

Understanding the morphological and genetic basis of regeneration may yield clues to unlocking regeneration in animals with limited or no regenerative abilities, like humans. Millions of people suffer from neurodegenerative diseases, spinal cord injuries, and limb amputations (Brown et al., 2005; Ziegler‐Graham et al., 2008; Mahabaleshwarkar and Khanna, 2014). Furthermore, aging and age‐related diseases will eventually affect everyone. Regeneration may slow the aging process and stem cells present one feasible way to combat a multitude of diseases and injuries (Rando and Wyss‐Coray, 2014). If regeneration is a stem deuterostome trait, it is likely that humans possess many, if not all, of the genetic switches controlling regeneration, but those switches have been modified or inactivated in some way over evolutionary time. It may, therefore, be possible to re‐activate those pathways in humans using information gained from studying genetic models made from animals with extensive regenerative capabilities.

We are seeking to identify the morphological and genetic underpinnings controlling regeneration in the solitary hemichordate, Ptychodera flava (Eschscholtz, 1825), which is a basal deuterostome and capable of regenerating all anterior and posterior structures when bisected (Rychel and Swalla, 2008; Humphreys et al., 2010). It is critical to know when internal structures regenerate to use genetic knock downs and knock outs, as well as overexpression, to characterize the function of genes directing the regeneration process. Moreover, hemichordates are the only deuterostome known to be able to regenerate an anterior head‐like structure and entire central nervous system (CNS). Ptychodera flava progresses from a fertilized embryo to a planktonic, feeding larva that can remain in the water column for up to 300 days (Hadfield, 1978; Lin et al., 2016). Upon metamorphosis, the larva develops into a juvenile worm that settles to the ocean floor to begin a benthic lifestyle. Solitary hemichordates are exclusively marine and adult animals have a tripartite body plan with an anterior proboscis that is used for digging and burrowing in the sand and mud, a middle collar region with a hollow, dorsal neural tube in ptychoderid hemichordates, a ventral mouth between the proboscis and collar, and a long posterior trunk with pharyngeal gill slits and gonads in the anterior trunk, a hepatic region in the mid‐trunk and a terminal anus (Balser and Ruppert, 1990; Brown et el., 2008; Luttrell and Swalla, 2014).

The hemichordate nervous system is quite interesting. Upon embryogenesis in direct developing hemichordates and metamorphosis in indirect developers, dorsal and ventral nerve cords develop along the full length of the trunk and nerve rings form around the base of the collar and the base of the proboscis (Balser and Ruppert, 1990; Kaul and Stach, 2010; Miyamoto et al., 2010; Miyamoto and Wada, 2013; Kaul‐Strehlow et al., 2015). Adult echinoderms also develop nerve cords throughout each arm and a circumferential nerve ring around the central disc; however, it still remains to be determined whether echinoderm and hemichordate nerve cords are homologous structures (Sly et al., 2002; Holland et al., 2013). In addition to nerve cords, P. flava also develops a hollow, neural tube that is positioned dorsally in the collar region. Our lab has shown this structure forms from ectoderm that invaginates and rolls up forming a hollow tube, similar to chordate neurulation (Morgan, 1894; Luttrell et al., 2012; Miyamoto and Wada, 2013; Luttrell and Swalla, 2014).

Lastly, hemichordates have a diffuse nerve net throughout the ectoderm, similar to the adult tunicate, Ciona intestinalis (Balser and Ruppert, 1990; Lowe et al., 2003; Dahlberg et al., 2009; Miyamoto et al., 2010; Kaul‐Strehlow et al., 2015). The adult acorn worm, in light of these similarities, has aspects of the different types of nervous systems that are present in the deuterostomes. Remarkably, P. flava is able to regenerate all of these structures, making hemichordates a model system to study nervous system evolution and regeneration in this clade of animals. Furthermore, as invertebrates, hemichordates lack the numerous genome duplications events present in the vertebrates (Dehal and Boore, 2005; Hughes and Liberles, 2008), thereby making functional studies of the regeneration genes more tractable in this animal.

Here, we report the spatial and temporal regeneration of internal body structures to complement the previously published external morphology regeneration data (Willey, 1899; Rao, 1954; Nishikawa, 1977; Rychel and Swalla, 2008; Humphreys et al., 2010). We also include analyses of the transcriptome from regenerating P. flava animals. In total, eight different stages of regeneration were sequenced, assembled, and annotated to track changes in early gene expression that direct regeneration of anterior structures. We document expression profiles and gene ontologies of hundreds of putative genes associated with anterior head regeneration. These data build a foundation for future experiments that may show what genes are sufficient and necessary to start the regeneration program in P. flava. Our morphological data shows the regenerative response to these early changes in gene expression and we document complete anterior regeneration of the proboscis, collar, and anterior trunk. Furthermore, we uncovered morphological differences between early development and regeneration of anterior structures, including the neural tube. The assembled transcriptomes reported here not only complement recent P. flava developmental transcriptomes (Shu‐Hwa et al., 2014; Tagawa et al., 2014; Simakov et al., 2015), but also open the door to systematic comparisons of embryonic processes to regenerative events in hemichordates specifically, and deuterostomes in general.