The FASEB Journal • Research Communication

Striving for normality: whole body regeneration througha series of abnormal generations

Ayelet Voskoboynik,*,1 Noa Simon-Blecher,†,‡ Yoav Soen,§ Baruch Rinkevich,†

Anthony W. De Tomaso,* Katherine J. Ishizuka,* and Irving L. Weissman**Department of Pathology, Stanford University School of Medicine, Stanford, California, USA;Department of Biology, Hopkins Marine Station, Pacific Grove, California, USA; †National Instituteof Oceanography, Oceanographic and Limnological Research, Tel-Shikmona, Haifa, Israel; ‡Facultyof Life Science, Bar Ilan University, Ramat Gan, Israel; and §Department of Biochemistry, StanfordUniversity School of Medicine, Stanford, California, USA

ABSTRACT Embryogenesis and asexual reproduc-tion are commonly considered to be coordinated devel-opmental processes, which depend on accurate pro-gression through a defined sequence of developmentalstages. Here we report a peculiar developmental sce-nario in a simple chordate, Botryllus schlosseri, wherein anormal colony of individuals (zooids and buds) isregenerated from the vasculature (vascular budding)through a sequence of morphologically abnormal de-velopmental stages. Vascular budding was induced bysurgically removing buds and zooids from B. schlossericolonies, leaving only the vasculature and the tunic thatconnects them. In vivo imaging and histological sectionsshowed that the timing and morphology of developingstructures during vascular budding deviated signifi-cantly from other asexual reproduction modes (theregular asexual reproduction mode in this organismand vascular budding in other botryllid species). Sub-sequent asexual reproduction cycles exhibited gradualregaining of normal developmental patterns, eventuallyleading to regeneration of a normal colony. The con-version into a normal body form suggests the activationof an alternative pathway of asexual reproduction,which involves gradual regaining of normal positionalinformation. It presents a powerful model for studyingthe specification of the same body plan by differentdevelopmental programs.—Voskoboynik, A., Simon-Blecher, N., Soen, Y., Rinkevich, B., De Tomaso, A. W.,Ishizuka, K. J., Weissman, I. L. Striving for normality:whole body regeneration through a series of abnormalgenerations. FASEB J. 21, 1335–1344 (2007)

Key Words: vascular budding � blastogenesis � development� tunicate

Embryogenesis and asexual reproduction are twodisparate developmental processes that share essentialdevelopmental stages including the establishment ofbody axes, morphogenetic patterning, organ forma-tion, and cell differentiation (1–6). The life cycle ofBotryllus schlosseri, a colonial tunicate (family Styelidae,subfamily Botryllinae), includes both sexual and asexualreproduction pathways (1–3). Despite differences in

initiation, these two reproduction modes give rise toessentially the same adult body plan (1–7). Sexualreproduction starts with egg fertilization and progressesthrough classic embryonic stages into a tadpole larvafeaturing chordate characteristics such as a tail, noto-chord, neural tube, and striated musculature (8). Uponhatching, the motile tadpole swims off from the natalcolony, settles on a suitable substratum, and metamor-phoses into the sessile body plan (9, 10). In thisinvertebrate form, B. schlosseri constitutes a colony ofindividuals (zooids and buds) undergoing cycles ofasexual reproduction by budding. All zooids and budsare connected through a vasculature network and areembedded within a gelatinous matrix (tunic). Thecommon vasculature terminated in sausage-shaped pro-trusions (ampullae; 1, 2; Fig. 1; Supplemental VideoS1). A mature colony includes three overlapping gen-erations: an adult zooid, a primary bud, and a second-ary bud. Each zooid is an autonomous, filter-feedingindividual comprising a heart, digestive tract, endostyle,branchial sac, neural complex, oral and atrial siphonsand gonads (1, 2, 11). Asexual (palleal) budding beginswith the formation of a small vesicle that breaks offfrom the parent epidermis and peribranchial epithe-lium and segregates into a blastula-like structure (1). Asthe cell proliferates, the vesicle undergoes a series ofinvaginations, differentiates, and develops into an adultzooid (1, 12, 13); Fig. 1; Supplemental Video S1). Thereplacement of zooids’ generation occurs through a syn-chronized wave of massive apoptosis in which the adultzooids are destroyed and taken over by their primary buds(14, 15; video S1). Palleal budding is synchronizedthroughout the colony and is divided into four majordevelopmental stages (A–D; ref. 16; Fig. 1; SupplementalVideo S1), a cycle that continues throughout the life spanof the colony. Thus, unlike most species, where the bodyis long-living and maintained by cellular replacements(e.g., ref. 17), B. schlosseri regenerates new colonial units

1 Correspondence: Department of Pathology, Stanford Uni-versity School of Medicine, Stanford, CA 94305, USA. E-mail:ayeletv@stanford.edu

doi: 10.1096/fj.06-7337com

13350892-6638/07/0021-1335 © FASEB

on a weekly basis while its connective vasculature andtunic remains intact (1, 2, 14, 15).

Previous studies in B. schlosseri (18–21) and in someother botryllid tunicates (22–28) revealed another re-generation pathway known as vascular budding, involv-ing an asexual regeneration of colonies from bloodvessels (rather than regeneration from buds and zooids).In Botryllus primigenus, vascular budding begins with theformation of a hollow blastula-like structure originatingfrom vascular epithelium and cells circulating in theblood (23). This structure develops into a vascular budcapable of regenerating an entire colony (23). In somespecies, vascular budding occurs concurrently with pal-leal budding (Botryllus primigenus and Botryllus delicates;refs. 23, 28), whereas in others it is initiated in theabsence of palleal budding when the colony recoversfrom hibernation or aestivation (Botrylloides leachi andBotrylloides gascoi; refs. 22, 24–27). Normal patterns andstages of development that resemble asexual reproduc-tion (palleal budding) patterns were observed in thesestudies (23, 24, 26–28).

Unlike other botryllid tunicates (23–28), vascularbudding in B. schlosseri is an induced phenomenon thatrequires the removal of zooids and buds (18–21). Thestages of this regeneration process have never beencharacterized. Using in vivo imaging and histologicalsections, we analyzed the regeneration of vascular bud-ding from initiation and over successive generations,and compared it with palleal budding. Unlike pallealbudding and vascular budding in other botryllid spe-cies, which maintain normal patterns of developmentthroughout the process (23, 24, 26–28), we found that

vascular budding in B. schlosseri involves progressionthrough a sequence of abnormal generations thatundergo gradual conversion toward a morphologicallynormal colony. Through various experimental manip-ulations, we studied the parameters required to inducevascular budding. We discovered that successful regen-eration requires colonies to have an intact vasculature,a minimal colony size, and an active blood flow. Inaddition, vascular budding can be induced only whenbuds and zooids are removed during a specific stage ofthe asexual life cycle. Incomplete removal of all budsand zooids or the presence of intact zooids and budsnext to the treated colonies prevents the regenerationof zooids from vascular origin. This indicates a suppres-sion mechanism mediated by soluble signals.

MATERIALS AND METHODS

Animals

Colonies of Botryllus schlosseri (Pallas) were collected from theMonterey Marina (Monterey, CA, USA). Hatched larvae weresettled and maintained as described previously (29). Colonieswere raised on 5 � 7.5 cm2 glass slides and kept vertically inslots of glass staining racks (3 slides per rack), with up to 18colonies per 17 L standing seawater aquariums. Colonies werekept at a 14:10 h light:dark regimen under constant 20°Ctemperature and fed four times a week with Liquifry Marine(Liquifry Co., Dorking, UK). Vascular budding experimentswere performed on 12 healthy colonies that reached theminimum size of 10 zooidal systems. These colonies werefurther subcloned and divided into 10 experimental groups.

Figure 1. Normal palleal budding in B. schlosseri. Stereomicroscopic images of palleal budding cycle are shown in panels A–Dcorresponding to developmental stages A–D, respectively. Coincident development of primary and secondary buds is outlinedin panels E–H (microscopic images). In stage A colonies (A, E), the primary buds almost completed organogenesis and thesecondary buds initiated as a thickening of the atrial wall of the primary bud (see also Fig. 3A). In stage B (B, F), the heart beatsin the primary bud and the secondary bud forms a closed sphere that will soon begin a series of invaginations reminiscent ofgastrulation. Stage C (C, G) is characterized by growth of the primary bud and organogenesis in the secondary bud. In stage D(D, H), zooids die in a wave of apoptosis while the primary buds mature and replace them. amp, ampulla; sb, secondary bud;res, zooid-resorbing zooid; bv, blood vessel; end, endostyle; h, heart; ds, digestive system; bs, branchial sac. Scale bars: A–D) �1 mm, E–H) 100 �m.

1336 Vol. 21 May 2007 VOSKOBOYNIK ET AL.The FASEB Journal

Each of the four largest colonies (1225c, 1196j, 1301d, and1210d) had one or more subclones in any of the experimentalgroups. Each of eight colonies (1105e, 1185d, 1255a, 1248a,1114a, 1129a, 1264a, 1264d) had subclones in four or moredifferent experimental groups.

Experimental design and surgical manipulations

We have studied the process of vascular budding in B.schlosseri by surgically removing buds and zooids with awheeler dissection knife (Miltex, York, PA, USA). All surgicalmanipulations were performed under a stereomicroscope(Wild Heerbrugg M7A, Gais, Switzerland). The removal ofbuds was confirmed using an inverted microscope (Diaphot200, Nikon, NY, USA). The influence of different conditionson vascular budding was examined using 10 experiments,each having only one factor changed (compare with therelevant control). All other conditions (tank size: 4 L, tem-perature: 20°C, light/dark regimen: 14/10 h, frequency ofcleaning and water replacement: once in every 1–2 days,feeding regimen: no feeding) remained invariant. In experi-ment 1, both zooids and buds were removed from 10 coloniesat stage D, with the number of zooids �8. Experiment 2tested different developmental stages (n�7, stages A–C, num-ber of zooids�8), the effect of the size of a colony was testedin experiment 3 (n�6, stage D, number of zooids�8).Experiments 4 and 5, respectively, tested the removal of theperipheral ampullae and the central vasculature in additionto the removal of zooids and buds (stage D: n�4 and numberof zooids�8, each). Experiment 6 was designed to examinethe effect of the presence of intact colonies in the same tankswith the manipulated colonies. In this experiment zooids andbuds were removed from 12 subclones (8 different genotypes,stage D; colony size�8 zooids). Manipulated and intactcolonies were placed in six different 4 L plastic tanks, with twomanipulated colonies and one intact colony in each tank. Intwo of the six tanks, the intact colony were placed on the same

slide as the manipulated colonies at a distanced of 2 cm. Inthe other four tanks, the intact and manipulated colonieswere placed on separate slides. Experiments 7 and 8 exam-ined the effects of partial removal of buds or zooids onvascular budding by removing only the buds in experiment 7(stages A–C: n�10, number of zooids�8) and removing allzooids and primary buds, leaving up to four secondary budsintact in experiment 8 (stages B–D: n�10, number of zo-oids�8). In experiment 9, cells were drawn from the vascu-lature of colonies in stage D. Between 1000 to 5000 of thesecells were injected into the vasculature of isogeneic subclonesat stages A–C (n�8, number of zooids�8). In experiment 10,buds were removed from colonies at stage D, leaving only theresorbing zooids (stage D: n�8, number of zooids�8; exper-iments 1–9, Table 1; experiment 10 described in the text).

Manipulated colonies were placed in 4 L plastic tanks (upto three colonies per tank). When the vascular buds becamezooids (filter feeding individuals), they were moved into 17 Ltanks and fed. Stereomicroscopic imaging was performedusing a stereomicroscope (Wild Heerbrugg M7A, Gais, Swit-zerland) coupled to a CCD camera (coolpix995 Nikon, NY,USA). Pictures and video clips were taken once a day duringthe first 2 wk and once every 3–7 days thereafter. The resultswere statistically analyzed using t test and Friedman ANOVAfor nonparametric data.

Time-lapse imaging

Time-lapse imaging was performed by an automated micros-copy (ImageXpress, Molecular Devices Corp., Palo Alto, CA,USA). After manipulation, colonies (stages B–D: n�10, num-ber of zooids�8) were kept on standard glass slides placed inholders that contained 10 ml of seawater. The holders weremounted onto the ImageXpress stage, and kept at roomtemperature. Regeneration areas of interest at each colonywere identified and imaged by an automated custom (VisualBasic) script. Phase contrast images at varying magnifications

TABLE 1. Experimental manipulation and regenerative potential (vascular budding) of Botryllus schlosseri coloniesa

Exp. Number/exp.manipulation: tissuesthat were left

Exp. manipulation:tissue excised

Blastogenicstage

Size(zooid

number)

Numberof

colonies

Noregeneration

only (%)

Initialbud

formationonly (%)

Abnormalzooid

formationonly (%)

Successfulvascularbuddingonly (%)

1. Vasculature and tunic Zooids and buds D �8 10 0 30 30 402. Vasculature and tunic Zooids and buds A–C �8 7 43 57 0 03. Vasculature and tunic Zooids and buds D �8 6 34 66 0 04. Central vasculature and

tunicZooids, buds, and

peripheralampullae

D �8 4 50 50 0 0

5. Peripheral vasculature(peripheral ampullae)and tunic

Zooids, buds, andcentralvasculature

D �8 4 0 100 0 0

6. Vasculature and tunic.Intact zooids in thesame slide/tank

Zooids and buds D �8 12 42 58 0 0

7. Vasculature tunic andzooids

Buds A–C �8 10 Buds regenerate from zooids developednormally

8. Vasculature tunic andfew (up to 4)secondary buds

Zooids andprimary buds

B–D �8 10 Degenerated and died, 30%Secondary buds developed into situe

inversus, 20%Secondary buds developed normallsy, 50%

9. Vasculature and tunicand stage D injectedcells (1000–5000)

Zooids and buds A–C �8 8 50 50 0 0

a Exp., experiment.

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were performed every 20–30 min during the first 2–3 daysand every 60 min on days 4–7. Images were stored in theImageXpress database. Time-lapse sequences were generatedfrom consecutive images taken from the same location.

Microinjections

Glass needles were prepared using a micropipette puller(Sutter Instruments, Movato, CA, USA). The tapped area wascut diagonally, forming a 50–60 �m diameter sharp tip.Hemolymph samples of 1–3 �l, each containing 1000–5000cells, were drawn from peripheral ampullae (stage D colo-nies) using air compressed microinjector (PLI-188, Nikon,NY, USA). The micropipette content was counted under themicroscope (Diaphot 200, Nikon, NY, USA) and immediatelyinjected into peripheral ampullae of isogeneic subclones atblastogenic stages A, B, or C (n�8, number of zooids�8).Microinjections were done under a microscope (Diaphot 200,Nikon, NY, USA). Incorporation of cells into the colonytransparent vasculature was confirmed visually using 200�magnification.

Circulation parameters measurements

Images of the colony marginal blood vessels and video clips ofcirculating hemocytes in the marginal blood vessel were takenusing a CCD camera (coolpix995 Nikon, NY, USA) coupled toa microscope (Diaphot 200, Nikon, NY, USA; 100� magnifi-cation). Number of circulating hemocytes and rates of bloodflow were measured and counted using ImajeJ software,version 1.32j (NIH, USA). The results were statistically ana-lyzed using t test and correlation tests.

Histology

Vascular budding samples (from days 1, 2, 3, 6, 8, 12, 16 ofdevelopment; n�40) were fixed in 2% paraformaldehyde for20 –30 min at room temperature, dehydrated in a gradedethanol series (70 –100%), and embedded in paraplast(Sigma-Aldrich, MO, USA). Sections (5 �m) were cut byhand-operated microtome (Leica, IL, USA) and stained withAzan Heidenhains (30) for general morphology.

RESULTS

Abnormal development through vascular budding

We studied the process of vascular budding in B.schlosseri by surgically removing all developing buds and

zooids and analyzing regenerating structures over suc-cessive generations of zooids. Time-lapse imaging re-vealed that, within a few hours after surgery, regenera-tion began in a few ampullae that harboredaggregations of undifferentiated lymphocyte-like bloodcells (as opposed to all other ampullae harboringaggregations of pigment cells; Fig. 2A; SupplementalVideo S2). Histological sections of these vascular budsfrom the first regenerating stages revealed uniquevesicle structures, each comprising an outer endothe-lial cell layer and an inner mass consisting mainly oflymphocyte-like cells (Fig. 3D). Buds developed withinthe ampullae lumen and remained connected to thecolony marginal blood vessels (Fig. 2; SupplementalVideo S2). While growing, each regenerating ampullaewas changed morphologically from a sausage-like struc-ture to an oval shape (Fig. 2B). A beating heart-likeorgan was observed in the developing bud within 1–2days after surgery (Fig. 2C, D). An endostyle-like, pig-mented structure extending on the ventral face of thedeveloping bud, stigmata-like structures, and aggrega-tions of lymphocyte and macrophages were observed inthe bud 3 days after removal of zooid and bud (Fig. 2F).

The timing and morphology of developing structuresduring vascular budding and thereafter deviated signif-icantly from the normal development of palleal bud-ding. The heart-like organ was the first to develop andwas observed in the vascular bud 1–2 days after removalof zooids and buds. This organ controlled blood flow toand from the regeneration area and was necessary forvascular bud development; vascular zooids were devel-oped only from vascular buds that developed hearts(Supplemental Video S3). Vascular buds at days 3–4were extensively colonized by macrophages (Fig. 2F,histological sections not shown); by day 6 of vascularbudding, organs (digestive system, endostyle, branchialsac; Fig. 3E) were fully developed. In contrast, duringregular palleal budding in this species, heart beatingwas observed only at day 10 of bud development (Fig.1B). Likewise, colonization by macrophages and organdevelopment in palleal budding occurred later than invascular budding (days 15 and 8, respectively). Histo-logical sections of regenerating buds revealed unusual

Figure 2. Vascular bud formation. Images weretaken at various times during the first 73 h of thevascular bud formation (ventral view). A) Regener-ation area 1 h after zooid and bud removal from acolony in stage D. Budding was initiated and devel-oped in ampullae 1 and 2. The tips of these ampul-lae contained aggregations of lymphocyte-like cells(unlike other ampullae in the colony). B) 7 h aftermanipulation, the tip of ampulla 1 grew andchanged its shape (vascular bud 1). A functionalheart was observed in ampulla 1 after 14 h (C). Thebud grew in size (D). An endostyle-like, pigmentedstructure was observed on the ventral face of thedeveloping bud after 54 h (E). F) 73 h vascular buds(1 and 2) contained a heart, endostyle-like, stigmata-like structures, and aggregations of lymphocyte andmacrophages. h, hour; vb, vascular bud; endo, en-dostyle-like; amp, ampulla; bv, blood vessel; mac,macrophages. Scale bar � 100 �m.

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epithelial cell morphology and abnormal cell-cell junc-tions (Fig. 3C vs. Fig. 3E, F and Fig. 4A, C vs. Fig. 4B, D).In addition, most of the animal’s cavities lined by theepithelia (e.g., the atrium) were loaded with circulatingcells; in palleal budding, cavities were empty (Figs. 3,Fig. 4). Vascular buds also had abnormal size, morphol-ogy, and orientation compared with normal buds (Fig.5). By day 6, regenerating buds were significantly larger(0.28�0.26 mm2, n�5, after bud and zooid removal vs.0.055�0.006 mm2, n�7, in normal, palleal budding ttest, P�0.005). The fully developed vascular buds (atthe completion of organogenesis) were smaller thanregular buds (1.46�0.33 mm2, n�5, vs. 2.58�0.31mm2, n�6, respectively t test, P�0.005), with an orien-tation rotated by 45o to 90o vs. orientation of a normalbud (Fig. 5E vs. F). Completion of vascular bud devel-opment occurred � 12 days after surgery with theformation and opening of spontaneously contractingsiphons. Thus, by the end of first regeneration cycle,the structure, size, and histology of the regenerated

colony were significantly different from those of theunperturbed colony.

From that point on, regeneration proceededthrough subsequent cycles of budding from regener-ated buds, rather than budding from the vasculature.However, the structures and locations of these budsremained different for several generations of asexualreproduction by palleal-like budding. The bud of thesecond generation originated from the body wall of thevascular bud; unlike normal palleal budding, the initi-ation site was located near the endostyle region ratherthan the lateral wall of the zooid (the normal pallealbudding site). Since the vascular bud’s position in thetunic was perpendicular to a normal palleal bud, thesecond-generation bud, had the same net orientationas unperturbed colony with respect to the colonyvasculature. As in vascular bud, the development of thesecond-generation bud was accelerated and exhibitedunexpected timing of organogenesis. The second-gen-eration bud took over the vascular zooid within 2 to 4days (Fig. 6B) before completion of organ develop-ment. After takeover, this bud completed its develop-ment and gave rise to a third-generation bud, whichalso originated near the endostyle area. The thirdgeneration developed over 6 days. In 40% of regener-ation cases, the morphology, size, and orientation ofsucceeding generations of buds and zooids gradually

Figure 3. Histological analysis of normal blastogenesis (A–C)vs. vascular budding (D–F) sections were stained with azanheidenhain, which stains nuclei and cytoplasm in red, colla-genic and reticular fibers as well as mucus in blue or darkblue. A) Day 2 palleal bud. The bud arises from the lateral wallof its parent, creating an epidermal disc that will later pinchoff to form a sphere. B) Day 6 palleal bud (encircled).Subdivisions of 3 small spheres have recently been completed;2 of the subdivisions can be seen in this section. C) A fullydeveloped bud (day 14) before takeover. Its organs—en-dostyle, branchial sac, and digestive system—can be seenhere. D) Day 1 vascular bud within an ampulla connected tothe colony marginal blood vessel. The bud (encircled) com-prises an outer single layer of epidermis and an inner mass oflymphocyte-like cells. E) Day 6 vascular bud exhibiting earlyformation of organs (e.g., digestive system, branchial sac,stigmata, and endostyle); all have an abnormal structure(compare to panel C). F) First-generation vascular zooid: 2days after it opened its siphons are already in a deterioratingstate. The zooid is rotated 45o relative to normal position. Asecond-generation bud (encircled) formed near the en-dostyle of the vascular zooid. This bud looked abnormal, too.bv, blood vessel; mbv, marginal blood vessel; hemoblastlymphocyte cell aggregation; ec, endothelial cell; full mac,full macrophagel; endo, endostyle; stig, stigmata; ds, digestivesystem; bs, branchial sac; cl, cloacal. Scale bars: A, D) 10 �m;B, C, E, F) 100 �m.

Figure 4. Histological sections of the digestive system andendostyle in normal zooid (A, C) vs. vascular bud (B, D). A)Cross section of normal endostyle near the endostyle groovethat contains a series of distinct groups of columnar epithelialcells and a cell free cavity. B) Cross section of the vascular budendostyle. The epithelial cells of the endostyle groove exhibitunusual morphology and organization as well as irregularcell-cell connections. The putative cavity region containsmany cells. C) Cross section of normal digestive systemepithelial cells. D) Cross section through the vascular buddigestive system exhibiting fewer organized epithelial struc-tures. Stain, azan heidenhain; scale bar � 50 �m.

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became normal. Within an average of 7 � 0.8 buddingcycles, the developed buds no longer differed morpho-logically from regular palleal budding (Fig. 6; success-ful vascular budding experiment 1, Table 1). In 30% ofthe cases of regeneration, successive generations re-mained abnormal and the colonies died within 60–70days after surgery (“abnormal formation only” in exper-iment 1, Table 1; supplemental Fig. 1). In the remain-ing 30%, only initial buds were formed, but organs(heart, digestive system, branchial sac, endostyle) failedto develop (“initial bud formation,” experiment 1 inTable 1; supplemental Fig. 1).

Successful regenerating colonies survived under ourculturing conditions for 221–260 days after surgery,thereby demonstrating conversion of abnormal bud-ding process into an unperturbed body plan of B.schlosseri.

Conditions for a successful vascular budding

To characterize the conditions required for successfulvascular budding, we removed buds and zooids atdifferent blastogenic stages and conditions. In some,the vasculature was further manipulated by removingthe peripheral ampullae or the central vasculature.Experiments were carried out on colonies containingone to four zooidal systems. Removal of buds andzooids yielded four types of responses (Table 1): 1) noregeneration, 2) initiation of a vascular bud that did notcomplete its development (initial bud formation, sup-plemental Fig. 1) and bud initiation, followed by suc-cessive generations of abnormal budding 3) with or 4)without conversion into a normal colony (successful

Figure 5. Light micrographs of palleal budding (A, C, E;dorsal view) vs. vascular bud development (B, D, F; dorsalview). A) Day 4 palleal bud (outlined in circle) consists of aclosed double-layer vesicle comprising an outer single layeredepidermal sphere and an inner single-layered vesicle of atrialepithelium. The space between the layers is filled with bloodcells. B) Day 3 vascular bud (outlined in circle) is significantlylarger and different in structure. C) Day 6 palleal bud looksempty compared with day 6 vascular bud. D) Day 6 organ-containing vascular bud. E) Day 13 normal zooid that hasrecently opened its siphons. The endostyle, which is extend-ing medially on the zooid ventral face, is marked by a dottedline. Its primary bud is located on the zooid lateral wall. F) Day12 vascular zooid has recently opened its siphons and differsin orientation, size, and pigmentation pattern. The midsagit-tal plane of the zooid is perpendicular to normal orientation;as a result, the endostyle (marked in dotted line) is perpen-dicular to its normal orientation. Its bud is budding from theendostyle area. mbv, marginal blood vessel; amp, ampulla; b,bud; endo, endostyle; os, oral siphon; as, atrial siphon. Scalebars: A–C) 125 �m, D–F) 1 mm.

Figure 6. Gradual acquisition of normal patterns in a regen-erating colony. This colony, whose vascular bud formation ispresented in Fig. 5B, D, F, became normal after eight abnor-mal generations. A) A fully developed vascular bud, itsorientation, shape, size, pigmentation, and organ morphol-ogy are all abnormal. The next generation bud is developingnear the endostyle (dotted line). B) First-generation zooidand its bud: the second-generation bud is taking over thevascular zooid only 3 days after its siphon opening (comparedwith 6–7 days normal duration) and before completion of itsorgan development. The bud endostyle (dotted line) orien-tation is rotated in 90o relative to normal endostyle orienta-tion. C) Fifth-generation zooid (still unusual). D) Seventh-generation zooid: the shape, size, and general morphologyappear more normal. Orientation of the zooids is slightlyrotated relative to normal (�25o); the next generation stillbuds from an unusual position. E) Ninth generation. Thecolony is heavily pigmented but otherwise appears to benormal; endostyle orientation (dotted line) is normal. F)Eleventh generation exhibits a normal colony. endo, en-dostyle; amp, ampulla; as, atrial siphon; os, oral siphon; sec,secondary. Scale bar � 1 mm.

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vascular budding, and abnormal zooid formation, re-spectively). Successful vascular budding was observedonly when all buds and zooids were removed duringblastogenesis stage D (the apoptotic stage, Fig. 1D).The chance of zooid formation after bud and zooidremoval during this stage was 70% (n�10, experiment1, Table 1). Removal of buds and zooids during blasto-genic stages A–C, though capable of inducing initialvascular bud development (�57%, n�7, experiment 2,Table 1), failed to develop a functional heart, andregeneration then ceased. Successful vascular buddingalso depends on colony size, it did not occur in colonieswith less than 9 zooids, not even in colonies in stage D(n�6, experiment 3, Table 1). Removal of either theperipheral ampullae (leaving only the central vascula-ture; n�4, experiment 4, Table 1) or the centralvasculature (leaving only the peripheral ampullae’sn�4, experiment 5, Table 1) completely inhibitedsuccessful regeneration, indicating the importance ofvasculature integrity in this type of vascular budding.The potential of regeneration in colonies with �8zooids whose buds and zooids were removed at stage D(Table 1, experiment 1) was significantly higher(P�0.05, Friedman 2-way analysis of variance test fornonparametric data) than those with buds and zooidsremoved under any other experimental condition (Ta-ble 1, experiments 2–6, 9).

Inhibition of a successful vascular budding by zooidsand buds

Since vascular budding does not occur under normalconditions, we tested the effects of intact or ectopicallyintroduced zooids or buds on the regeneration process.Placing intact zooids or buds next to (on the sameslide) or in the same tank (4 L, on a different slide)with stage D zooid- and bud-excised colonies, inhibitedvascular budding (n�12, experiment 6, Table 1), sug-gesting that diffusible factors may be involved in itssuppression. Similarly, removal of buds alone duringblastogenic stage A, B, or C while leaving the zooids andthe vasculature intact resulted in exclusively normalregeneration of the remaining zooids via palleal bud-ding (n�10, experiment 7, Table 1). Keeping stageB–D secondary buds after the removal of primary budsand zooids (n�10, experiment 8, Table 1) led in somecases to the development of normal and left-rightinverted (situes inversus viscerum, n�2 of 10) coexist-ing zooids, but never to complete vascular budding.Finally, removal of all primary and secondary budsduring the takeover phase led to a reversed takeoverprocess, where resorbing zooids reattached to the col-ony marginal vessel and budding of the body wall of thedegenerating zooid occurred (n�8). We therefore con-cluded that the presence of any type of buds, zooids, ordeteriorating zooids in the same tank, even withoutphysical contact with the colony, prevents regenerationof vascular zooids through vascular budding.

Correlation between circulation parametersand regeneration

Bud initiation was associated with active angiogenesisand remodeling of the colony vasculature manifestedby extensive migration, fusion, and branching of non-regenerating ampullae (Fig. 7A–C vs. Fig. 7D–F; Supple-mental Video S4).

Comparative examination of blood flow rate in col-onies that failed to regenerate with successful regener-ating colonies revealed that the regeneration is associ-ated with higher flow rates and rapid changes in thecolony vasculature architecture (Supplemental VideoS4A vs. S4B). Average flow rate measured in regener-ating colonies 1 day after bud and zooid removalwas �10-fold higher (t test, P��0.005) than in coloniesthat failed to regenerate (192�30 cells/min vs. 17�6cells/min, respectively Fig. 8). Correlation coefficientof r � 0.97 was measured between regeneration poten-tial and rate of blood flow. We examined the numberand flow rates of hemocytes in blastogenic stage D vs.stages A–C to test the accountability of stage-dependentcirculation features for the dependence of regenera-tion on developmental stage. The number of circulat-ing hemocytes in stage D was 4-fold higher than instages A–C (6.1�1.9 cells/1000 �m2, and 1.6�0.35cells/1000 �m2, respectively, with P�0.005 in t test);flow rate was �10-fold faster in stage D (509�180cells/min vs. 50�32 cells/min, respectively; t test,P�0.005, Supplemental Video S5A vs. S5B).

To test the possible contribution of stage-specific cellcomposition (e.g., stage D-specific presence of circulat-ing stem cells) to successful vascular budding, weremoved buds and zooids at stages A–C and injected1000–5000 circulating cells of stage D collected fromisogeneic colonies (number of injected cells was deter-mined according to Laird et al., ref. 31). The engraftedcells did not enable development of vascular zooidsthrough vascular budding in colonies at stages A–C(n�8; experiment 9, Table 1), suggesting that stage-specific composition of circulating cells is not sufficientto explain the inability to regenerate via vascular bud-ding in stages A–C.

DISCUSSION

This study describes the induction and progression of awhole body regeneration phenomenon in a simplechordate, Botryllus schlosseri, in which the entire colonyof interconnected reproduction units (buds and zooids)regenerated from the vasculature. Unlike vascular bud-ding in other botryllid ascidians (23–27), we demon-strated that vascular budding in B. schlosseri progressesthrough multiple generations of abnormal intermedi-ate body forms, exhibiting gradual conversion into anormal colony.

1341STRIVING FOR NORMALITY

Striving for normality: an alternativedevelopmental pathway

Conversion into a final configuration of body form is auniversal theme in developmental systems and is com-monly regarded as a precisely orchestrated programthat is deeply embedded in the genome (32, 33). Thisprogram relies on progression through defined states

and is not expected to withstand extreme arbitraryperturbations. For example, when pluripotent stemcells are detached from their normal, regulative envi-ronment, they often give rise to progressively abnormalstructures (e.g., teratoma, cancer; refs. 34–36). There-fore, the gradual conversion into a normal colony in B.schlosseri most likely reflects induction of an alternative,embryonic-like, developmental program that is invokedonly in the absence of buds and zooids. Our time-lapserecords and histological sections suggested that at leastsome of the ampullae contained a blastogenic tissuethat is triggered to develop upon removal of all budsand zooids. The remarkable differences in vascularbudding organogenesis compared with palleal budding(e.g., the expedited heart development, and the differ-ent histological structures) are also consistent withinduction of an alternative developmental pathway.

The differences between vascular and palleal bud-ding developmental programs might reflect in part thedifferences between the environment inside ampullaeand the environment inside buds (or zooids), respec-tively. Whereas in palleal budding the axes and orien-tation of a newly formed bud are influenced by posi-tional information provided by the parent bud (12, 21,37, 38), in vascular budding the first bud responds tocues in the ampullae. Indeed, the vascular system iscapable of affecting bud axes; the posterior end of

Figure 7. Vascular remodeling during successful (A–C) and unsuccessful vascular budding (D–F) A) Ampullae and marginalblood vessel in a stage D colony 1 h after complete removal of buds and zooids. Ampullae exhibit a sausage-like shape; eachampulla is connected through a short blood vessel to the colony marginal blood vessel. B) 2 days later, the ampullaemigrated within the tunic and created a new vascular connection by fusing with each other. C) 3 days after zooid and budremoval, the ampullae fuse with the marginal blood vessel and form a wider vessel able to support rapid cell movementto the regeneration area. D) Ampullae and blood vessels in a stage C colony 1 h after zooid and bud removal. Ampullaeand blood vessels contain fewer cells and differ in shape from stage D vessels (plate A). E) One day later, fewer ampullaefusions were formed compared with successful regeneration. F) On day 4, the vasculature is degenerated and blood flowceases. Scale bar � 100 �m.

Figure 8. Comparison between average blood flow rates ofcolonies that regenerated vs. colonies that failed to regener-ate. Blood flow rates were measured at the vasculature 1 dayafter bud and zooid removal.

1342 Vol. 21 May 2007 VOSKOBOYNIK ET AL.The FASEB Journal

zooids developed from an early-stage bud engraftmentinto a vascularized tunic was localized at the intersec-tion of the bud and the entering vessel (21). In vascularbudding, the second-generation buds initiate from theinitial (vascular) bud. Thus, the second generation isexposed to different, presumably closer to normal,“bud-like” cues. Subsequent generations are exposed toenvironments that gradually, from one generation tothe succeeding one, appear to be closer to the normalenvironment, eventually converging into the genera-tion of a normal colony. Thus, the gradual establish-ment of normal axes and body form might reflect theconvergence of positional information set by eachparent.

Repression of an unfavorable developmental pathway

The long delay between the onset of vascular buddingand establishment of normal colonies might render itunfavorable compared with other budding modes. As-suming that reproduction by vascular budding in B.schlosseri is a viable but unfavorable pathway reservedfor loss of other budding modes, there should be amechanism to prevent it under normal circumstances.We found that when leaving even a single bud in thecolony, or if untreated colonies with zooids are placedin the same 4 L tank, no vascular budding occurs. Theprevention of vascular budding induced by distant budsor zooids suggests that suppressive diffusible factors areinvolved. These suppressive factors may be activated bythe buds or zooids themselves or could also be activatedby the vasculature (or even the tunic) affected by thepresence of normal buds or zooids. Inhibition of onebudding mode by groups of cells or by a different modehas been reported in colonial tunicates (39, 40). More-over, diffusible substances secreted by parent zooidshave been shown to affect the behavior of growing buds(41–43). The defined substances thyroxine and thio-urea have been implicated in either suppression oracceleration of budding, respectively (44). Tunicates,the closest living relative of vertebrate (45), evolvevarious patterns of development. While solitary tuni-cates propagate exclusively by sexual reproduction,colonial tunicates reproduce both sexually and asexu-ally, and are capable of producing buds (2, 7). Colonialtunicates use different modes of budding for bothproliferation and survival (5, 6). Two budding featuresdifferentiate botryllid ascidians from other tunicates:Botryllid ascidians are the only tunicates with thecapacity to produce palleal buds (in which the bud isderived from the lateral wall of the parent and involvesthree layers: atrial epithelium, blood, and epidermis).This subfamily is also the only one in which thedevelopmental stages of buds are synchronized in thecolony (2). Whereas in some botryllid ascidians vascularbudding occurs spontaneously (either simultaneouslywith palleal budding: Botryllus primigenus and Botryllusdelicates; see refs. 23, 28) or when the colony recoversfrom aestivation (Botrylloides sp; refs. 24–27), in Botryllusschlosseri vascular budding is strictly an inducible pro-

cess. Under normal laboratory and field conditions,palleal budding is the only mechanism of asexualdevelopment observed in this species.

Role of blood circulation in supporting the programof vascular budding

The high correlation between blood flow rate and theability to regenerate via vascular budding suggest thathigh flow rate is essential for successful regeneration.Dependency on high flow rate might account for thefailure of regenerating at stages A–C, which have slug-gish blood flow rates compared with stage D (Fig. 1D;video S5). Enhanced blood flow can encourage regen-eration by increasing the supply of oxygen and nutri-ents to the regenerating area. High demand for oxygenand nutrients by the developing bud can also suggestthe necessity of heart development at early stages ofregeneration in the first 2 days of vascular buddingcompared with 10 days during normal palleal budding.

In the absence of buds and zooids, the ampullae (46,47; Supplemental Video S1) and the vasculature keepthe circulation by synchronized contractions (Supple-mental Video S1). These contractions may be sufficientto drive the initial blood circulation required for theformation of a heart. Further regeneration is likely todepend on a proper blood circulation maintained bythe heart. This need for high flow rate can explain theaccelerated heart formation, as observed in this work.

Multiple routes to the same body form: B. schlosseri asa model for alternative embryonic programs

B. schlosseri appears to exhibit at least three indepen-dent embryonic programs (i.e., sexual reproduction,asexual palleal budding and vascular budding), allprogressing through distinct stages and body forms andleading eventually to indistinguishable colonies. Theintriguing convergence of three distinct programs intoa single morphological module, makes it an attractivemodel for studying alternative specifications of posi-tional information. This includes mechanisms for con-text-dependent regulation of developmental process,and mechanisms for switching between reproductionmodes (e.g., from palleal budding to sexual reproduc-tion or to vascular budding).

The relatively high abundance of colonies that fail tocomplete the program of vascular budding (86% n�51Table 1, experiments 1–6, 9) is likely to assist inunderstanding the mechanisms underlying the variousstages of vascular budding. Further characterization ofthe stepwise variations in the spatial patterns of posi-tional clues and hox gene expression in B. schlosseriwould likely shed new light on the mechanisms formaintaining and switching between alternative modesof embryonic development.

We thank Elizabeth Moiseeva for histological work of art,Karla Palmeri, Amir Voskoboynik, and Stefano Tiozzo forcritical reviews of the manuscript, and Libue Jerberak, Chris

1343STRIVING FOR NORMALITY

Patton, and Randy Will for technical and mariculture assis-tance. This study was supported by the National Institutes ofHealth (R01-DK54762) and by a grant from the U.S.-IsraelBinational Science Foundation (2003/010). N.S.-B. was adoctoral fellow of the Ch. Clore Foundation.

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Received for publication September 27, 2006.Accepted for publication December 25, 2006.

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