homework report scientific articles

2010 Zoological Society of JapanZOOLOGICAL SCIENCE 27: 91–95 (2010)

Regeneration in the Hemichordate Ptychodera flava

Tom Humphreys1*, Akane Sasaki2, Gene Uenishi1, Kekoa Taparra1,

Asuka Arimoto2 and Kuni Tagawa2

1Pacific Biomedical Research Center, University of Hawaii at Manoa, HI 96822, USA 2Marine Biological Laboratory, Graduate School of Science,

Hiroshima University, Hiroshima 722-0073, Japan

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial

regions are severed from the body of the worm. After transection, the body wall contracts and heals

closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blast-

ema grows rapidly and begins the process of differentiating into a head with a proboscis and collar.

At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the form-

ing proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens

ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encir-

cle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to

fit the worm’s body, has grown attached to the severed site. At about this time the animal regains

apparently normal burrowing behavior. After the head is formed, a second blastema-like area

appears between the new head and the old body and a new branchial region is inserted by regen-

eration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented

and whitish such that in-situ hybridization can be used to study the expression of genes during

the formation of new tissues.

Key words: animal model, brain evolution, SEM, gill, in-situ hybridization, regeneration, acorn worm

INTRODUCTION

Hemichordates are among animals such as hydra,

planaria, and starfish that have amazing regenerative

capabilities and can form a complete animal from random

portions cut from the body. Hemichordate regeneration is

primarily epimorphic, with the regenerated portion fit to the

original body parts to form a complete whole. Among ani-

mals with a dorsal hollow neural structure, hemichordates

are the only group that exhibit such a flexible regenerative

capability (Cori, 1902; Dawydoff, 1907; Kuwano, 1902;

Packard, 1968; Rao, 1955; Spengel, 1893; Tweedell, 1961;

Willey, 1898), although some colonial tunicates can carry

out whole-body regeneration by forming a complete new

zooid from the vascular system left when the zooids of the

colony are excised (Rinkevich et al., 2007). Reports dating

from the end of the 19th century indicate that P. flava regen- erates a head when the anterior region is severed from a

portion of the body (Spengel, 1893; Willey, 1898). Although

reports vary, descriptions in general indicate that when the

body of a hemichordate is severed, a head and other ante-

rior missing parts are regenerated on an anterior facing

wound and tail parts on a posterior facing wound, with the

new structures filling out the complete body in a coordinated

fashion. We conducted an extensive survey of both anterior

and posterior regeneration when the body of P. flava is sev- ered at various locations along the anterior-posterior axis,

and conclude this species is able to regenerate the missing

anterior and posterior parts through an epimorphic process

wherever the body is severed.

In recent years, as an understanding of stem cells in

higher organisms has coalesced into a robust scientific field,

excitement about regeneration and regenerative medicine

has grown considerably. It seems reasonable that an under-

standing of the molecular gene circuitry that is activated and

deployed in animals that can regenerate may provide

medical science with clues about how to unlock latent

regenerative pathways in humans. Extensive studies in our

lab on molecular expression during development of P. flava (Tagawa et al., 2001) and in other labs on other hemichor-

dates (Lowe et al., 2003) have shown that the cohort of

genes involved in neurogenesis in hemichordates (Harada

et al., 2000; Harada et al., 2002; Tagawa et al., 2000;

Tagawa et al., 1998; Taguchi et al., 2000; Taguchi et al.,

2002) is the same as that specifying neural development in

chordates and humans. Although the diffuse nervous sys-

tem in hemichordates is in many ways much more ancestral

than the more evolved chordate nervous system, its most

centralized component is a dorsal hollow nerve track formed

by neurulation just as in chordates (Morgan, 1893), and the

organization of domains of expression of genes specifying

neural structures is very similar to that of chordates (Lowe

et al., 2003; Tagawa et al., 2000). Thus a molecular analysis

of head regeneration in P. flava is a good focus for pursuing the potential for invertebrate regeneration to provide keys to

* Corresponding author. Phone: +1-808-956-7862;

Fax : +1-808-956-4768;

E-mail : [email protected]

doi:10.2108/zsj.27.91

T. Humphreys et al.92

latent regenerative capabilities in the human neural gene

programs.

Already studies in the regeneration of lower animals,

especially planarians, are producing extensive and fascinat-

ing insights from molecular and genetic approaches for

understanding the gene circuitry involved in regeneration

(Birnbaum and Sanchez Alvarado, 2008; Sanchez Alvarado,

2006). In-situ hybridization is being used to show localiza-

tion of the expression of developmental genes in the regen-

erate (Agata et al., 2003; Rentzsch et al., 2007); RNAi has

proven valuable for establishing how specific genes are

involved in regeneration (Gurley et al., 2008; Petersen and

Reddien, 2008); expression profiling of regenerating tissue

is offering insights into the ontology of the gene sets mobi-

lized in the regenerative process (Rinkevich et al., 2007);

and the elucidation of the molecular features defining stem

cells (Jaenisch and Young, 2008; Morrison and Spradling,

2008) that are central to regeneration are all revitalizing and

revolutionizing regeneration research. As we learn to apply

powerful modern molecular techniques to regeneration in

animals, the study of regeneration in hemichordates will pro-

vide a unique perspective in the search for clues about

latent regenerative potential.

MATERIAL AND METHODS

Ptychodera flava were collected from areas of fine to coarse clean sand on the extensive flat shallow reef at Paiko, Oahu,

Hawaii, where tidal depths range from 0 to 0.7 meters. Worms were

exposed by disturbing the sand to a depth of about 4 to 10 cm with

a wave of the hand in the water over the sand. Worms ranged from

2 to 20 cm in depth. About 2% of the worms collected exhibited

signs of recent anterior regeneration irrespective of the season. The

animals were maintained in the laboratory in 60-liter seawater

aquaria filled with reconstituted sea water (Instant Ocean) at 26°C and equipped with 3-cm-deep coarse (1- to 3-mm grains) coral

gravel bottom filters driven by aeration. The aquaria were kept

clean, and 50% of the water was changed out every two weeks.

Intact animals or anterior pieces with heads that had posterior parts

removed by transection were kept in closed plastic boxes with tops

and bottoms of heavy 1-mm mesh screen covered with a layer of

fine clean sand (2 to 5 mm). Recently transected posterior bodies

were kept in clean glass bowls in the aquaria until about 12 days of

regeneration, at which point the animals regained burrowing behav-

ior and could crawl out of the bowls. They were then moved to the

closed plastic boxes with mesh tops and bottoms and the layer of

fine sand. In all experiments, freshly collected animals were sev-

ered at various body levels with an orthogonal cut by using sharp

surgical scissors.

Samples for scanning electron microscopy (SEM) were fixed in

4% glutaraldehyde in 0.1 M cacodylate buffer with 0.35 M sucrose,

washed in 0.1 M cacodylate buffer and 0.4 M sucrose, post fixed in

1% OsO4 0.1 M cacodylate buffer, critical point dried, mounted on

aluminum stubs, coated with gold/palladium, and observed with a

Hitachi S-800 Field Emission SEM with digital image capture in the

Pacific Biosciences Research Center Biological Electron Micros-

copy Facility, University of Hawaii at Manoa.

Fixation of samples and in-situ hybridization were carried out

as previously described for hemichordate embryos and larvae

(Lowe et al., 2004; Tagawa et al., 1998) using a PfSoxB1 probe (Taguchi et al., 2002).

RESULTS

In dozens of collections ranging from 50 to 300 P. flava individuals obtained over several years from the reef at

Paiko in Honolulu, about 1 to 10 of the animals would have

heads of white tissue that contrasted with the more pig-

mented orange, reddish, greenish or brown posterior portion

of the animal’s body. A few of these animals had only small

or rudimentary heads that rapidly developed into full-sized

heads when the animals were brought into the lab. These

light colored heads appeared to represent anterior regener-

ation of naturally wounded animals. A series of preliminary

experiments was conducted in the laboratory to define the

parameters of this regeneration. Tests were conducted to

determine the aquarium temperature optimal for the good

health of the animals and for regeneration. At 19°C the worms appeared healthy, but little or no regeneration was

observed. As the temperature was raised, the animals

remained healthy and regeneration became more robust in

both rate and extent. The optimal well being of the animals

appeared to be achieved between 26°C and 30°C. An aquarium temperature of 26°C was chosen for all experi- ments.

Animals maintained in lab aquaria were transected at

various levels and regeneration was observed. As shown in

Fig. 1, robust and morphologically normal anterior (Fig. 1 A)

and posterior (Fig. 1B) regeneration was observed in nearly

100% of animals severed at all levels. A general perception

from these experiments is that regeneration is epimorphic,

with new tissue added seamlessly onto the old tissue to pro-

vide the missing parts, without any regression or reorgani-

zation of the old tissue. Regenerates after about 12 weeks

regained normal pigmentation and could not be distin-

guished from animals that had not regenerated. Fig. 2

shows a striking example of the integration of tissue, where

a worm transected at mid-collar to remove the proboscis

and anterior collar regenerated new tissue, which was well

integrated with the old tissue, to replace the lost parts. Minor

exceptions to full and integrated regeneration were

observed. Anterior regeneration failed in about 20% of

pieces shorter than about 3 cm in length from the tail region,

and a small number of large worms severed at more anterior

sites produced two adjacent heads at the cut site. Animals

with severed branchial baskets often degraded the severed

portion of the branchial basket during anterior regeneration,

although maintenance of the severed branchial basket and

its integration into the epimorphic regenerate were also

often observed.

For standardized anterior regeneration experiments, we

used small to medium worms, 5 to 12 cm in length,

transected 1 to 3 mm behind the posterior terminus of the

gill basket, as marked in Fig. 3A. Upon transection, the pos-

terior of the animal ceases to show directed movement or

burrowing behavior. For about 48 hours, the wound remains

expanded, as shown in Fig. 3B, exposing the open gut cav-

ity surrounded by layers of the body wall. By 48 hours the

cut edges of the body wall are noticeably swollen, creating

a smooth, rounded edge of tissue. This rounded, swollen

edge of body wall can be seen in the semicircle of body wall

still extending from the partially closed wound shown in Fig.

3C. By 72 hours the wound has closed, as shown in Fig. 3D,

and an indistinct whitish spot, the forming blastema, not vis-

ible in Fig. 3D, appears in the closure area, generally in a

slightly dorsal position on the cut end. When these freshly

accumulating blastema cells are examined by SEM they

Hemichordate Regeneration 93

appear to have exposed surfaces

with three distinct surface morphol-

ogies and with, at most, only a sin-

gle short cilium (Fig. 4A). The three

cell types, in order of abundance,

are cells with a blebbing or bubbly-

appearing surface, cells covered

with microvilli, and cells with a

smooth surface. These exposed

cells can be compared with the epi-

thelial cells from the original surface

nearby, which have a forest of cilia

(Fig. 4B), as will the surface cells of

the regenerate at later stages, and

as do the epithelial cells of P. flava wherever we have examined them

(not shown).

The accumulation of cells

increases rapidly to a well-devel-

oped blastema by 4 days (Fig. 3E).

By 5 days, as shown in Fig. 3F, the

rapidly growing blastema has trans-

formed into a flattened bulge of

almost heart shaped central tissue

(see also Fig. 5 of a fixed specimen

at 5 days), the nascent proboscis,

and two lateral ridges that will

develop into the collar. About this

time, the new pharyngeal opening

of the gut is evident as a nascent

mouth ventral to the blastema. Dur-

ing the next few days, the proboscis

assumes its definitive shape, and the lateral ridges of the

blastema expand ventrally to surround the mouth and dor-

sally to complete the collar. The appearance at 7 days is

shown in the photograph in Fig. 3G and the SEM image in

Fig. 5. The new head, attached directly to the tissue at the

original cut surface, continues to grow rapidly and attains a

size appropriate for the size of the worm by about 12 days,

as seen in Fig. 3H. The animal, without any evidence of a

regenerating new branchial region, begins to exhibit normal

burrowing behavior and intake of sand grains.

The first sign of regeneration of the missing pharyngeal

region appears soon after the completion of the head. First

there seems to be an insert of white tissue between the

apparently completely regenerated head and the old body.

This insertional blastema grows rapidly, as shown at 17

days in Fig. 3I, into a new pharyngeal region and branchial

basket over the next few weeks, as illustrated at 25 days in

Fig. 1A.

To determine whether gene expression in the regener-

ating tissue could be examined by in-situ hybridization, we

Fig. 3. Progression of anterior regeneration after transection of P. flava just posterior to the gill basket. (A) Intact worm marked at site of transection by arrowhead at posterior end of branchial

basket. (B) Posterior body of severed worm just after transection. (C–I) Progression of regenera-

tion from 2 days to 17 days. (C) Two days after transection, showing a bulge on one side of the

swollen edge of the severed body wall as the wound is pulling closed. (D) Three-day transected

anterior end, which has healed closed and exhibits a tiny blastema not apparent in this photo. (E)

Four-day regenerate with closed wound and a growing blastema. (F) Five-day regenerate with a

slightly flattened middle that is the nascent proboscis and small ridges on each side which will

become the collar. (G) Seven-day regenerate with nascent proboscis and prominent collar ridges

on each side. (H) Full-sized head grown to fit body diameter at 12 days. (I) Seventeen-day regen-

erate showing the early stages of formation of the branchial region as an insert of tissue between

the regenerated head and the old body.

Fig. 1. Anterior regeneration at 25 days (A) and posterior regeneration at 22 days (B)

after transection just posterior to the branchial basket. Arrowheads mark site of original

transection in each photograph. In (A), the regenerated whitish tissue consists of the

newly formed proboscis and collar as well as the differentiating branchial region with the

nascent gill basket with gill slits. In (B), the new posterior part of the worm that has

formed behind the branchial basket, as marked by the arrowheads, has developed pigmen-

tation and a differentiating hepatic region. c, collar; b, branchial region; h, hepatic region.

Fig. 2. Worm transected mid-collar at 11 days

post-operation. Note that the new tissue

integrates with the old tissue and only replaces

the portion of the collar and the proboscis that

was removed. Arrowhead marks site of transec-

tion.

T. Humphreys et al.94

performed this procedure following protocols developed for

P. flava embryos and larva using a probe for the PfSoxB1 gene (Taguchi et al., 2002). It appears, as shown in Fig. 6,

that cells throughout a 3-day blastema exhibit a SoxB1 sig- nal when incubated with an antisense PfSoxB1 probe. The blastema tissue shows a strong signal for the expression of

SoxB1, while there is no evidence of a signal in the original tissue upon which the blastema formed. There is also no

signal from a sense-strand control probe in tissue cut from

the site immediately after transection or in a 3-day blastema

(data not shown). Standard in-situ hybridization procedures

can be adapted to study the course of gene expression dur-

ing regeneration in P. flava.

DISCUSSION

These observations support and extend the many prior

observations that extensive regeneration occurs in

hemichordates (Cori, 1902; Dawydoff, 1907; Kuwano, 1902;

Rao, 1955; Rychel and Swalla, 2008; Spengel, 1893; Willey,

1898). We observed universal regeneration whereever we

transected the worm. Prior reports have indicated certain

limits to regeneration after transection. These in some cases

could represent species differences, but our experience, not

discussed in detail, indicates that the health of the animals,

and clean aquaria and holding conditions for the wounded

animals, are critical for success in observing universal

regeneration at all levels.

Our observations that an average of about 2% of ani-

mals at our collection site exhibit signs of anterior regener-

ation, and that visible signs of regeneration lasted for about

3 months in the regenerated animals in our aquaria, suggest

that about 10% of P. flava individuals in this location lose their heads every year.

The rapidity and completeness of regeneration in P. flava is quite striking. The process of anterior regeneration can be divided into three phases. During the first three days

after the wounding that removes the anterior parts, the ante-

rior tissue swells and is mobilized, and the wound closes

and heals shut. When the wound closes after about three

days is the first suggestion of a group of cells assembled

into a blastema to begin the process of reforming the lost

parts of the body.

The second phase involves a very rapid increase in the

cell mass of the blastema, without any evidence of remod-

eling of original tissue, and the epimorphic molding and dif-

ferentiation of the blastema cells into the new head over the

next 10 days. We could not determine whether the cells for

the new head are derived from cell division or from mobili-

zation of cells migrating out of the original body parts. Stain-

ing with Proliferating Cell Nuclear Antigen antibody has sug-

gested there is rapid cell division at this time (Rychel and

Swalla, 2008). If the cells are derived from the old body, we

have seen no evidence that suggests this involves the

release of cells or remodeling from the old tissues in the

original body. The new head appears to just be added to the

stump of the original body, which maintains its original

appearance, as far as visual observation can tell.

The third phase of regeneration is the formation of a

new branchial region. This begins at about 12 days after the

new head is made and the animal begins to exhibit burrow-

ing behavior. At that time, an insert of blastema-like tissue

Fig. 4. Scanning electron microscope images of regenerating

blastema cell surfaces and adjacent epithelial surface. (A) Exposed

blastema cells at 3 days have a few single cilia; the blastema

appears to have cells with at least three surface textures: b, bleb-

bing cell; m, microvillar cell; s, smooth cell. (B) Cells of the original

surface are covered with cilia, as are the surfaces we have

observed on all older P. flava epithelia.

Fig. 5. Seven-day blastema with nascent proboscis (P) with lateral

ridges (LR) on each side that will grow, extend around the mouth

(M), and fuse to form the collar.

Fig. 6. In-situ hybridization, with a PfSoxB1 probe, of 5-day regen- erating head. The cells in the blastema tissue, which is organized as

a central nascent proboscis and at this stage is flattened and some-

what heart shaped, and the two lateral ridges that will form the col-

lar, show signals for SoxB1 expression. No signals were evident in

the cells of the old tissue upon which the blastema formed. p,

nascent proboscis; c, nascent collar ridges; o, original tissue.

Hemichordate Regeneration 95

appears between the new head and the old body. This insert

expands rapidly and differentiates into a branchial region

with a new gill basket. The full restoration of the missing

body parts is achieved about 5 weeks after original transec-

tion. It is another 5 weeks before the regenerated tissue

regains enough pigmentation such that visual evidence of

regeneration is lost.

The source and nature of the cells in the hemichordate

blastema, a central issue in the more studied regeneration

systems over the years, will only be resolved with further

studies. The high expression of the SoxB1 gene in the P. flava blastema cells supports the idea that blastema cells are multipotent stem-like cells, since SoxB1 is now recognized as a gene important for producing iPS cells (Takahashi, et al.,

2007). In hydra and planarians, the stem cells can be recog-

nized cytologically and are known to be scattered throughout

the tissue and to move to the site of regeneration to establish

the blastema (Birnbaum and Sanchez Alvarado, 2008), which

then grows by proliferation. However, the regeneration blast-

ema in amphibians appears to be formed by the partial dedi-

fferentiation of cells in the tissue into stem cell-like states, and

these cells then enter the blastema and form the regenerated

tissue (Kragl et al., 2009).

The demonstration that gene expression during

hemichordate regeneration can be approached by in-situ

hybridization is encouraging. The finding that the SoxB1 gene is expressed in the blastema cells, but not in the tissue

upon which the blastema forms, tends to confirm the general

idea that the blastema is an accumulation of stem cells

derived by mobilization of such cells from their niches in the

adult tissue or are cells induced to differentiate into stem

cells by the wounding process. This result confirms the

potential of in-situ hybridization and other powerful molecu-

lar approaches coupled with the genomic information that

will soon be available from sequencing of the P. flava genome (Tagawa et al., in preparation) to contribute to sig-

nificant progress in the study of regeneration in this system.

ACKNOWLEDGMENTS

This work was supported by a KAKENHI (Grant-in-Aid for

Scientific Research) on Priority Areas “Comparative Genomics” from

the Ministry of Education, Culture, Sports, Science and Technology of

Japan and by funds from the University of Hawaii Research Founda-

tion. We thank Tina Carvalho and Marilyn Dunlap of the PACIFIC Bio-

sciences Research Center, Biological Electron Microscope Facility, for

assistance with SEM preparations and interpretations.

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(Received October 26, 2009 / Accepted October 28, 2009)