Phylogeny of Thymus and Bone Marrow - Bursa Cells, R.K. Wright and E.L. Cooper eds.

@ Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands

THE IIEUATOGENIC TISSUES OF TIJNICATES

Thomas H. Ernak. DepartmenL of Physlology

Unlver.sltY of Callfornia !tredical CenterSan Fiancisco, Cal-lfornla 94143

INTRODUCTIOTiI

Tunlcates are unique among lnvertebrates in that they share such chordaLe

characterl-stics as a notochord, dorsal tubul-ar nerve cord, and pharyngeal- gill

slits with the vertebrates. The ascidlans, a group of sessile tmicates' are

generally considered to have glven rLse Lo the vertebrates via a pelaglc tadpole

latval. RecenLly, biol-ogists have turned toward these fllter feeding mrlne

animals 1n an attempt to understand the origin of the lunune t""pot"t2' 3' 4'

Speclal- attention has focused on the ascidian lynphocyte, a relatively undiffer-

entiated cell that l-s comon to all ascldian species and generates all other

blood ce1l ayp."5, 6'7. circulatlng lF'phocytes are reported to participate ln

the rejectlon of allografts2, fot rosettes wlth sheep red blood cells8, and

respond to phytohenagglutinlng.

ByautoradiograPhywlthtrltiatedthyflidine,bloodcel].sengagedlnPremitotic

DNA synthesis have been localized Ln the tlssue s of Stgela "7"n"IO. Ascidian

bl-ood cells, which constitute a renewing ceL1 populatlon' proliferate in clrcu-

lating blood and in lyaph nodules throughout the body' The ultrastructure of

several- types of bl-ood celLs has been described in blood "p".."ll'

12' 13 b.a toa

in a lynph nodule. For thLs study, a postlarval ascldian Juvenile and several

adul-t ascldians were exposed to tritlated thyrnldinel4' 15' 16 for a short time

l-nterval in order to localize sites of hematoge[ic activlty. In StgeTa clava,

the organlzation of a pharyngeal Lymph nodul"e was exanined by electron micros-

copy" "nd

the putative DNA synthesizing ce1ls described'

HEMATOGENESIS IN A POSTLARVAL JUVENILE

The Ciona intestinalis juvenile (Fig. 1) is considered to most closely

45

6

resemble the ancestral ascidi-"rr18' L9

Af ter metamorphosis, it has a beati-ng

widely spaced excurrent siphons, and

mata) on each side of the ph"ryrr*2o.

left of the pharynx.

i'and is essentially a sirnpltf ied adult.

heart , a si-ngle incurrent siphon, two

a pair of primitive gill slits (protostig-

The gut loop lies slightly behind and to the

Numerous hemocytoblasts, undlfferentiated ce1ls hrith a large, nucl"eolated

nucleus and little basophil-lc cytoplasm, and a few presmbly differentiated ce11s

cluster in the pharyngeal wall around the protostigmata and near the endostyle.

Hemocytoblasts originate fron cel1s of the archent..or2l and are especlalLy

numerous below germinal cells in the stignatlc eplthellul[. Clusters of these

prinitive blood celLs do not occur around the postpharyngeal gut. In autoradio-

grams, hemocytoblasts are labeled with tritlated thymidine (fig. 2). With

continued dlvislon and by nlgratlon, the pharyngeal hemocytoblasts w111 give rise

to henatogenLc tissue in the pharynx and around the gut of an adult Cjona. They

will probably also suppty gerninal cel-Is for the ovary and testis22.

DISTRIBUTION OI' I{EMATOGENIC SITES IN ADIJI,T ASCIDIANS

In adult ascidians, the pharynx is a greatly enlarged organ perforated by a

multltude of ciliated gill s11ts (stigmata). HemaLogenlc tissue occurs 1n the

pharyngeal wa1l, around the di-gestive tract, and anong advanced solitary species,

ln the body walI, Each ascidian fanlly exhiblts a characteristic relationshlp

befween pharynx, postpharyogeal gut (esophagus, stomach, and intestlne), and

gonads, and the distrLbution of henatogenic tlssue foLlows accordingly.

An adult Ciona intestinal.is has several of the most prinltlve ascidian body

features. The postpharyngeal gut lies slightly behind the pharlmx, and the

gonads f-ie withln the curvature of the dlgestive tract. Hematogenic tissue ls

abundant in the pharyngeal wa1l and around the gut-l-oop. A few clueters also

occur in the body lralf at the posterlor end of the body6 and where the pharynx i.s

attached to the body wa11, either under the endostyle or by mesenteries. Except

in these reglons, blood formlng tissue is absent from the body wa11.

Henatogenic cel-le Ln the pharynx of Ciona are abundant ln transverse bars

47

(Flg. 3) and scattered in longitudinal bars and al-ong the endostyle. Blood cells

are arranged 1n smalJ-, diffuse clusters adjacent to the pharyngeal ePitheliun;

ce11s labeled vlth trirlated thymidine are scattered throughout the tissue (Fig.

4). Most non-dividing cel1s are presumed to be Partially or fu1ly differentiated

blood cells. Around the gut, hematogenic tj-ssue is aLso diffuse and associated

wlth connectlve tissue or the external gut linlng. Clustered blood cells may be

so plentiful as to 1ie cLose to the stomach ePltheliun. In regions rthere the

gonads are present, hemaEogenic tissue is sparse.

Ta cheTgosona productum and Ascidia ceratodes, other solltaly ascidians whose

gonads lie within the gut LooP' most hemtogenic tissue ls around the gut and

organized into sna1l nodules containing several cel-ls whlch incorPorate tritiated

thynidlne. Lynph nodules are assoclated with connective tissue and blood chan-

nels but not the external or internal gut eplthelial nodules also frequently occur

near the gonade.

The gut-loop of most colonial ascldlans is U-shaped and lies behind the

pharyu, a delLcate structure contalning lLtt1e hernatogenlc tissue. In

PoTgcTinum p-lanum and Euherd.nania cTavitotmis, clusters of labeled blood celLs

are most abundant in the region of the gut-loop where they occur Ln small Patches

ln the connective tlssue and adjacent to the eplcardium, a singl,e pouch alongsLde

the gut but not llning it.

The gonads of advanced sPecles 1le withln the body wall' and it Ls only ln

these specles that lynph nodules are abundant ln this regl-on. In StgeJ.a c-Zava'

nost lynph nodules occur 1n the pharyngeal and body walls; few occur around the

.10postpharyngeal gut--. Although no nodules have bee4 observed below the stomach

eplthellurn, a ferr scattered ones are seen in the connectlve tissue around the

intestinal epitheliun. Lymph nodules 1n the body wall l"le next to the atrial

epithelLum and in the connectlve tissue linlng blood channels (Fig. 5)' Some

nodules also occur in the endocarps, projections of the atrial epltheliun filled

with connective tissue and blood cells. In Stye-la, a nodule conslsts of one or

two groups of ce1ls Labeled wlth trltiated thynidine surrounded by several un-

labeled ce11s. Many of the DNA syntheslzing ceLl-s are hemocytoblasts with a

4

dj-sLinct nucleolus. In lftolgufa verrucifera and Pguza haustot, where both the gut

and gonads lie withl-n the body wa1l, distlnct l,ynph nodules occur both in the

connectlve tlssue and adjacent to the atrlal epitheLLun. In the connective

tissue, nodules frequently border on blood channels (Fig. 6).

ORGA}IIZATION OF A LYMPH NODULE

,aAccording to Peres--, henocytoblasLs 1n an ascidlan lynph nodule produce

lymphocytes which Lhen give rise to all other blood celL types, I{emocytoblasts

are dlstinguished by the presence of a nucleolus. The nucl-eus of the smaller,

less basophilic lynphocyte is reported to contaln, instead, patches of chromatLn.

Contrary to Peresr findings, electron nlcroscopy of a pharyngeal lynph nodule

from StgeJa cl.ava indicates that most differentiating blood cells arise directly

from hemocytoblasts, not through a lynphocytic internediate.

Henocytoblasts, the putative DNA synthesizing cel1s of autoradiograms, occur

in clusters in the center of a lynph nodule (tr'ig. 7); a few of these cells also

occur on the perlpheral marglns. Each group of hemocytoblasts is surrounded by

maturing blood ce11s in varlous stages of dlfferentlation. Only a fev of lhe

perlpheral cells, which incLude phagocytes, compartment cells, and several types

of granular anoeboeytesl3, appear full-y matured; apparently most conpletely dif-

ferentiated cells are j-n circulating b1ood. Connective tissue flbers surround a

Fig. 1. Phase contrast light micrograph of a Ciona juvenile, six days afterfertllization. EN, endostyle; ES, esophagus; P, protostigrna;

' S, stomach. Bar in Figs. 1-6 equals 50 yn.

Tig. 2. Autoradiogram of longitudinal section through a Ciona juvenile shoninglabeled hemocytobl-asts (arrows). P, protostigna; S, stomach.

Fig. 3. Autoradiograrn of longitudinal sectlon through transverse bar (TB) andrms of stigna (ST) in pharynx of adult Ciona intestinaTis.

Fig. 4. Autoradiogram of henatogenic tissue in transverse bax of ciona showinglabeled DNA syntheslzing ceJ-ls.

Flg. 5. Autoradiogram of cross section through body wa1l of stgela ciava showingl-abeled lyrrph nodules next to atrLal eplthel-lun (AE) and in connectivetlssue. E, epldermlsl M, muscle.

I'ig. 6, Autoradlogram of lynph nodules belorr l-ntestlnal eplthellum of Pguraiaustor. Nodules border on blood channels (BC).

50

nodule and separate outLying blood cells from the pharyngeal epitheliun (Fig. 7).

Hemocytoblasts border dlrectly on one another, giving them angular outlines

(Tig. S). They measure about 5 Um in dlameter and have a large, spherical

nucleus contalning one or trrro nucleoli and 1ittle chromatin. A nucleolus nay

occur ln the center of a nucleus or adjacenL to the nuclear nembrane. Most cyto-

plasmic organelles cluster on one side of a cell. This region contains several

round or oval mitochrondria and a small Golgi apparatus. A pair of centrioles

lies on the concave side of the Golgi clsterns, usually between the cisterns and

the nucleus (Fig. 9). Uost of the cytoplasn Ls fllled with numerous polyribosomes

but a few clsterns of rough endoplasmic reticulum and an occaslonal srnall dense

granule (Flg. 8) are also present.

DifferentLatlng leucocytes around the central hemocytoblasts lose their

prominent nucleolus as the amount of chronatin i.ncreases. Electron dense

granules usually appear in the cytoplasm, however, before lhe nuclear changes are

conpleted. As one procedes away from the center of a nodul-e, naturing blood

cells Lncrease in slze; their dense granules becone larger and more numerous.

Cel-l dLfferentlatlon is also narked by the loss of polyribosones and the develop-

ment of elongate nltochondria, a larger Golgi apparatus, and long cisterns of

rough endoplasmic reticulum.

Several i.Itrphocyte-like cell's (Ffg. f0) also occur ln the peripheral parts of

a nodule. They are similar to henocytoblaats in their cytoplasmic features, but

they are usually somelrhat smal1er, measuring about 4 }lltr' The nucleus lacks a

consplcuous nucleolus, and patches. of chromatln occur al-ong the nuclear menbrane

and in the interior of the nucleus. Sl-nce the nucl-eolus is apparently Lost

durlng cell division24, it is posslbte that some of these cel1s could be sten

ce11s which Just completed cell division. Thls would account for Eheir snaller

eize ln a nodule.

Fig. 7. Electron micrograph of lyoph nodule in transverse bar froa pharyu ofstgeTa cLava. C, connectlve tlssue fibers; GR, differentiatLng granularblood ce11s; II, henocytoblast; PE, pharyngeal epithellun.

52

DISCUSSION

In prlnitive adult ascldiang, hemopoietLc tlssue occurs mainly in the

pharyngeal and gut rda1ls, This distribution j-a 11ke that ln lower vertebrates

(e.g., lanpreys), where a deflnitive thynus and spleen are "b".rrt25. In advanced

ascldians, hematogenic tissue is organized lnto distlnct nodules, where, as in

Stgela, a nodule is composed of one or two centers of divlding hemocytoblasts

surrounded by non-dividlng, naturing blood cell-s. Lynph noduLes lle within

connectlve tissue, either belolr bl-ood channels or epithella (pharyngeal,

epicardial, or atrial, but usually not digestive epithella). Those in the body

wall- of advanced specles probably followed the gonads as they nigrated anterlorly

from the gut loop. NoduLes could also have been lntroduced into the body wa1l by

way of pharyngeal mesenterles.

Ascldl-an blood cells are a renewi-ng cel1 population composed of stem, translt-

iona1, and nature cells. They have a rapid late of proliferation which, at the

steady state, is bal"anced by the rate of ce1l loss. Since lnvertebrate chordates

are the.only deuterostomes nhose blood ce1ls are renewed, tt ts likel-y that the

phylogenlc orlgl-n of verteblate hemopoietlc tissue lles lrithin the Trnicata. In

echinoderns, blood cells have the characterlstics of expandirrg popr.r1"tioo"26,

that is, a stem cel-l compartment whlch gives rise to other blood cel1 types does

not occur. Sea urchln amoebocytes have a slow rate of proliferation and show a

slight increase in the percentage of labeled ceI1s with tLme; some cells which do

not incorporate trLtiated thynldine possibly orlgl"nate from the parietal perito-

,r",-26. The starfish axlal organ, Ttedmann bodies, and other organs of the henal

systen which roere once considered to produce amoebocytes, are apparently not

specialized for henopoiesis2T.

In stgela cJava, bl-ood ce1ls are renewed within several re.k"l0. By 20 days

Fig. 8. Section through henocytoblasts 1n center of nodule. DG, dense granulelM, mitochondrion; Nu, nucleolus.

Flg. 9. Cytoplasnlc organelles Ln henocytoblast. CN, cmtrioles; G, Golgicisterne; M, rnltochondrion; N, nucleusl PR, polytibosomes.

Fig. 10. Lynphocyte-like cell surrounded by dlfferentiating blood ce11s.

il

after the LnJection of tritiated thymidlne, cells in the perlpheral parts of a

nodule ate now labeled. Cel1s in the interior of a nodule are no J-onger labeled;

presumabJ-y they have differentiated into Eaturing blood ceL1s. By 60 days' a

nodule as well- as most circulatlng blood cells are no longer labeled.

Blood cells in the periphery of a nodule are ln transitional stages of dlf-

ferentlatlon. Ful1y natured blood cells are apparently circulating Lhroughout

the body. In other renewing populations of stgela c7ava, i.e,, the gutl5 and

-28gonads-", where germinal and mature cel1s are easily distinguished because they

are spatially separated, fully differentiated cells do not proliferate. It ls'

thus, probable that most fu1ly dlfferentiated blood cells do not dlvide. Vacuo-

lated ce1ls ln stge"la are not l-abeled at one hour but are labeled in both J-ynph

nodules and circulatlng blood after 20 d"ytl0. kL Petophora annectens, compart-

ment cells and phagocytes but not other fu11y natured blood cells are reported

to be labeled by tritiated thynldine29.

Although sone differentiated bLood ce11s can apParently divlde, nost DNA syn-

thesizing ceLLs are probably henocytoblasts. These cells, which also divide in

clrculating blood, have a high nuclear-cytoplasnic ratio, a Prominent nucleolus'

sparse chromatin, numerous polyribosomes, and few cytoplasmic organeJ-1es. These

features are also shared by undl-fferentlated geminal cells in Ehe ascidian gut

eplthelirml4' 30. As stem celJ-s, hemocytoblasts are capable of reproducing then-

selves. as well as producing several- other cell types. They are specl-allzed for

ce11 division as shown by the lncorporation of tritiated thynldine lnto newly

synthesized DNA. The large nucleolus and abundant polyribosomes suggest'

respectively, active RNA and protein synthesis. Polyribosomes are considered to

be sites for the synthesls of slructural- prot"its3l, apparently to meet the

requirements of a rapidJ-y divlding cel1.

Henocytoblasts in a juvenil,e and in an adult, although both stem cells, can be

distingulshed by their kinetic behavior. In a juvenile, nost ce1l prol"iferation

ls probably for the production of more hemocyLoblasts whereas that in an adult

is for the differentiation of mature blood ceLls.

Although the cell type that gives rise to all other ascidian bLood cells has

55

traditionally been call-ed a lymphocyte, th18 terninology hae led to confuslon

among irnnunologists. Based upon vertebrate henatoLogy24, ascidian basophil-lc

blood ce1ls with a relatlvely J-arge nucJ-eus, prominent nucJ-eolus, and sparse

chromatin which have been referr<ld to as 1)rmphocyt."ll' 12' 13' 29 ^t" 'ot"

approprLately cal-Ied stem ".11"32,

hemoblasLs22, or hemocytoblasts. The sensi-

tlvity of these cells to *-r"y"29' 33 i" " characteristic shared by all relative-

J-y undifferentiated cells of vertebrates, whether ln blood, dLgestive tfacta, or

gor.d"34. The role of ascidj-an bLood ce1ls in graft re3ection2, rosette forrna-

tion8, or the response to mitogensg is not well understood. !'urther studies

deflning the functlonal characterl-stlcs of the ascidian hemocytoblast shoul-d

therefore' contribute to our understanding of the phylogeny of the vertebrate

imtrune syatem.

ACIG'IOWLEDO.{ENTS

part of thls research (a11 autoradiography and fixatlon of tissues forel-ectron nlcroscopy) was conducted at the scripps Institution of oceanography'La Jol-la, California. For support and guidance at this institution' I am grate-ful- to Dr. Nicholas D. HolLand. For his suggestlons and many stimulati-ngdiscussions, I thank Dr. Richard K. Wrlght. I am also grateful to Dr' Stephen S'

Rothman for suPPort during the preParation of this paper' The author ispresently a NIiI post-doctoral fellow at the University of Callfornla, San

Francisco.

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,*t-d

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