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Getting a Head with Ptychodera flava Larval Regeneration.

Introduction

One of the great marvels in biology is the ability to regenerate a fully functional nervous system after damage from disease or injury. Scientists have studied this remarkable process for decades, but it is still largely a mystery how some animals accomplish this incredible feat. Humans have limited regenerative abilities, particularly in the central nervous system (CNS) (Chernoff et al., 2002; Stocum, 2006; Poss, 2010). Some peripheral neurons can regenerate to a certain degree; however, damage to the brain or spinal cord usually results in permanent, catastrophic impediments that are not corrected though regenerative mechanisms (Yannas, 2001). Animal models that are capable of extensive and complete nervous system regeneration are needed to effectively make strides in understanding the molecular mechanisms underlying neural regeneration. Moreover, models that are closely related to humans will likely provide greater insight into achieving extensive mammalian CNS regeneration, because many of the same genes, gene networks, and developmental programs are shared between the deuterostomes (Davidson and Erwin, 2006; Swalla, 2006. 2007).

The ability to regenerate neural tissue is widespread among numerous animal phyla, such as Ctenophora, Cnidaria, Playhelminthes, Annelida, and Nemertea, to name a few (Alvarado, 2000; Brown et al., 2009; Bely and Nyberg, 2010; Somorjai et al., 2012; Giangrande and Licciano, 2014). Within the deuterostome clade, however, few animals are capable of complete nervous system regeneration after total neural ablation. Echinoderms (sea stars, sea urchins, sand dollars, sea cucumbers, and sea lilies) have impressive regenerative abilities. Every class in this group of animals has members that are able to regenerate certain tissues and structures, including nerve cords, innervated guts, and even the central disk, or radial nerve cord (Vickery and McClintock, 1998; Vickery et al., 2002; Mashanov and Heinzeller, 2008; Carnevali et al., 2009). Some sea star and sand dollar larvae regenerate entire body halves after surgical bisection, demonstrating regeneration continuity between the larval and adult life stages (Vickery and McClintock, 1998; Vickery et al., 2002). The echinoderm nervous system and body plan, however, are highly derived, display radial symmetry, and lack many features consistent with vertebrate body plans. Notably, echinoderms do not develop a neural tube in either the larval life stage or the adult life stage, similar to vertebrate neurulation (Mashanov et al., 2007; Garner et al., 2016). Furthermore, echinoderms lack an anterior head and instead possess a radial, oral-aboral body axis, as opposed to the anterior-posterior body axis of chordates (Minsuk et al., 2005; Wygoda et al., 2014). In light of these morphological and developmental differences, we believe that echinoderms may not be the most informative model with which to decipher mechanisms of CNS regeneration in the deuterostomes.

Certain chordates are also capable of robust regeneration; however, these models have unknown barriers blocking complete CNS regeneration. The caudal tail and anterior regions of cephalochordates regenerate with high fidelity, but as the amputation plane moves toward the pharynx region, the regenerative abilities decline and are completely absent in the pharynx (Somorjai et al., 2012). Bisection within this region results in death for both halves of the animal. Some chordate ascidians undergo whole-body regeneration (Schultze, 1899; Brown et al., 2009; Rinkevich et al., 2010; Jeffery, 2014; Kassmer et al., 2016; Zondag et al., 2016); however, ascidian tadpole larvae, which possess several vertebrate features, including a dorsal, hollow neural tube, do not regenerate during this early life stage (Jeffery, 2014). The remarkable regenerative abilities of ascidians appear to be activated after metamorphosis. When ascidians metamorphose from their tadpole larval stage and are competent for whole-body regeneration, the majority of their neural tube undergoes apoptosis, and only the anteriormost part remains and becomes a neural gland situated between the incurrent and excurrent siphons of the adult (Manni et al., 2005; Horie et al., 2011). The adult ascidian also forms a rich nerve net throughout the ectoderm, but it lacks a head, neural tube, and centralized nervous system, similar to the vertebrates (Sasakura et al., 2012). Teleost fish, newts, salamanders, lizards, and tadpole larval, anuran amphibians can regenerate portions of their spinal cord, but this is generally restricted to the caudal end of the spinal cord, found in the tail of the animal (Singer et al., 1979; Beattie et al., 1990; Duffy et al., 1992; Chemoff, 1996; Sirbulescua and Zupanca, 2011; Kizil et al., 2012; Ghosh and Hui, 2016). Furthermore, bisection of the animal in the transverse plane results in death, and no vertebrate has been shown to regenerate an entire body half or head region.

Ptychoderid hemichordates undergo vertebrate-like neurulation (Luttrell et al., 2012) and yet have remarkable CNS regenerative abilities that may provide insight into achieving more extensive neural regeneration in humans (Luttrell et al., 2016). Hemichordates are marine invertebrates and a sister group to the echinoderms (Wada and Satoh. 1994; Cameron et al., 2000). Hemichordates share numerous developmental and morphological traits with the chordates, such as pharyngeal gill slits and a Hox-specified anterior-posterior body plan (Aronowicz and Lowe, 2006; Brown et al., 2008; Luttrell and Swalla, 2014). Indirect-developing acorn worms begin life as planktonic, feeding, tornaria larvae that can remain in the water column for up to 300 days (Hadfield, 1978; Nielsen and Hay-Schmidt, 2007). The larva progresses through various developmental stages throughout the long planktonic phase, from a newly hatched Heider stage larva to a Metschnikoff, Krohn, Spengel, and, last, Agassiz stage larva, or what is commonly known as the swimming head larva (Fig. 1; Rao, 1954; Nishikawa, 1977; Tagawa et al., 1998; Nakajima et al., 2004; Lin et al., 2016). The larva becomes competent for metamorphosis at the Spengel stage and transforms to a juvenile worm through the Agassiz stage (Fig. 1; Lin et al., 2016). The tripartite body plan of the worm becomes apparent, and the metamorphosing larva settles to the benthos for the remainder of its lifespan. The tripartite worm is composed of an anterior proboscis, or head, a middle collar region, and a long posterior trunk (Luttrell et al., 2016). Interestingly, the solitary hemichordate Ptychodera flava has a dorsal, hollow neural tube in the collar region that we, and others, have shown develops in a similar fashion to the chordate neural tube (Luttrell et al., 2012; Miyamoto and Wada, 2013). Even more striking, P. flava reliably regenerates all body structures, including its complete neural tube, after total ablation (Rychel and Swalla, 2008; Humphreys et al., 2010; Luttrell et al., 2016). It remains to be determined, however, at what point during development this regeneration process is activated. It may be that regeneration is initiated after the animal undergoes metamorphosis from a planktonic larva into a juvenile worm, or it may be that the regeneration process becomes active at some point before metamorphosis. Does the metamorphosis process somehow initiate regeneration, or is this capacity active at all stages of development in P. flava, similar to the regeneration process in some echinoderms?

The present study shows that Krohn stage P. flava tornaria larvae are capable of robust regeneration in the sagittal, coronal, and axial planes (Figs. 2-4). We found that proliferating cells are abundant at the cut site in P. flava larvae and throughout the regenerating tissue. We also show that bisected larvae regenerate their nervous system and are indistinguishable from healthy, intact larvae, but the visible phenotype is consistent with the previous developmental larval stage. Functional studies aimed at uncovering the genetic and molecular mechanisms controlling neural regeneration may, in certain cases, be more tractable in the larvae due to their small size, transparency, and relatively simple body plan and tissue architecture compared to adult acorn worms.

Materials and Methods

Animal collection and management

Ptychodera flava Eschscholtz, 1825 larvae were obtained from the lab of Y-HS at Academia Sinica in Taiwan, where they spawned gravid adult worms and reared the embryos through hatching (Lin et al., 2016). Larvae were transported in a container of seawater from Taipei, Taiwan, to the University of Washington Friday Harbor Laboratories in Friday Harbor, Washington. Larvae were contained in 2-1 Erlenmeyer flasks filled with 0.2 [micro]m of filtered seawater (FSW) and kept at room temperature. Air was continuously bubbled into each flask, and the water was changed every four days with fresh FSW. Larvae were fed after each water change with Rhodomonas sp. and Isochrysis galbana algae.

Larval regeneration

Healthy, intact Krohn stage larvae were bisected through the midline in the sagittal, coronal, or axial planes, with a sterile microsurgical blade (Figs. 2-4). A total of 25 larvae were bisected through each plane and allowed to regenerate at room temperature in 6-well plates filled with FSW. Left and right halves, dorsal and ventral halves, and anterior and posterior halves were separated into individual wells. Each well contained no more than 10 larval halves, and all were fed starting from day 1 post-bisection, with one drop each of concentrated Rhodomonas sp. and I. galbana algae. FSW and algae were changed every 2 days during regeneration, over the course of 28 days.

In a second set of experiments, we removed the anterior apical region in healthy Krohn stage larvae by using a microsurgical blade, to determine the time required for sensory organ regeneration. All live, regenerating larval pieces were photographed on a Nikon (Melville, NY) SMZ 1500 dissecting microscope and photographed with a QImaging (Surrey, British Columbia. Canada) MicroPublisher Real-Time Viewing camera.

EdU (5-ethynyl-2'-deoxyuridine) labeling

The apical region of Krohn stage larvae was removed, and both pieces were allowed to regenerate at room temperature, about 24 [degrees]C. After 2, 5, 8, and 15 days, the regenerating larvae were immersed in 2 ml of 10 [micro]mol [l.sup.-1] EdU in FSW for 30 min at room temperature (RT). Larvae were then fixed in 3.2% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at RT and then washed 3 times in PBS with 1% Tween (PBST) for 15 min each. Larvae were then blocked for 30 min in 5% goat serum in PBST on a rocker at RT. Larvae were incubated for 2 h in Click-iT reaction mixture (Invitrogen, Carlsbad, CA), per the manufacturer's guidelines, at RT on a rocker and then washed 3 times in PBST for 10 min each (Salic and Mitchison, 2008). Larvae were subsequently stained with antibodies, mounted onto slides in VECTASHIELD (Vector Laboratories, Burlingame, CA) with 4',6-diamidino-2-phenylindole (DAPI), and photographed on a Nikon Eclipse E800 confocal fluorescent microscope or a Zeiss LSM 800 confocal microscope (Carl Zeiss, Jena, Germany).

Immunocytochemistry

After EdU staining, larvae were incubated overnight (or in some cases up to 2 days) with 1:1000 rabbit polyclonal anti-serotonin (Sigma-Aldrich, St. Louis, MO). Control larvae were incubated overnight in PBST without primary antibody. The following day, larvae were washed 5 times in PBST for 30 min each and then incubated overnight in a 1:1000 dilution of Alexa Fluor 568 goat anti-rabbit immunoglobulin G (IgG) antibody (Invitrogen. Eugene, OR). Larvae were then washed again 5 times for 30 min each in PBST and then mounted onto slides in VECTASHIELD with DAPI (Vector Laboratories).

Results

Sagittal, coronal, and axial regeneration

The Krohn stage of larval development is characterized by pronounced tentacular buds on the ciliary bands, making it particularly easy to identify (Fig. 1). We bisected larvae through three different planes to verify whether different regions of the larvae regenerate better than others, similar to what has been shown in some echinoderm larvae (Vickery and McClintock. 1998; Vickery et al., 2002). Because echinoderms and hemichordates are sister groups, it is probable that certain aspects of the regeneration process are shared between the two groups. Furthermore, if regeneration rates vary between different larval regions, this may help to identify a potential stem cell niche. We defined complete regeneration as having a complete gut and exhibiting normal feeding, normal morphology, and normal swimming behavior.

Larvae that were cut through the sagittal plane into left and right halves displayed the most robust regeneration (Fig. 2). In total, 25 Krohn stage larvae were bisected, and 74% of the halves regenerated normal larval morphology and were feeding and swimming normally after 28 days (Figs. 2, 3). There was a slight difference between the overall numbers of fully regenerated halves, with the right half outperforming the left. The right half of the bisected larvae experienced an 88% regeneration rate, with 22 of the 25 halves regenerating a complete gut and beginning feeding within 2 weeks (Figs. 2, 3). Normal morphology and swimming were acquired for some of the larval halves within 3 weeks, but the final count was assessed after 28 days because some halves were feeding but had not yet acquired normal morphology. The left half displayed a 60% regeneration rate, with 15 of the 25 halves meeting fully regenerated benchmarks at 28 days (Figs. 2, 3). Nearly all of the larvae that achieved complete regeneration were feeding by two weeks post-cut. The small differences in regeneration rates between the left and right halves could be attributed to slight cutting errors, with one half of the larva receiving more tissue than the other.

Larvae that were cut through the coronal plane, separating dorsal and ventral halves, displayed dramatically different regeneration rates between the two halves. The overall rate for this cut was 44%, with the ventral half drastically outperforming the dorsal half (Fig. 2). The ventral half of the animal regenerated normal morphology, feeding, and swimming behavior in 17 of the 25 halves, or 68% of the cut larvae (Figs. 2, 3). This was in stark contrast to the dorsal half, which achieved a 20% regeneration rate; only 5 of the 25 halves regenerated a complete gut and began feeding to achieve normal morphology within 28 days (Figs. 2, 3). While systematic cutting errors might have played a role, there is probably some other facet to the ventral side that confers a regenerative advantage. One likely possibility is that the ventral half contains the mouth and thereby enables the regenerating larva to feed much sooner than the dorsal half.

Axially cut larvae, separating the anterior and posterior halves, displayed the lowest total regeneration rate, at 40%. due largely in part to a nearly total lack of regeneration in the anterior half (Figs. 2, 4). Posterior halves completely regenerated a gut, fed, formed eyespots, and developed normal swimming behavior 72% of the time. Eighteen of the 25 halves appeared normal after 28 days, and some had regenerated eyespots at the apical tuft after only 17 days post-cut (data not shown). This was in stark contrast to the anterior half, which fully regenerated in only 8% of the cuts. Only 2 of 25 anterior halves regenerated a complete gut and telotroch or a ciliated band at the posterior end of the animal (Fig. 4). All of the remaining halves, 23 of 25, healed at the cut wound site and persisted through 2-4 weeks, but they never regained complete guts and eventually shrank in size to small balls of cilia, and degraded. The disparity seen between anterior and posterior regeneration in axial cuts may be attributed to one or more different possibilities. Feeding times likely play a role, and/or there may also be an unknown stem cell niche that resides in the posterior half region.

Wound healing for nearly all bisected larvae occurred within the first two days after amputation (Figs. 3, 4). It is worth noting that for some samples the tissue at the cut site was pinched together during bisection. In these cases, the larva was completely bisected; however, the wound at the cut site for one or both halves was sealed together, and it did not appear to reopen. It was not possible to accurately determine wound healing time in these cases because there was no open wound after cutting. The majority of animals, however, had open wounds that healed within two days, including larval halves that ultimately did not regenerate. This indicated that the wound healing process may be different from the regeneration process in Ptychodera flava. Interestingly, all of the cut larval halves that fully regenerated, regardless of the plane of bisection, displayed the morphology of the previous larval developmental stage (Figs. 3-5). At the end of 28 days, all regenerated Krohn stage larvae lacked tentacular buds on the ciliary bands. Collar and trunk coeloms that were present in the uncut animals were notably absent in the regenerated larvae. They retained the protocoel. though not well developed in many specimens. These phenotypes were consistent with the Metschnikoff larval stage (Figs. 1, 5), and this suggested that there was de-differentiation occurring within the regeneration process in P. flava larvae.

Apical organ regeneration

The nervous system of the larva consists of an anterior apical organ situated below the eyespots that contains a rich complex of serotonin-positive cells (Nakajima et al., 2004: Burke et al., 2006; Byrne et al., 2007). The apical organ also stains positive for 1E11, a monoclonal antibody against sea urchin synaptotagmin protein (Nakajima et al., 2004; Burke et al., 2006; Byrne et al., 2007). Synaptotagmin is a calcium-binding factor of synaptic vesicles and is expressed exclusively in neurons in some invertebrates (Littleton et al., 1993; Nonet et al., 1993; Augustine, 2001; Bai et al., 2002; Katsuyama et al., 2002). The four corners of the apical organ connect to ciliary bands that wrap around the larva and function to bring food into the mouth and move the animal through the water column. The ciliary bands also stain positive for 1E11 (Nakajima et al., 2004). We hypothesized that the apical organ would regenerate similar pre-amputation levels of neuronal cell composition around the same time that sensory eyespots form. To test this, we excised the apical organ and stained for cell proliferation and serotonin-positive cells as the tissue regenerated.

Apical regeneration closely resembled axial regeneration; however, the anterior apical piece did not regenerate to a fully functional animal in any of the cut larvae. The apical organ did not die after the initial cut and persisted through wound healing and in a few samples up to 25 days post-cut, depending on the original size of the fragment (Fig. 6). The overall size of the apical tissue gradually degenerated over time and eventually became a small ball of ciliated cells, typically after two or three weeks post-amputation. Serotonin-positive cells were found in the apical tuft after 15 days and did not appear to undergo apoptosis and degrade, although the tissue itself diminished in size throughout the time points (Fig. 6). Proliferating cells labeled with EdU (Salic and Mitchison, 2008) were detected throughout all time points, but the number of dividing cells declined over time and was severely reduced by day 15 post-amputation (Fig. 6).

Regeneration from the posterior site was markedly different from that of the anterior piece. Significant cell proliferation was detected throughout the tissue at the cut site after 2 days post-amputation, indicating that active cell division was contributing to wound healing and promoting regeneration (Fig. 7). These dividing cells coalesced toward the center of the posterior piece, where new tissue was replacing structures lost in amputation (Fig. 7). Serotonin-positive cells were detected at 2 and 5 days post-cut in the portions of ciliary bands that remained in the posterior pieces after amputation, while serotonin-positive cells were absent in the center of the posterior piece where the apical organ was excised at these same time points (Fig. 7). Two distinct populations of serotonin-positive cells were found near the midline in the regenerating presumptive apical organ at 8 days post-cut (Fig. 7). These populations of cells likely merged around 15 days, forming a condensed, serotonin-rich apical organ containing approximately 62 serotonin-positive cells (Figs. 7, 8). Uncut, non-regenerating Krohn stage larvae contain about 100 serotonin-positive cells in the apical organ (Fig. 8). Although the regenerating apical organ only contained slightly more than half of the amount of serotonin-positive cells as an uncut larva, eyespots developed and were visible at 15 days post-cut (Figs. 7, 8). At this time point, the regenerating larvae exhibited normal swimming and feeding behavior, indicating functional sensory structures. Cell proliferation increased dramatically throughout all tissues at the 15-day time point, implying that significant growth was probably occurring after required sensory structures regenerated (Figs. 7, 8).

Discussion

This study shows that the regeneration process is active in Ptychodera flava before metamorphosis during the larval stage. This is consistent with the regeneration process in some echinoderm larvae. Certain species of sea urchin, sand dollar, and sea star larvae fully regenerate after bisection through the axial plane (Vickery and McClintock, 1998; Vickery et al., 2002). Both the anterior and posterior halves completely regenerate, with no mortality, within 12-14 days in the sea stars Luidia foliolata and Pisaster ochraceus and the sea urchin Lytechinus variegatus (Vickery and McClintock, 1998: Vickery et al., 2002). The sand dollar Dendraster excentricus also regenerates after bisection through the axial plane, but the anterior half experiences reduced regeneration compared to the posterior half (Vickery et al., 2002), similar to the axial regeneration observed in this study. The regeneration timescale and overall capacity are comparable between echinoderm larvae and P. flava larvae, indicating that regeneration was probably an ancestral character in the Ambulacraria clade and could possibly extend to the common ancestor of the deuterostomes in general.

We also show that certain regions of the larva regenerate more efficiently than others. The dorsal half of the larva experiences reduced regeneration rates compared to the ventral half. This may indicate that having a mouth facilitates regeneration, by allowing the animal to bring food into the body cavity at earlier times than the dorsal half of the larva. The gut is full of algae at 7 days post-bisection in some of the ventral halves of the larvae, while the dorsal half is feeding after 2 weeks (Fig. 3). It is possible that bisection of the halves was not completely equal on the midline, conferring an advantage to the half with more tissue and with likely more gut present, resulting in less regeneration time to feeding. It is also possible that the ventral half, by morphology (Fig. 2C), has more gut tissue present than the dorsal half and therefore requires less time to regenerate a complete gut. This results in earlier feeding times and supports the hypothesis that feeding facilitates regeneration by supplying energy to supplement the high metabolic cost of regeneration.

Larvae that were bisected in the axial plane displayed the most disparate regeneration rates, which also could be attributed to feeding ability. Posterior halves of the larvae retain the majority of the gut at bisection. They possess the midgut, posterior gut. and anus; barring slight deviations from the midline during cutting, they need to regenerate only the anterior-most part of their tripartite gut and mouth. The anterior half, on the other hand, must regenerate the midgut, hindgut, and anus in order to maximize the full benefit of feeding. The two anterior halves that completely regenerated were able to bring food into their incomplete guts at two weeks post-bisection (Fig. 4), which may have aided them in achieving complete regeneration. While feeding does seem to support robust regeneration in P. flava larvae, there may be other factors contributing to regeneration success.

The low regeneration rate observed in anterior halves and the lack of regeneration in anterior apical fragments may also be due to the amount of cells present and/or the cellular composition of the tissue. Apical fragments may be too small and simply not possess enough cells, or total energy, to support regeneration. Proliferating cells are detected by EdU staining in the apical tissue from 2 to 15 days post-amputation, but the amount of cell division diminishes over time. This implies that regenerative mechanisms may be activated on amputation; but because the tissue is not feeding, there is no compensation for the energetic requirements of regeneration and growth, and, therefore, these processes halt over time. The other possibility is that there may be some population of cells in the posterior region that is required for regeneration. Rychel and Swalla (2008) showed that significant cell proliferation also occurs in adult P. flava acorn worms during anterior regeneration. It is an open question whether these dividing cells are originating from de-differentiated somatic cells that are returning to a progenitor cell state and then acquiring new cell fates in the newly regenerated tissue, or whether dividing cells are bona fide stem cells occupying an unknown niche. If the latter is true, then it is probable that the larvae also possess an unknown stem cell niche, which would likely be in the posterior region. This hypothesis would explain why anterior pieces of apical cut larvae do not regenerate and why only a small fraction of the anterior pieces of axial cut larvae regenerate. It is possible that those cuts were made off-center and that each anterior piece retained a small portion of the posterior stem cell niche. Current work in our lab is aimed at using stem cell markers to answer this intriguing question.

We also provide evidence that neurons in the apical sensory organ regenerate from posterior pieces after complete ablation. Eyespots are visible above the apical organ at 15 days post-amputation (Figs. 7, 8) and confirm that sensory structures reliably regenerate from posterior halves of the larvae. The number of serotonin-positive cells in the apical organ after 15 days of regeneration was around half the number of cells in uncut Krohn stage larvae. This evidence, combined with the significant increase in cell proliferation shown at the 15-day time point (Fig. 7), suggests that sensory structures are likely being further elaborated. The functionality of these structures was not tested experimentally; however, the larvae displayed normal swimming behavior and motility through coordinated ciliary action. This implies that around two weeks is the minimum time required to regenerate a rudimentary anterior sensing and coordinating structure under these environmental conditions. This establishes a baseline for future functional studies aimed at exploring neuronal regenerative mechanisms in P. flava larvae.

Finally, we show that Krohn stage larvae might display positional memory deficits during regeneration. Ptychodera flava larvae metamorphose at the Agassiz stage of development (Fig. 1), and this could be a strategy that would allow an injured larva to remain in the water column longer to repair wounds and grow to optimal sizes prior to metamorphosis. The final regeneration size of every larva was significantly smaller than the animal prior to cutting. This size difference can be explained, however, by a lack of feeding during the early stages of regeneration. Adult acorn worms display the same size disparity after regeneration, and this was attributed to a lack of feeding during the first week of regeneration after the anterior region, including the mouth and anterior gut, was amputated (Luttrell et al., 2016). Another possibility for the phenotypic difference is that the regenerated larvae are technically still in the Krohn stage of development, but they have lost collar and trunk coeloms and tentacular buds on the ciliary bands due to drastic alteration of the tissue during amputation. These results suggest that de-differentiation is occurring, although additional studies with other larval stages and developmental molecular markers will be needed to confirm which cell types are undergoing de-differentiation during larval regeneration in P. flava.

In summary, Krohn stage P. flava larvae regenerate all structures, including their nervous system, eyespots, gut, mouth, and ciliary bands. This study provides a framework for future research aimed at uncovering the cell types and genetic mechanisms underlying the regeneration process in this basal deuterostome. Adult P. flava acorn worms also regenerate: however, their large size and opacity make them difficult to use in a variety of molecular assays. Sectioning is required to detail internal molecular markers, and even then it is difficult to gain a clear picture of what is happening throughout all tissues. These obstacles can now be overcome by using the larvae to investigate regenerative mechanisms. Their tiny size means a smaller amount of reagents can be used, resulting in a lower experimental cost. More importantly, the larvae are transparent and easy to image by using confocal microscopy. In situ hybridization protocols and many other techniques have been published for the larvae, and the signal is easy to detect because of their clear tissues. Drug trials aimed at inhibiting regeneration to detect required genetic pathways are more feasible with the larvae because they require considerably less volume to submerge. Culturing techniques have been established for the larvae, and, therefore, they can easily be kept healthy and maintained in a small area of the lab for the duration of the larval stage. Methods to induce metamorphosis have proven highly successful (Lin et al., 2016), and, therefore, the larvae provide a system to study regeneration through all life stages in the lab. The P. flava model has the potential to reveal key molecular and genetic mechanisms underlying regeneration in the deuterostomes. The relatively simple body plan and tissue architecture of the larva compared to the adult will undoubtedly simplify future regeneration studies.

Acknowledgments

We thank the US National Science Foundation in partnership with the Taiwan Ministry of Science and Technology (MOST) for supporting the East Asia and Pacific Summer Institute (EAPSI) program. We thank Tzu-Pei Fan for providing tornaria larvae and care and maintenance protocols. We thank Drs. Olivia Bermingham McDonogh and Sharlene Santana of the University of Washington for their helpful comments on the manuscript. Drs. James Truman and Lynn Riddiford generously provided help with antibody staining techniques and allowed us to use their Zeiss LSM800 confocal microscope. This material is based on work supported by the National Science Foundation (NSF) Graduate Research Fellowship Program, grant 1256082 to SL. and BEACON NSF Science and Technology Center, grant 948 to SL and BJS. Y-HS is supported by MOST 103-2311-B-001-030-MY3 and 106-2321-B-001-039.

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SHAWN M. LUTTRELL (1,2,3), YI-HSIEN SU (3), AND BILLIE J. SWALLA (1,2,*)

(1) Department of Biology, University of Washington, Seattle, Washington 98195-1800; (2) Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington 98250-9299; and (3) Institute of Cellular and Organismic Biology, Academia Sinica, No. 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan

Received 9 September 2017: Accepted 25 April 2018: Published online 15 June 2018.

(*) To whom correspondence should be addressed. E-mail: bjswalla@uw.edu.

Abbreviations: CNS, central nervous system: DAPI, 4'.6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine.
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Author:Luttrell, Shawn M.; Su, Yi-Hsien; Swalla, Billie J.
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Date:Jun 1, 2018
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