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Ermak styela clava hematogenic tissues 1976


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Ermak styela clava hematogenic tissues 1976

  1. 1. 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 Center San Fiancisco, Cal-lfornla 94143INTRODUCTIOTiI Tunlcates are unique among lnvertebrates in that they share such chordaLecharacterl-stics as a notochord, dorsal tubul-ar nerve cord, and pharyngeal- gillslits with the vertebrates. The ascidlans, a group of sessile tmicates aregenerally considered to have glven rLse Lo the vertebrates via a pelaglc tadpolelatval. feeding mrlne RecenLly, biol-ogists have turned toward these fllter 3 4animals 1n an attempt to understand the origin of the lunune t""pot"t2Speclal- attention has focused on the ascidian lynphocyte, a relatively undiffer-entiated cell that l-s comon to all ascldian species and generates all other 67. circulatlng lFphocytes are reported to participate lnblood ce1l ayp."5, 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 12 13 b.a toa several- types of bl-ood celLs has been described in blood "p".."ll in a lynph nodule. For thLs study, a postlarval ascldian Juvenile and several 15 16 for a short time adul-t ascldians were exposed to tritlated thyrnldinel4 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" the putative DNA synthesizing ce1ls described "nd HEMATOGENESIS IN A POSTLARVAL JUVENILE The Ciona intestinalis juvenile (Fig. 1) is considered to most closely 45
  2. 2. 6 i L9resemble the ancestral ascidi-"rr18 and is essentially a sirnpltf ied adult.Af ter metamorphosis, it has a beati-ng heart , a si-ngle incurrent siphon, twowidely spaced excurrent siphons, and a pair of primitive gill slits (protostig-mata) on each side of the ph"ryrr*2o. The gut loop lies slightly behind and to theleft of the pharynx. Numerous hemocytoblasts, undlfferentiated ce1ls hrith a large, nucl"eolatednucleus and little basophil-lc cytoplasm, and a few presmbly differentiated ce11scluster in the pharyngeal wall around the protostigmata and near the endostyle.Hemocytoblasts originate fron cel1s of the archent..or2l and are especlalLynumerous below germinal cells in the stignatlc eplthellul[. Clusters of theseprinitive blood celLs do not occur around the postpharyngeal gut. In autoradio-grams, hemocytoblasts are labeled with tritlated thymidine (fig. 2). Withcontinued dlvislon and by nlgratlon, the pharyngeal hemocytoblasts w111 give riseto henatogenLc tissue in the pharynx and around the gut of an adult Cjona. Theywill 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 amultltude of ciliated gill s11ts (stigmata). HemaLogenlc tissue occurs 1n thepharyngeal wa1l, around the di-gestive tract, and anong advanced solitary species,ln the body walI, Each ascidian fanlly exhiblts a characteristic relationshlpbefween pharynx, postpharyogeal gut (esophagus, stomach, and intestlne), andgonads, and the distrLbution of henatogenic tlssue foLlows accordingly. An adult Ciona has several of the most prinltlve ascidian bodyfeatures. The postpharyngeal gut lies slightly behind the pharlmx, and thegonads f-ie withln the curvature of the dlgestive tract. Hematogenic tissue lsabundant in the pharyngeal wa1l and around the gut-l-oop. A few clueters alsooccur in the body lralf at the posterlor end of the body6 and where the pharynx i.sattached to the body wa11, either under the endostyle or by mesenteries. Exceptin these reglons, blood formlng tissue is absent from the body wa11. Henatogenic cel-le Ln the pharynx of Ciona are abundant ln transverse bars
  3. 3. 47(Flg. 3) and scattered in longitudinal bars and al-ong the endostyle. Blood cellsare 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 differentiatedblood cells. Around the gut, hematogenic tj-ssue is aLso diffuse and associatedwlth connectlve tissue or the external gut linlng. Clustered blood cells may beso plentiful as to 1ie cLose to the stomach ePltheliun. In regions rthere thegonads are present, hemaEogenic tissue is sparse. Ta cheTgosona productum and Ascidia ceratodes, other solltaly ascidians whosegonads lie within the gut LooP most hemtogenic tissue ls around the gut andorganized into sna1l nodules containing several cel-ls whlch incorPorate tritiatedthynidlne. Lynph nodules are assoclated with connective tissue and blood chan-nels but not the external or internal gut eplthelial nodules also frequently occurnear the gonade. The gut-loop of most colonial ascldlans is U-shaped and lies behind thepharyu, a delLcate structure contalning lLtt1e hernatogenlc tissue. InPoTgcTinum p-lanum and Euherd.nania cTavitotmis, clusters of labeled blood celLsare most abundant in the region of the gut-loop where they occur Ln small Patchesln the connective tlssue and adjacent to the eplcardium, a singl,e pouch alongsLdethe gut but not llning it. The gonads of advanced sPecles 1le withln the body wall and it Ls only lnthese specles that lynph nodules are abundant ln this regl-on. In StgeJ.a c-Zavanost lynph nodules occur 1n the pharyngeal and body walls; few occur around the .10postpharyngeal gut--. Although no nodules have bee4 observed below the stomacheplthellurn, a ferr scattered ones are seen in the connectlve tissue around theintestinal epitheliun. Lymph nodules 1n the body wall l"le next to the atrialepithelLum and in the connectlve tissue linlng blood channels (Fig. 5) Somenodules also occur in the endocarps, projections of the atrial epltheliun filledwith connective tissue and blood cells. In Stye-la, a nodule conslsts of one ortwo 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. 4. 4dj-sLinct nucleolus. In lftolgufa verrucifera and Pguza haustot, where both the gutand gonads lie withl-n the body wa1l, distlnct l,ynph nodules occur both in theconnectlve tlssue and adjacent to the atrlal epitheLLun. In the connectivetissue, nodules frequently border on blood channels (Fig. 6).ORGA}IIZATION OF A LYMPH NODULE ,a According to Peres--, henocytoblasLs 1n an ascidlan lynph nodule producelymphocytes which Lhen give rise to all other blood celL types, I{emocytoblastsare 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 nodulefrom StgeJa cl.ava indicates that most differentiating blood cells arise directlyfrom hemocytoblasts, not through a lynphocytic internediate. Henocytoblasts, the putative DNA synthesizing cel1s of autoradiograms, occurin clusters in the center of a lynph nodule (trig. 7); a few of these cells alsooccur on the perlpheral marglns. Each group of hemocytoblasts is surrounded bymaturing blood ce11s in varlous stages of dlfferentlation. Only a fev of lheperlpheral cells, which incLude phagocytes, compartment cells, and several typesof granular anoeboeytesl3, appear full-y matured; apparently most conpletely dif-ferentiated cells are j-n circulating b1ood. Connective tissue flbers surround aFig. 1. Phase contrast light micrograph of a Ciona juvenile, six days after fertllization. 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 shoning labeled hemocytobl-asts (arrows). P, protostigna; S, stomach.Fig. 3. Autoradiograrn of longitudinal sectlon through transverse bar (TB) and rms of stigna (ST) in pharynx of adult Ciona intestinaTis.Fig. 4. Autoradiogram of henatogenic tissue in transverse bax of ciona showing labeled DNA syntheslzing ceJ-ls.Flg. 5. Autoradiogram of cross section through body wa1l of stgela ciava showing l-abeled lyrrph nodules next to atrLal eplthel-lun (AE) and in connective tlssue. E, epldermlsl M, muscle.Iig. 6, Autoradlogram of lynph nodules belorr l-ntestlnal eplthellum of Pgura iaustor. Nodules border on blood channels (BC).
  5. 5. 50nodule 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, sphericalnucleus contalning one or trrro nucleoli and 1ittle chromatin. A nucleolus nayoccur 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 severalround or oval mitochrondria and a small Golgi apparatus. A pair of centrioleslies on the concave side of the Golgi clsterns, usually between the cisterns andthe nucleus (Fig. 9). Uost of the cytoplasn Ls fllled with numerous polyribosomesbut a few clsterns of rough endoplasmic reticulum and an occaslonal srnall densegranule (Flg. 8) are also present. DifferentLatlng leucocytes around the central hemocytoblasts lose theirprominent nucleolus as the amount of chronatin i.ncreases. Electron densegranules usually appear in the cytoplasm, however, before lhe nuclear changes areconpleted. As one procedes away from the center of a nodul-e, naturing bloodcells 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 ofrough endoplasmic reticulum. Several i.Itrphocyte-like cells (Ffg. f0) also occur ln the peripheral parts ofa nodule. They are similar to henocytoblaats in their cytoplasmic features, butthey are usually somelrhat smal1er, measuring about 4 }lltr The nucleus lacks aconsplcuous nucleolus, and patches. of chromatln occur al-ong the nuclear menbraneand in the interior of the nucleus. Sl-nce the nucl-eolus is apparently Lostdurlng cell division24, it is posslbte that some of these cel1s could be stence11s which Just completed cell division. Thls would account for Eheir snallereize ln a nodule.Fig. 7. Electron micrograph of lyoph nodule in transverse bar froa pharyu of stgeTa cLava. C, connectlve tlssue fibers; GR, differentiatLng granular blood ce11s; II, henocytoblast; PE, pharyngeal epithellun.
  6. 6. 52DISCUSSION In prlnitive adult ascldiang, hemopoietLc tlssue occurs mainly in thepharyngeal and gut rda1ls, This distribution j-a 11ke that ln lower vertebrates(e.g., lanpreys), where a deflnitive thynus and spleen are In advanced "b".rrt25.ascldians, hematogenic tissue is organized lnto distlnct nodules, where, as inStgela, a nodule is composed of one or two centers of divlding hemocytoblastssurrounded by non-dividlng, naturing blood cell-s. Lynph noduLes lle withinconnectlve tissue, either belolr bl-ood channels or epithella (pharyngeal,epicardial, or atrial, but usually not digestive epithella). Those in the bodywall- of advanced specles probably followed the gonads as they nigrated anterlorlyfrom the gut loop. NoduLes could also have been lntroduced into the body wa1l byway 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 thesteady state, is bal"anced by the rate of ce1l loss. Since lnvertebrate chordatesare the.only deuterostomes nhose blood ce1ls are renewed, tt ts likel-y that thephylogenlc orlgl-n of verteblate hemopoietlc tissue lles lrithin the Trnicata. Inechinoderns, 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 doesnot occur. Sea urchln amoebocytes have a slow rate of proliferation and show aslight increase in the percentage of labeled ceI1s with tLme; some cells which donot incorporate trLtiated thynldine possibly orlgl"nate from the parietal perito-,r",-26. The starfish axlal organ, Ttedmann bodies, and other organs of the henalsysten which roere once considered to produce amoebocytes, are apparently notspecialized for henopoiesis2T. In stgela cJava, bl-ood ce1ls are renewed within several re.k"l0. By 20 daysFig. 8. Section through henocytoblasts 1n center of nodule. DG, dense granulel M, mitochondrion; Nu, nucleolus.Flg. 9. Cytoplasnlc organelles Ln henocytoblast. CN, cmtrioles; G, Golgi cisterne; M, rnltochondrion; N, nucleusl PR, polytibosomes.Fig. 10. Lynphocyte-like cell surrounded by dlfferentiating blood ce11s.
  7. 7. ilafter the LnJection of tritiated thymidlne, cells in the perlpheral parts of anodule 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 anodule 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 Lhroughoutthe body. In other renewing populations of stgela c7ava, i.e,, the gutl5 and -28gonads-", where germinal and mature cel1s are easily distinguished because theyare spatially separated, fully differentiated cells do not proliferate. It lsthus, 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-ynphnodules and circulatlng blood after 20 d"ytl0. kL Petophora annectens, compart-ment cells and phagocytes but not other fu11y natured blood cells are reportedto 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 inclrculating blood, have a high nuclear-cytoplasnic ratio, a Prominent nucleolussparse chromatin, numerous polyribosomes, and few cytoplasmic organeJ-1es. Thesefeatures are also shared by undl-fferentlated geminal cells in Ehe ascidian gut 30. As stem celJ-s, hemocytoblasts are capable of reproducing then-eplthelirml4selves. as well as producing several- other cell types. They are specl-allzed force11 division as shown by the lncorporation of tritiated thynldine lnto newlysynthesized DNA. The large nucleolus and abundant polyribosomes suggestrespectively, active RNA and protein synthesis. Polyribosomes are considered tobe sites for the synthesls of slructural- prot"its3l, apparently to meet therequirements of a rapidJ-y divlding cel1. Henocytoblasts in a juvenil,e and in an adult, although both stem cells, can bedistingulshed by their kinetic behavior. In a juvenile, nost ce1l prol"iferationls probably for the production of more hemocyLoblasts whereas that in an adultis for the differentiation of mature blood ceLls. Although the cell type that gives rise to all other ascidian bLood cells has
  8. 8. 55traditionally been call-ed a lymphocyte, th18 terninology hae led to confuslonamong irnnunologists. Based upon vertebrate henatoLogy24, ascidian basophil-lcblood ce1ls with a relatlvely J-arge nucJ-eus, prominent nucJ-eolus, and sparse 13 29chromatin which have been referr<ld to as 1)rmphocyt."ll 12 ^t" ot"approprLately cal-Ied stem hemoblasLs22, or hemocytoblasts. The sensi- ".11"32, 33tlvity of these cells to *-r"y"29 i" characteristic shared by all relative- "J-y undifferentiated cells of vertebrates, whether ln blood, dLgestive tfacta, orgor.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 studiesdeflning the functlonal characterl-stlcs of the ascidian hemocytoblast shoul-dtherefore contribute to our understanding of the phylogeny of the vertebrateimtrune syatem.ACIGIOWLEDO.{ENTS part of thls research (a11 autoradiography and fixatlon of tissues forel-ectron nlcroscopy) was conducted at the scripps Institution of oceanographyLa 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 SRothman for suPPort during the preParation of this paper The author ispresently a NIiI post-doctoral fellow at the University of Callfornla, SanFrancisco.REFERENCES I. Berrill, N. J. (1955) The Origin of Vertebrates, oxford university Press, London. 2. Reddy, A. L., Bryan, B. and Hildemann, W. H. ( 1975) Immunogenetics 1 , 584- 590. 3. Wright, R. K. 4) J. Invertebr. Pathol . 24, 29-36 (Lg7 4. Wright, R. K. (1975) Amer . ZooL. 15 , 2I-27 and Cooper, E. L. 5. George, W. C. (1939) Quart. J. Mi-croscop. Sci. 81, 39L-428 6. Mj-llar, R. H. (1953) L.M.BC. Mem. Typ. Br. I"Iar. P1. Anim 35 7. GoodbodY, I. (L974) Adv. Mar. Bio1. 12, 1-150. 8. Hildemannr W. H. and Reddy, A. L. (1973) Fed. Proc. 32, 2188-2194. 9. Hildemann, W. I{. and Uhlenbruck, G. (L974) in Progress in Immunology II Vol. 2 (Brent , L. and Holborow, J., eds.), pp. 292-296, North-Holland Ams terdam.
  9. 9. 5610. Emak, T. H. (f975) Experlenria 3f, 837-839.11. Kalk, M. (1963) Quart. J. llicroscop. Sci. 104, 483-493.12. Overton, J. (1966) J. Morph. 119, 305-326.13. Dudley, P. L. (1968) J. Morph. 124, 263-282.14. Ernak, T. H, (1975) Ph.D, Dlssertation, University of California, San Dlego.15. Ermak, T. H. (1975) J. Exp. ZooI. 194, 449-466.16. Ermak, T. H. Ms. in preparatlon.17. Tissues fixed ln 3% gJ-utaraldehyde in 0.1 M phosphate buffer with 0.7 U sucrose; postfixed ln lZ OsOO ln same buffered sucrose. SectLons examined on a JEM 1008 electron mi-croscope.I8. Berrlll, N. J. (1936) Phi1. Trans. Roy. Soc. London 226, 43-70. ,*t -d19. Millar, R. H. (1966) in Some Conremporary Studies in Marine Science (Barnes, H., ed.), pp. 519-534, Al1en and Unwin, Ltd., London.20- Berrill, N. J. (1950) The Tunicata, Ray Soclety, London,2L. Cowden, R. R. (1968) Trans. Amer. Mlcroscop. Soc. 87, 52I-524.22. Mukai, H. and Watanabe, H. (1976) J. Morph. I48, 337-362,23. Peres, J. M. (1943) Ann. Inst. Oceanogr, 2L, 229-359,24. Bessis, !1. (1973) Living Blood Ce1ls and Their Ultrastructure, Springer- Vgrlag, Heidelberg.25. cood,.R. A., Finstad, J., Pol1ara, 8. and Gabrielsen, A. E, (1966) in Phylogeny of Imunlty (Soith, R. T,, Miescher, P. A. and Good, R. A., eds.), pp. 149-170, Universlty of Fl-orida Press, GaLnesville.26. Ho1land, N. D. (1965) Biol. Bul"L. 128, 259-270.27. trerguson, J. C. (1966) Trans. Amer. Microscop, Soc. 85, 2OO-209.28. Errnak, T. H. (1976) Tiss. CelJ., in presa.29. Freenan, G. (1970) J. Reticuloendothel. Soc. 7, 183-194.30. Thonas, N. !L (1970) J. Mar. Biol. Ass. U.K. 50, 737-746.31. Palade, G. (1975) Science f89, 347-358.32. Snith, M. J. (1970) Bio1. 3u11. 138, 354-378.33. Freenan, c. (1964) J. Exp. Zoo7. 156, 157-184.34. Patt, H. M. and Quastler, H. (1963) Physiol. Rev. 43, 357-396. I