1. a-
Separatum TXPERITNTIA 3/, 837 (1975)
Birkhduser Verlag, Basel (Schweiz)
An Autoradio$raphic Demonstration of Blood Cell Renewal in Styela claaa (tJrochordata:
Ascidiacea)
The blood cells of ascidians circulate in the blood chan- Mnterials and meth,ods. Specimens of Styeta claua were
nels and wander throughout the tissues and the tunic. collected from Mission Bry, San Diego, California, IJSA
Although some blood cell types are common to all asci- and inj ected intra-atrially with 1 pCi of tritiated thymidine
dians, other blood ceIl types often differ from species to (New England Nuclear corp.) per g fresh weight. The
species. The number of blood cells described in any one aqueous solution of tritiated thymidine (specific activitv
species also varies with the morphological criteria of the 6.7 Cilrrrlw) was diluted with an equal volume of 2 times
authors. With iight microscopy, 8 types have been describ- concentrated sea water before use" 3 individuals were
ed in Styela claual while 5 types have been described in sacrificed by fixation in Bouin's fluid at each of the follow-
Styela pl'icataz " ing time intervals : J- h, La, 20 and 60 days. The body wall
The origin and renewal of ascidian blood cells have been and digestive tract were dissected out, dehydrated,, and
the subject of controversy. The neural gtand3 and haemo- embedded in paraffin" 7 pm sections were cor.ered with
blasts in the connective tissue a have been reportecL as Kodak Nuclear Track Emulsion type NBT-2 by the
sites of biood cell formation. Several authors have dipping method and stored at 4 oC for periods of 2 weeks
remarked on the absence of mitotic figures in the blood to 2 months" Autoradiograms were developed in Kodak
spaces 5, 6" There is general agreement that the lymphocyte D-l9 developer (3 min), and sections were stained through
is the progenitor biood cell type. Vhether l1'mphocyte cell the emulsion with hematoxylin"
division occurs only in the lymph nodules of the body wall Resr't'lts. 3 types of blood cells can be recognrzed in
and digestive tract6 or both in the lymph nodules and in autoradiograms: the lymphocyte, the leucocyte, and the
the circulating blood 1, 2 has not been established. In vacuolated cell. A11 3 blood cell types occur free in the
either case, the lymphocytes are presumed to differentiate circulating blood and clustered in lymph nodules within
into the other cetrI types by the loss of the nucleolus, an the connective tissue of the body. In the body wall, the
increase in the amount of crrtoplasm, and the acquisition lymph nodules occur in patches immediateiy adjacent to
of various cytoplasmic inclusions and vacuoles. The the atrial epithelium (the internal lining of the body wa1l).
transformations of one blood cell type to another have In the digestive tract, they are most concentrated in the
been deduced from morphological criteria alon e2, ? . branchiai wall"
rn the present investigation, autoradiography with
tritiated thymidine was used to locahze sites of blood cell
proliferation and determine possibie blood cell transforma- r'W. C. GronGE, Q. J1. microsc. Sci. Bl,391_ (1939).
tions in the ascidian Styela claua. A short exposure to 2 T" onuvn, Sci. Rep. Res. Insts Tohuku t]niv., Bio1. l l, rgr (1936).
3 L. Cuf Nor, Archs ZooI" exp. gdn. g, 13 (189j ).
tritiated thymidine labeled blood cells engaged in pre- * J. M. Pfnis, Annis Inst. oc6anogr., Monaco 2/,229 (19,t3).
mitotic DNA synthesis. By sampling tissues at increasing 5 M. Korr.lra.x, Annls Sci. nat., Zoo7.8,
1 (1908).
time intervals after this short exposure, the fate of the 6 R" H" Mrrran, L.h'I.B.c. {ern. typ.Br. mar. pl.
Anim. ss, (1953).
proliferating cells was f ollowed "
7 R" ExDEAN,
Q. Jl.microsc. Sci. /0l , M (1960).
"u* *=*"+*n
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F ig. 1. An autoradiogram of the lymph nodules in the body wall of Fig. 2. An autoradiogram of the lymph nodules in the body wali of
Styela claua t h after the injection of tritiated thymidine. x 600" Styela claua 60 days after the injection
of tritiated thymidine. The
blood celis are no longer labeled although several atrial epithelial
celis (arrows) are still labeled. m, muscle. x 600.

2. /-
838 Specialia ExpBnrBNrrA 3tl7
Lymphocytes are small, round to oval cells approximate- labeled, but few lymphocytes were labeled any longer.
ly 6-8 pm in diameter with a large nucleolated nucleus. Most of the labeled cells, both in the lymph nodules and
The nucleus fills most of the cell and is surrounded by a in the circulating blood, were leucocytes and vacuolated
small amount of basophilic cytoplasm. Several lymphocy- cells. At 60 days after the administration of tritiated
tes frequently cluster together in the interior of the lymph thymidine, most of the blood cells in the lymph nodules
nodules. Cell boundaries are difficult to distinguish as the were unlabeled (Figure 2). llowever a few scattered
Iarge basophilic nuclei of adjacent cells crowd each other labeled leucocytes and vacuolated cells occurred in the
with little intervening cytoplasm. connective tissue below the atrial epithelium and in the
Leucocytes are large blood cells about L}-L+ pm in blood channels.
diameter with a small, basophilic nucleus which is eccen- D'iscuss'ion. The blood cells of. Styela claua constitute a
trically displaced and lacks a conspicuous nucleolus. renewing 8 cell system with a renewal time on the order of
Most of the cell is filled with slightly basophilic cytoplasm. several weeks. As suggested by other authors 1' 2, blood
Leucocytes range in shape from spherical to oval and cell proliferation occurs both in the lymph nodules and in
may have several cytoplasmic processes. The cytoplasm the blood channels. The possible blood cell transforma-
may be granular or transparent and may contain a large tions are shown in Figure 3. Although both lymphocytes
basophilic inclusion. Leucocytes probably include several and leucocytes proliferate, the lymphocyte is probably
cell types which are difficult to distinguish from each the more primitive blood cell type. It is ultrastructurally
other. In the lymph nodules, the abundant cytoplasm the most undifferentiated blood cell type e and may also
around each nucleus produces regions of light basophilia be capable of differentiating into germinal cells 10 and
and makes the leucocyte nucleus easily distinguished from somatic cells other than blood cells 11. Presumably the
the lymphocyte nucleus. lymphocytes differentiate into leucocytes. llowever,
Vacuolated cells are the largest of the blood cells, being since the leucocytes divide and also probably represent
roughly 16-18 pm long. The nucleus is eccentrically several separate cell types, the leucocytes themselves
placed and lacks a conspicuous nucleolus. Within the might be composed of stem, dividing transit, or non-
cytoplasm, the vacuolated cells contain numerous vacu- dividing transit 12 components. Since the differentiated
oles which are clear or yellow in hematoxylin stained vacuolated cells do not divide, they must be differentiating
sections and which have a high index of refraction. from a precursor cell type; whether this precursor is a
Vacuolated cells frequently lie embed d ed between the lymphocyte or a leucocyte could not be determined in the
atrial epithelium and the connective tissue of the body present investigation. Based upon morphological criteria,
wall. however, vacuolated cells have been reported to differen-
A t h exposure to tritiated thymidine labeled many tiate from intermediate cell types and not from lympho-
blood cells in the body. Blood cells were labeled. both in cytes in other ascidians 7 , L3.
the lymph nodules and in the blood channels. In the Blood cells are renewed in insects 1a and mammals 15
Iymph nodules, both lymphocytes and leucocytes wero but not in echinoderms 16 where blood cells have charac-
labeled (Figure 1), and labeled nuclei were frequentlv teristics of expandirg* cell populations. In mammals,
clustered in small groups. Probably no vacuolated cells proliferating cells are most concentrated in the bone
were labeled at this time ; however, positive reaction s marrow, lymph nodes, and spleen, but, like tn Styela, they
were difficult to detect in autoradiograms as the vacuoles also occur in the circulating blood and in the connective
appear very similar to out-of-focus silver grains. ti$sue. In Styela, other elements of the vascular system
By 20 days after injection, most of the labeled cells in (the heart and connective tissue lining the blood channels)
the lymph nodules occurred in the peripheral parts of the comprise expanding cell populations 17.
nodules. Some vacuolated cells were now unequivocally
Surnrnary. The blood cells of Styela claua were shown
by autoradiography r,vith tritiated thymidine to be renew-
ed after several weeks. Proliferating lymphocytes and
leucocytes occurred in the lymph nodules and blood
channels of the body. Vacuolated cells did not proliferate
but differentiated from a precursor cell type.
T. H. ERnrax 18
D'iu'is'ion, of Marine B'iology,
Scri,pps Inst'itut'ion of Oceanography,
La Jolla (Cali,forn'ia 92037, USA), 5 March 7 975.
I f, 8
e
B. MBSSTER and C. P. LBnroND, Am. J. Anat. 106,247 (1960).
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10 A. T. NBwBERRv,
11 G. F'nnBnreN,
t'J. E. Cr,Bevnn,
J. Morph.726, t23 (1,968).
J. exp. Zool.756,I57 (7964).
Thyrnid,ine Metabol,ism and, CeII, Kinetics (John
Wiley & Sons, fnc., New York 1,967).
13 A. SansA.nrN, Arch. ital. Anat. Embriol . 60,33 (1955).
14 E. P. CnoNKrrE, V. P. BoNn, T. M. Fr,rBnNnn and
J. R. RunrNr,
Lab. fnvest. 8,263 (1959).
15 S. C. SsnrvASrAvA and A. G. RrcsARD, Biol. Bull. 728,337 (1965).
16 N. D. Holr,AND,
J. H. Pnrr-r,rps and A. C. Grnsr, Biol. Bull. 128,
zse (Le6s).
L7 T. H. Ennar, Ph. D. Diss. (1975).
Fig. 3. Transformation of blood cell typesin Styela claaa. A, lympho- 18 Present address: Department of. ZooIogy, University of California,
cyte; B, leucocyte; C, vacuolated cell. Berkeley, California 94720, USA.