Ermak renewing cells of ascidians 1982Document Transcript
AMER. ZOOL., 22:795-805 (1982) The Renewing Cell Populations of Ascidians1 THOMAS H. ERMAK Department of Physiology, University of California, San Francisco, California 94143 SYNOPSIS. Renewing cell populations are tissues or groups of cells which rapidly prolif- erate and whose cell division is balanced by cell loss. The rapid proliferation of cells can be determined in autoradiograms by the uptake of tritiated thymidine into DNA synthe- sizing cells, and the migration and loss of cells can be followed by taking samples of tissues at increasing time intervals after exposure to the radioisotope. In ascidians, renewing populations are the testis, ovary, blood cells, and epithelial lining of the digestive tract. They are made of subpopulations called compartments: 1) germinal compartments of Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 relatively undifferentiated, dividing stem cells (these cells are labeled at short time inter- vals), 2) mature compartments of fully differentiated, nondividing cells (these become labeled with time by the migration of stem cells), and 3) transitional compartments of cells in intermediate stages. The stomach groove cell population is a model system for cell differentiation and renewal in the digestive tract. Germinal compartments can be distin- guished from mature compartments by their morphology (pseudostratified vs. simple columnar epithelia), by the uptake of tritiated thymidine in autoradiograms, by histo- chemical staining, by their surface features in the scanning electron microscope, and by their intracellular organelles when viewed in the transmission electron microscope. Stem cells differentiate into absorptive cells and zymogen cells. In differentiating, stem cells increase in size and lose their abundant free ribosomes. Absorptive cells develop longer microvilli, a long cilium, smooth apical vesicles, and large supranuclear lysosomes. Zym- ogen cells produce abundant rough endoplasmic reticulum and numerous zymogen gran- ules. The renewing cell populations of blood (lymph nodule) and digestive tract are im- portant model systems for studying cell differentiation, morphogenesis, and the phylogeny of vertebrate hemopoietic, immune, and digestive systems. INTRODUCTION that the turnover of these renewing cell From a developmental perspective, a re- populations could truly be established andnewing cell population is an adult tissue or followed. The pioneering work was per-group of cells which maintains a subpop- formed by Leblond and his associates withulation of embryonic-like cells which con- a variety of radioisotopes to label dividingtinually replace aging differentiated cells cells and follow their fate with time (Le-during the lifetime of the animal. These blond and Stevens, 1948; Leblond andrenewing populations serve a variety of Walker, 1956). They found that tritiumfunctions in the body. For example, they gave the best resolution in autoradiogramsmay form a protective barrier of cells and that thymidine preferentially labeledagainst the external environment, such as replicating DNA. Although thymidine isin the digestive tract or skin, or they may not normally a precursor of DNA, it canprovide a constant supply of a cellular nonetheless enter the biosynthetic pathwayproduct, such as the blood cells or ga- and label DNA during both pre-mitotic andmetes. pre-meiotic DNA synthesis (for review, see Cleaver, 1967). When administered, the The presence of these embryonic-like tritiated thymidine is available for only acells, today called stem cells (Cairnie et ai, short period of time, and only those cells1976), in rapidly dividing tissues was rec- that are synthesizing DNA at the time ofognized for years based upon histological injection and immediately afterwards areobservations. However, it was not until the labeled. After DNA synthesis, all cells pre-use of autoradiography in the late 1940s sumably undergo cell division. The fate of the DNA synthesizing cells can be followed 1 From the Symposium on The Developmental Biology in autoradiograms by taking samples of theof the Ascidians presented at the Annual Meeting of tissues at increasing time intervals after thethe American Society of Zoologists, 27-30 December initial exposure period.1981, at Dallas, Texas. 795
796 THOMAS H . ERMAK cell extrusion Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 cell cell absorption division differentiation and secretion BFIG. 1. The compartments of renewing cell populations as shown for a columnar epithelium. A. Germinalcompartment with mitotic figure along the lumenal edge. B. Transitional compartment. C. Mature compart-ment of fully differentiated cells. Based upon the uptake of tritiated thy- compartment system, and a third com-midine and its change in cells with time, partment of transitional cells can be rec-Leblond classified the cell populations of ognized as cells in the process of matura-mammals into three categories: static, ex- tion. The stem cell compartment, also calledpanding, and renewing cell populations the germinal compartment, is composed of(Messier and Leblond, 1960). Static pop- relatively undifferentiated, rapidly divid-ulations do not divide and expanding pop- ing cells that can also be subdivided ac-ulations have a slow rate of proliferation. cording to their position in the cell cycle.Renewing populations have a rapid rate of Cells undergoing mitosis (mitotic figures)cell proliferation and most cell production can be morphologically distinguished fromis used for the replacement of lost cells cells in interphase (Fig. 1A). Interphaserather than for growth. A large percentage cells undergoing DNA synthesis can be la-of the cells are labeled with tritiated thy- beled with tritiated thymidine and detect-midine, and with increasing time intervals ed with autoradiography, as explainedafter exposure, the percentage of labeled above. Other cells in interphase, unlabeledcells drops to zero due to loss of labeled in autoradiograms, are presumed to be incells from the tissue. In some tissues, one of two resting stages, one between mi-migration of groups of labeled cells can ac- tosis and DNA synthesis and the other af-tually be followed (for example, the epi- ter DNA synthesis and before mitosis.thelium of the digestive tract or sperma- The mature compartment is composedtogenic cells). The system is thus in a steady of fully differentiated cells, such as absorp-state with cell production balanced by cell tive or secretory cells of the digestive tractloss. (Fig. 1C). These cells do not usually take Renewing populations are made up of up tritiated thymidine (thus, they do notseveral compartments based upon mor- usually divide). With time, they eventuallyphological as well as kinetic criteria (Fig. become labeled by the migration and dif- 1). Stem cells and mature cells create a two ferentiation of stem cells. Aging mature
RENEWING CELL POPULATIONS OF ASCIDIANS 797 TABLE 1. Compartments of asddian renewing cell populations classified according to morphology and cell kinetics. Cell population Type of cell Type of compartmentTestis Spermalogonia germinal Spermatocytes dividing transitional Spermatids transitional Spermatozoa matureOvary Oogonia germinal Primary oocytes dividing transitional Oocytes I and II transitional Oocytes III mature Follicle cells dividing transitional Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 Test cells matureBlood cells Hemoblasts germinal Leukocytes matureDigestive tract Basophilic cells germinal Absorptive, zymogen and ciliated cells mature * Each compartment originates from the preceding compartment except in the ovary, where germinal cellsgive rise to both oocytes and follicle cells.cells are lost from the population by cell the gonads occurs in all animal phyla (butextrusion, cell death, or cell excretion. not all adult species) and the morphologi- The time that elapses between a cell en- cal events characterizing cell differentia-tering a compartment and that cell or its tion are familiar to most biologists. I will,progeny leaving it is its transit time. I use therefore, only briefly review the kineticsthis term to denote the shortest time for a of renewal in ascidians. On the other hand,labeled stem cell to migrate through the the renewal of blood cells and digestivetransitional and mature compartments. tract are less common, occurring mainly in Renewing cell populations have been ex- the vertebrates and a few invertebrates.tensively investigated in mammals and in- These populations provide importantclude the epidermis, the epithelial lining models for the differentiation of somaticof the alimentary canal, the hemopoietic cells in the adult and are important sys-cells of bone marrow, the lymphatic cells tems for studying the phylogeny of verte-of thymus, spleen, and lymph nodes, the brate hemopoietic, immune (Wright, 1977),spermatogenic cells, and the uterine and and digestive systems (Ermak, 1981). Forvaginal epithelial cells (Messier and Le- these populations, the kinetics of cell re-blond, 1960). The list of renewing cell newal will be described as well as the over-populations in ascidians is almost as im- all aspects of cell differentiation duringpressive: the epithelial lining of the diges- maturation and migration of cells.tive tract (Ermak, 1975a, c, 1976a, 1981),the hemopoietic cells of lymph nodules and GONADScirculating blood (Ermak, 19756, 1977), the In the gonads, germ cells in both pre-spermatogenic cells (Ermak, 19766), and meiotic and pre-mitotic DN A synthesis canthe ovarian cells (Ermak, 19766). As in be labeled with tritiated thymidine and de-mammals, these populations can be sub- tected with autoradiography (Ermak,divided into germinal, transitional, and 19766). In the testis, each compartment ismature compartments (Table 1). I have ex- in a direct transit line with the next. Sper-amined the kinetics of cell renewal in a sin- matogonia (stem cells) form the basal layergle, solitary species, Styela clava (Ermak, around the circumference of the follicle.1975a, b, c, 1976a, b, 1977), and compared Spermatocytes and spermatids form con-them to those in a variety of other ascidian tinuous layers within the basal layer andspecies (Ermak, 1977, 1981). Renewal of spermatozoa fill the lumen. In Styela, the
798 THOMAS H. ERMAKtransit time from DNA synthesis in the pri- geal or atrial epithelium and along bloodmary spermatocyte to the appearance of channels.labeled spermatozoa is about 10 days (Er- In Styela, blood cells are renewed withinmak, 19766). A one-hour exposure to tri- several weeks (Ermak, 19756). A one-hourtiated thymidine labels many spermato- exposure to tritiated thymidine labels he-gonia and spermatocytes, and after 10 days moblasts in the centers of nodules and inspermatids and spermatozoa become la- circulating blood. By 20 days after expo-beled. With even longer times (20-30 days), sure, cells in the peripheral parts of a nod-labeled spermatozoa appear in the sperm ule become labeled whereas cells in the in-ducts. terior are no longer labeled. By 60 days, a Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 In the ovary, the germinal epithelium nodule as well as most circulating bloodgives rise to oocytes in sequential stages cells are no longer labeled.(usually noted as I, II, and III) as well as Although some differentiated blood cellsfollicle cells surrounding the oocytes can apparently divide, most labeled cells(Tucker, 1942; Kessel and Kemp, 1962). are probably hemoblasts. These cells areTest cells are presumably derived from also sensitive to X rays (Freeman, 1964,follicle cells. DNA synthesizing cells are 1970), a characteristic shared by stem cellsoogonia and preleptotene primary oo- of vertebrates (Patt and Quastler, 1963).cytes. The smallest oocytes lie within the Most fully differentiated blood cells prob-ovarian wall, and larger oocytes become ably do not divide. Vacuolated cells in Stye-progressively displaced from the germinal la are not labeled at early time intervalsepithelium as they grow larger, become less (Ermak, 19756). In Perophora annectens,basophilic, and acquire accessory cells. Al- compartment cells and phagocytes but notthough not followed for more than 60 days, other fully differentiated cells are report-the total time period for oogenesis is prob- ed to be labeled with tritiated thymidineably on the order of several months (Er- (Freeman, 1970).mak, 19766). At one hour to 20 days after The lymph nodule of Styela is a modelexposure, cells in the germinal epithelium system for the differentiation of blood cellsas well as follicle cells around the oocytes (Ermak, 1977). Hemoblasts are relativelyare labeled. By 60 days, a few stage I oo- undifferentiated, spherical cells which havecytes and test cells are labeled. Test cells a large nucleus containing one or more nu-are presumed to originate from follicle cells cleoli and little chromatin. The cytoplasmwhereas oocytes are presumed to originate is filled with numerous polyribosomes, afrom gonial cells in the ovarian wall as few cisternae of rough endoplastic reticu-originally proposed by Tucker (1942). lum, and an occasional small, dense gran- ule. A pair of centrioles (probably involved BLOOD CELLS in centromere formation during mitosis) is The hemopoietic tissue of ascidians is sometimes observed near the Golgi com-organized into clusters of stationary cells plex.called lymph nodules and circulating cells Differentiating leukocytes around the(George, 1939; Millar, 1953). Lymph nod- central hemoblasts lose their prominentules occur in the pharyngeal wall, around nucleus as the amount of chromatin in-the digestive tract, and in the body wall of creases (Ermak, 1977). Large electronadvanced species (Ermak, 1977). In prim- dense granules usually appear in the cy-itive ascidians, hemopoietic tissue appears toplasm before the nuclear changes arediffuse whereas in advanced species, it is completed. As one proceeds away from theorganized into distinct nodules. In Styela, center of a nodule, maturing blood cellsa nodule is composed of a few clusters of increase in size and their dense granulesdividing hemoblasts (the stem cells) sur- become larger and more numerous. Cellrounded by non-dividing, maturing blood differentiation is also marked by the losscells (Ermak, 1977). Lymph nodules lie of polyribosomes and the development ofwithin connective tissue below the pharyn- elongate mitochondria, a larger Golgi
RENEWING CELL POPULATIONS OF ASCIDIANS 799complex, and long cisternae of rough en- the underlying connective tissue throughdoplasmic reticulum. the transition zone and up the sides of the folds into the non-proliferating mature DIGESTIVE TRACT zone of absorptive and zymogen cells. The Most of the epithelia lining the digestive mature zone forms a simple, columnar ep-tract are renewing cell populations. In Styeki ithelium and represents about 80% of thethey are the dorsal tubercle, dorsal lamina, height of the fold. Migrating labeled cellsbranchial bars, zone 1 of the endostyle, reach the top of the fold in about 16 daysstigmata, esophagus, stomach, and intes- (Ermak, 1976a). Aging mature cells aretine (Ermak, 1975c). In the postbranchial presumably extruded into the gut lumendigestive tract of most other ascidians, re- at so-called "extrusion zones" at the top of Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010newing populations are also a characteris- the fold (at the junction with the crest pop-tic feature of the esophageal and stomach ulation of ciliated mucous cells). Migrationepithelia, but expanding populations rates in other regions of the digestive tractsometimes cover all or part of the intestine range from 10 days for the stigmata to 5(Ermak, 1981). The renewing populations weeks in the intestine (Ermak, 1975c).are adapted to different degrees of body Germinal regions can be distinguishedsize, organization, and evolutionary ad- from mature regions at the light micro-vancement, and with an increase in body scopic level with the use of histochemicalsize, the digestive organs undergo exten- stains. With routine hematoxylin and eo-sive folding and multiplication of pairs of sin, the stem cell is characterized by baso-germinal and mature compartments (cell philic cytoplasm, and with toluidine bluerenewal units). Colonial ascidians gener- on epon sections, only the lysosomal re-ally have a single cell renewal unit per cell gions of mature cells are stained. The ma-population in their small organs whereas ture region also gives a strong positive re-solitary ascidians have highly folded gut action for acid phosphatase3 which is absentepithelia with multiplication of cell renew- from the cytoplasm of stem cells (Fig. 3).al units (Ermak, 1981). The groove pop- The brush border at the base of the grooveulation in the stomach of Styela provides a usually shows only a weak alkaline phos-model system for cell renewal and differ- phatase reaction in comparison to the in-entiation in the digestive tract. This epi- tense reaction on the sides of the folds (Er-thelium covers the majority of each stom- mak, 1975a). Finally, the cytoplasm of theach fold as shown in the scanning electron germinal region takes up large amounts ofmicrograph2 of the stomach wall (Fig. 2). tritiated leucine (Ermak, 1975a), a reflec-Germinal zones occur at the base of each tion of active protein synthesis in these cells.fold and form a pseudostratified columnar Germinal regions can also be distin-epithelium (Ermak, 1975c). A one-hour guished from mature regions in surfaceexposure to tritiated thymidine labels about view with the scanning electron micro-10% of the groove height (Ermak, 1976a). scope (Fig. 4). Stem cells appear free ofStem cell nuclei migrate to the cell apex surface organelles marking differentia-and undergo mitosis there. Mitotic figures tion, namely cilia or long microvilli. In-are labeled within several hours after ex- stead the germinal compartment is markedposure (Ermak, 1975c). With time, stem by smooth, short microvilli. At the top ofcells migrate as a band of labeled cells over the fold, the "extrusion zone" can be iden- - For scanning electron microscopy, stomach tissue 3was fixed in 2% glutaraldehycle in 74% sea water for To test for the presence of acid phosphatase, tis-24 hr (Holland and Jespersen, 1973; Nemanic and sues were prepared according to the standard cou-Pitelka, 1971) and dried in a Freon critical point dryer. pling azo dye technique (Pease, 1968) using frozenSpecimens were coated with about 15 nm of gold- sections incubated at room temperature with sodiumpalladium alloy and examined with a Stereoscan S4 a-naphthyl phosphate, poly vinyl pyrrolidone, and fastscanning electron microscope. garnet CBC salt in 0.1 M acetate buffer at pH 5.0.
800 THOMAS H. ERMAK Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010FIG. 2. Stomach folds of Styela clava as viewed with the scanning electron microscope. The stomach wall hasbeen cut across with a razor blade and viewed from within. On the cut surface of the folds, the demarcationbetween the inner columnar epithelium and the underlying connective tissue and blood spaces is distinct.Crest cell populations are conspicuous on the top of each fold, x 100.FIG. 3. Section through the base of a stomach groove stained for acid phosphatase. Germinal cells arerelatively unreactive. Mature cells (probably lysosomal regions of absorptive cells) give a strong positive re-action. X125.Fit;. 4. The base of a stomach groove showing the surface features ot the germinal (g) and mature (in)regions. Germinal /ones with short microvilli appear "naked" in comparison with mature regions with nu-merous long cilia and longer microvilli. x 1,000.
RENEWING CELL POPULATIONS OF ASCIDIANS 801tified as the junction between the groove population and the ciliated crest cell pop-ulation (Fig. 5). The differentiation of stem cells into ab-sorptive cells and zymogen cells (Fig. 6) hasbeen followed by transmission electronmicroscopy4 in the stomach groove popu- lation of S. clava (Ermak, 1975a; Thorn- dyke, 1977) and on the stomach folds of Ciona intestinalis (Thomas, 1970). These mature cells appear to be characteristic of Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 the stomach and digestive diverticulum ofother ascidians as well (Degail and Levi, 1964; Burighel and Milanesi, 1973). Stem cells (Fig. 6A) are smaller in size, have a higher nucleus-to-cytoplasm ratio, and are relatively undifferentiated in structure (Fig. 7). Their most chracteristic features, like those of the hemoblast in alymph nodule, are a nucleus containing aprominent nucleolus and little chromatin(Thorndyke, 1977) and numerous free ri-bosomes in the cytoplasm (Fig. 8). The api-cal ends of the cells bear several microvilliwhich are shorter (about 3 /xm) and lessnumerous than those on the absorptive cellsand several small, dense granules. Below A BCthe cell apex, mitotic figures are occasion- FIG. 6. Schematic diagrams of the stem cell (A), ab-ally observed (Fig. 7). There are no cilia sorptive cell (B), and zymogen cell (C) in the stomachextending into the stomach lumen, but a groove cell population of Styela clava. c, cilium; dc,pair of diplosomal centrioles is located just diplosomal centrioles; go, Golgi apparatus; ly, lyso-below the microvilli. These organelles are some; mb, multivesicular body; rer, rough endoplas- mic reticulum; sg, secretory granule of stem cell; v,presumably involved in centromere for- apical vesicles; z, zymogen granule.mation during mitosis and probably alsogive rise to the cilia of absorptive cells. During differentiation into absorptivecells (Fig. 6B) and zymogen cells (Fig. 6C), ules and numerous free robisomes. Thestem cells lose their dense secretory gran- nucleus of the absorptive cell loses its prominent nucleolus, but it is retained in the zymogen cell. 4 For transmission electron microscopy, stomach Absorptive cells (Fig. 6B) predominatetissue was fixed in 3% glutaraldehyde in 0.1 M phos- in the mature region and are characterizedphate buffer (pH 7.3) with 0.70 M sucrose for 1.5 hr by long microvilli (about 8 /xm long), a longfollowed by postfixation in ice cold 1% OsO4 in thesame sucrose-buffered medium for 1 hr. Samples were cilium, apical smooth vesicles, elements ofembedded in Epon 812 and examined in a JEM-100B rough endoplasmic reticulum, and a stackelectron microscope. of large supranuclear lysosomes (Figs. 9 —FIG. 5. Junction (asterisk) of a groove population (gp) with a crest population (cp) at the top of a stomachfold. Cell extrusion presumably occurs at this point. Crest population also shows differentiation between"naked" germinal (g) regions and ciliated mature (m) regions, x 1,000.
802 THOMAS H. ERMAK iV/< •? / Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 •a* 8FIG. 7. Transverse section through portions of the germinal (g) and transitional (tr) regions of the stomachgroove population of Styela clava. Stem cells contain small, dense granules. Differentiating zvmogen cellscontain larger granules ot light electron density. The germinal zone also contains mitotic figures (mi). x4,5(>0.FK.. 8. Detail of secretion granules and cvtoplasm of stem cells and transitional zvmogen cells from Figure7, The cytoplasm contains numerous polyribosomes. x 15,000.
RENEWING CELL POPULATIONS OF ASCIDIANS 803 • ••xXiyi. . * *• .^m. ^BLJ^ . ^ .-.••/?".- ^~ x">-: Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010FIG. 9. Apical portion of transitional absorptive cells showing intermediate stage of apical vesicle (v) accu-mulation and short, blunt cilia (c). The latter appear to be true intermediates rather than tangentially sectionedlong cilia, x 9,000. 11). Transitional cells have smaller lyso- ments of rough endoplasmic reticulumsomes, an intermediate number of apical (Fig. 11).vesicles and profiles of short, blunt ciliasuggestive of ciliary growth (Fig. 9; see also CONCLUSIONSThorndyke, 1977). The lysosomes mea- The renewing cell populations of ascid-sure about 2-4.5 /u.m in diameter and are ians are characterized by a rapid rate offilled with membrane lamellae and clumps proliferation, with cell production bal-of light and dark granular material (Fig. anced by cell loss; thus, the system is in a 11). Although digestion in ascidians has steady state. These populations are un-been described as extracellular (van Weel, common among invertebrates, particularly 1940; Morton, 1960), this organelle might renewing blood cells and digestive epithe-be an important site of intracellular diges- lia. Considering the affinities of ascidianstion (Ermak, 1975a; Burighel, 1979). to vertebrates (Berrill, 1955), it is possible Differentiating zymogen cells are en- that renewing cell populations are char-countered just outside of the zone of stem acteristic of chordates as a whole. Thesecells prior to the development of lysosomes populations provide excellent model sys-in the absorptive cells (Figs. 7, 8). Mature tems for the study of cell differentiationzymogen cells (Fig. 6C) contain a large, ve- and have been useful in examining thesicular Golgi complex, numerous zymogen origins of cell renewal (Ermak, 1981) andgranules (1—2 yjm in diameter), both in the the phylogeny of the vertebrate immunecell apex (Fig. 10) and the vicinity of the system (Ermak, 1977; Wright, 1977). TheGolgi complex, and abundant tubular ele- role of these populations in morphogene-
804 THOMAS H . ERMAK .L. f Downloaded from http://icb.oxfordjournals.org by Thomas Ermak on April 24, 2010 i ; * *» • ,i m "•• * * •*-%„ .- / : •l~ft l i .<» ^v*FIG. 10. Apical portion of a mature zymogen cell (center) and two absorptive cells (left and right) containingnumerous apical vesicles (v). x 12,000.Fit.. I I . Supranuclear region of an absorptive cell (center) showing a single, large lysnsome (Iy) and azymogen cell (left) showing abundant rough endoplasmic retitulum (iei). x 18,000.
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