Cell Proliferation in the Digestive Tract of Styela clava(Urochordata: Ascidiacea) as Revealed byAu torad iography with Tri tiated Thymidi ne THOMAS H. ERMAK Scripps l n s t i t u t i o n of Oceanography, La Jolla, Californiu 92037 ABSTRACT The DNA-synthesizing cells i n the digestive tract of Styela clava were labeled with tritiated thymidine and detected at increasing time intervals with autoradiography. Most of the epithelia consist of renewing cell populations. These are the dorsal tubercle, dorsal lamina, branchial bars, zone 1 of the endo- style, stigmata, esophagus, stomach, intestine, and rectum. Expanding popula- tions are the branchid tentacles, unspeciafized pharyngeal cells, and pyloric tubules. In the renewing populations, cell proliferation occurs in restricted ger- minal zones of pseudostratified cells. I n the postbranchial digestive tract, these germinal zones usually occur at the bases of folds or grooves. Germinal cell nu- clei migrate into the cell apex and undergo mitosis there. With time, germinal cells migrate as a band of labeled cells over the underlying connective tissue into non-proliferating mature zones of ciliated, secretory, or absorptive cells. Aging mature cells are presumably extruded into the gut lumen, maintaining the system i n a steady state. Transit times are about ten days for the stigmata; two and a half weeks for the dorsal tubercle, esophageal mucous population, stomach crest population, and stomach groove population; five weeks for the intestine and rectum; and several months for zone 1 of the endostyle and esoph- ageal band population. To date, most of the work on gut cell midine and detected with autoradiography.proliferation employing tritiated thymidine The fate of the DNA synthesizing cells wasand autoradiography has been confined followed by taking samples of the digestiveto the renewing epithelia of mammals tract at increasing time intervals after the(Leblond and Messier, 58; Messier, 60; administration of tritiated thymidine.Leblond et al., 64). Comparable work hasbeen done on relatively few non-mamma- General orientationlian vertebrates and invertebrates (Davis The digestive tract of ascidians (shownand Burnett, 64; Anderson; 65; Holland, schematically in fig. 1A) is composed of a64). The sparsity of such information on branchial basket, the food collecting andascidians is especially unfortunate because respiratory apparatus, and a postbranchialof their vertebrate affinities. Previous work region, which functions in digestion andon ascidian gut cell kinetics is limited to a absorption of nutrients. The postbranchialcell formation test (as defined by Leblond region is made of an esophagus, stomach,et al., 59) for Halocynthia sp. (Mochan intestine, and rectum. The pyloric gland,et al., 71). However, from anatomical evi- a diverticulum of the gut, is given off thedence alone, the replacement of gut epi- main course of the digestive tract at thethelial cells has been proposed in several junction of the stomach and intestine. Seaascidians (Aros and Viragh, 69; Fouque, water enters the branchial basket through53; Relini-Orsi, 68; Thomas, 70). the branchial siphon, passes through the Therefore, the purpose of the present stigmata (gill slits) in the branchial walls,investigation is to elucidate cell prolifera- and leaves through the atrial siphon. Thetion patterns in the digestive tract of the major portion of the branchial basket isascidian Styela claua, a large solitary as- concerned with the secretion and move-cidian found in bays along the California ment of a mucous net across the branchialcoast. DNA synthesizing cells about to en- 1 Present address: Department of Physiology, Univer-ter mitosis were labeled with tritiated thy- sity of California, San Francisco, California 94143.J. ExP. ZOOL., 1 9 4 ; 44-66, 449
CELL PROLIFERATION I N ASCIDIAN GUT 451walls and with the creation of water cur- the lumenal edges of the pseudostratifiedrents to bring in plankton and suspended regions.organic matter. The mucous net is se- For more complete descriptions of thecreted by the endostyle and moves along mechanism of feeding in ascidians and thethe branchial bars by ciliary action. Water morphology of the digestive tract in Styela,currents are produced by numerous cili- the reader is asked to refer to McGinitie ated stigmata in the branchial walls. Par- (39), Berrill (50), Morton (60), Fouqueticulate matter is trapped by the mucous (5) and Relini-Orsi (68). 3,net and moves toward the anterior part MATERIALS A N D METHODSof the basket. Here, the mucous passesdown the dorsal lamina to the esophageal Styela c l a m is a club-shaped ascidianopening. Below the branchial siphon, the native to Korea and Japan and lives onbranchial tentacles exclude larger sus- docks, pilings, and boats in protected Cali-pended particles, but allow smaller parti- fornia bays (Abbott and Johnson, 72; Ab-cles to enter the branchial basket. The bott, 75). Individuals up to 5 cm in lengthdorsal tubercle, which projects into the are relatively clean, showing the brownbranchial basket, plays a role in chemore- wrinkled test, but larger ones usually haveception (Carlisle, 53). a coating of bryozoans, algae, and other The postbranchial digestive tract lies ascidians. Seventy specimens of S. clavaon the left side of the branchial basket. were collected from the docks at QuiviraThe esophagus joins the basket half way Basin of Mission Bay, San Diego, California,down the dorsal side. A t the entrance to and injected with 5 microcuries of tritiatedthe esophagus, the mucous net forms a thymidine (New England Nuclear Corp.)food cord, passes through the stomach and per gram fresh weight. Individuals rangedbecomes compacted in the intestine. Mu- in weight from one to four grams. Thecous secretions are added in the esopha- tritiated thymidine (specific activity 6.7gus; enzymes are secreted mainly in the curies per millimore) was diluted 1: 1 withstomach; and absorption occurs chiefly in two times concentrated sea water and in-the intestine (van Weel, 40; Morton, 60). jected intra-atrially. The animals were The internal lining of the digestive tract maintained at 18"C-2OoC until the timeof Styela is extensively folded, a feature of sacrifice. Ten individuals were sacrificedcharacteristic of solitary ascidian species. by fixation in Bouins fluid at each of theEach region of the digestive tract is cov- following time intervals: 1 hour, 3 days,ered by the atrial epithelium (an epidermal 10 days, 15 days, 20 days, 30 days, and 60specialization) on the outside and lined by days after injection.the digestive epithelium on the inside. In The digestive tract was dissected frombetween the epithelia occur blood spaces the body after at least one days fixationand connective tissue. In the branchial and the various organs dehydrated in alco-region, smooth muscles pass below the hol and embedded in paraffin. Sectionsbranchial bars, and in the postbranchial were cut at 7 microns and covered withregion, the pyloric tubules run below the Kodak Nuclear Track Emulsion type NBT-2digestive epithelium. by the dipping method. Autoradiograms Much of the digestive tract is lined by were exposed for periods from two weekssimple columnar epithelia, the majority to two months and developed in Kodak D-19of which are either ciliated, secretory, or developer (3 minutes) at 68°F. All slidesabsorptive in nature. In several restricted were stained through the emulsion withzones throughout the digestive tract, the hematoxylin.epithelia are modified into basophilic zones The following structures or organs wereof pseudostratified columnar cells (fig. 2D). examined: branchial tentacles, dorsal tu-These zones are most obvious at the bases bercle, endostyle, dorsal lamina, branchialof the folds in the esophagus and stomach bars, stigmata, esophagus, stomach, intes-and in the intestinal groove. These regions tine, rectum, and pyloric gland. T h o struc-have been cited as regions of intense cell tures in the branchial basket were notproliferation (Fouque, 53; Relini-Orsi, 68), studied: the penpharyngeal band, whichsince mitotic figures frequently occur along is continuous with the dorsal lamina, and
452 THOMAS H . ERMAKthe retropharyngeal band, whose mucous consisting of lateral and axial cells. A lat-cells and band cells are considered in the eral cell compartment lies on each side ofesophagus. the axial cell compartment and partly on RESULTS top of zone 2 (fig. 2A). At one hour after injection, only lateral cells are labeled Branchial tentacles (fig. 4). Although only a few cells are la- A t one hour after injection, no localized beled, this is one of the more heavily la-region of cell proliferation is observed in beled zones of the endostyle. By 20 dayscross sections of the tentacles, two or three after injections, no migration of cells isnuclei being labeled per section. No changes detected. After 60 days, however, severalare observed after longer time intervals. axial cells near the lateral cells are now labeled (fig. 5), indicating that the lateral Dorsal tubercle cells had sunk into the basal portion of The dorsal tubercle is a knob-shaped zone 1 and differentiate into axial cells.protuberance with a deep horseshoe-shaped Although zones 2 through 9 all have la-groove running along its surface (fig. 1C). beled cells at one hour after injection, theyThe exact shape of the groove, however, show no migration or loss of labeled cellsvaries among individuals. The groove is at increasing time intervals. Zones 2 andlined by the heavily ciliated funnel epithe- 4 are the lightest labeled populations whilelium while the surface is lined by the non- zone 3 is the heaviest labeled populationciliated lip epithelium (fig. 1D). Pseudo- in the endostyle, including zone 1 (seestratified regions occur at the base and at DI sc u s s ION).the top of the groove. Beneath the funnelepithelium is a band of acidophilic mate- Branchial bars and dorsal laminarial which appears to be supportive in The dorsal lamina and the ventral (en-function. dostylar) surface of each longitudinal bar At one hour after injection, many pseu- are covered by ciliated mucous cells whiledostratified cells are labeled, but no cells the transverse bars and the dorsal surfaceon the sides of the grooves or on the sur- of each longitudinal bar are covered by aface of the dorsal tubercle are labeled (fig. relatively unspecialized epithelium. The3). By 20 days after injection, cells have transverse bars are an integral part of themigrated onto the sides of the grooves branchial wall. The longitudinal bars, how-and onto the surface of the dorsal tubercle. ever, are only connected to the rest of the branchial wall at the junction with the Endostyle transverse bars. As looked at from the in- The endostyle consists of a deep furrow side of the branchial basket, each longi-running along the ventral surface of the tudinal bar bridges over the underlyingbranchial basket (fig. 1B). Most early ac- row of stigmata; either end of the bridgecounts recognize eight cell populations. is connected to a transverse bar.However, recent ultrastructural examina- At one hour after injection, many cili-tions of zone 5 on the endostyle of several ated cells on the dorsal lamina and on theascidians (Godeaux and Firket, 66, 69) ventral surface of the longitudinal bars arehave shown that this zone consists of two Fig. 2 A. A cross section through the endostyleseparate regions, making nine cell popula- showing the nine cell populations. B. A cross sec-tions in all. By the system of Barrington tion through the esophagus. C . The ventral (left)(57), zones 6 through 8 are now zones 7 and dorsal (right) internal surfaces of the stomach and descending part of the intestine showing thethrough 9 (fig. 2A). The nine cell popula- twisting longitudinal folds o n the ventral surfacetions in the endostyle of Styela clava are and the straight folds o n the dorsal surface of theroughly arranged in alternating glandu- stomach. D. A cross section through a pseudostrati-lar and ciliated regions. Zone 1 has long fied region. E. A cross section through the stomach. F. A cross section through the intestine in correctcilia which reach the top of the endostylar position in relation to the stomach in E. cb, bandfurrow. Zones 3, 5, 6, and 9, on the other cells; c m , mucous cells; r, raphe; b, bulb; ty,hand, have short cilia, while zones 2, 4, typhosole; f, folds; ig, intestinal groove; cc, simple columnar cells; mf, mitotic figure; tc, transitionaland 7 are glandular. cells; pc, pseudostratified columnar cells; gp, Sokolska (31) and Levi and Porte (64) groove population; cp, crest population; pt, py-distinguish two compartments in zone 1 loric tubules.
454 THOMAS H . ERMAKlabeled, but few unspecialized cells are fold (fig. 2B). Pseudostratified regions oc-labeled. On the longitudinal bars most la- cur at the base of each fold and on eachbeled cells occur close to the junction with side of the band cell population.the transverse bars. On the dorsal lamina, At one hour after injection, only thoseno distinct region of cell proliferation oc- nuclei which occur in a basal position incurs, the labeled nuclei being scattered the pseudostratified region are labeled.along the entire epithelium. Mitotic figures along the lumenal edge of A t increasing time intervals, there is a the pseudostratified region and mucousloss of labeled cells from both the longitud- cells and band cells on the sides of theinal bars and the dorsal lamina. Although folds are not yet labeled.the ciliated epithelia appear to be renewed, A t three days after injection, nuclei areno definite patterns of cell migration could labeled at all levels in the pseudostratifiedbe determined. There appears to be a gen- layer (fig. 8). Mitotic figures are also some-eral movement of cells towards the tip of times labeled, but no mucous cells or bandthe dorsal lamina and away from the at- cells are labeled. The labeling of mitotictached parts of the longitudinal bars figures by three days is a result of the mi-towards the free parts. Cell migration pat- gration of interphase nuclei from the basalterns are difficult to ascertain because to the apical portion of the cell in prepara-there appears to be no clear separation tion for mitosis. After mitosis, the daugh-into germinal and mature compartments. ter nuclei migrate back to the basal por-A t a l times after injection, the unspecial- l tion of the cell.ized epithelium on the branchial bars has By 10 to 20 days after injection mucousscattered positive reactions. cells but no band cells are now labeled. As shown in figure 9, there is quite a bit Stigmatic e p i t h e l i u m of variability in migration rate both be- Each stigma is lined by heavily ciliated tween animals and between folds in thecells which beat outward when producing same animal.water currents. Small pseudostratified re- By 60 days, mucous cells are no longergions of basophilic cells occur at the an- labeled, presumably because they are ex-terior and posterior end of each stigma. truded into the gut lumen; however, theA t one hour after injection, only pseudo- band cells are now labeled (compare figs.stratified cells are labeled (fig. 6). Labeled 10 and 11). The band cells only originatenuclei are not observed in sections of every from the pseudostratified region on thepseudostratified region, but usually a few side of the ventral groove. This pseudostra-nuclei are labeled. No cells on the sides of tified region is unique in that i t producesthe stigmatic walls are labeled. At ten days both mucous cells and band cells; mucousafter injection, cells on the side walls are cell production occurs on the opposite sidenow labeled (fig. 7) indicating that cells of the pseudostratified region from bandfrom the anterior end of each stigma have cell production.migrated posteriorly, and that the cells atthe posterior end have migrated anteriorly. StomachThe two bands of labeled cells have met The stomach has 25 to 30 longitudinaleach other on the side wall, leaving behind folds on its dorsal and lateral surfaces.lightly labeled cells in the pseudostratifiedregions. A t 20 days after injection, the cells Fig. 3 An autoradiogram of the lip and funnellining the stigmata are no longer labeled. epithelia of the dorsal tubercle one hour after in- jection. X 315.Thus, all those cells labeled at the time of Fig. 4 An autoradiogram of zone 1 of the en-injection as well as their immediate prog- dostyle 20 days (same as 1 hour) after injection.eny have been lost from the cell population. Only lateral cells are labeled. X 600. Fig. . An autoradiogram of zone 1 of the en- 5 Esophagus dostyle 60 days after injection. Axial cells are now labeled. X 600. The esophagus has five folds and is lined Fig. 6 An autoradiogram of several stigmataby two populations of cells. Mucous cells one hour after injection showing labeled pseudo-occur on every fold except the base of the stratified cells. X 600. Fig. 7 An autoradiogram of several stigmataventral fold where band cells occur. The ten days after injection showing labeled ciliatedband cells are restricted to one side of the cells on the side walls. X 600.
456 THOMAS H . ERMAKThe ventral surface is covered by a raphe By ten days after injection, mature cellswhich expands into a large bulb halfway on the sides of the grooves are now labeled.down the length of the stomach (fig. 2C) Both chief and zymogen cells are labeled,and continues as the typhosole in the in- and several pseudostratified cells are stilltestine. The stomach folds are covered by labeled (fig. 14). Animals killed 15 daystwo cell populations: the groove population after injection have several folds where thewhich occurs at the base of each fold, and entire groove height is labeled (fig. 15).the crest population, which occurs on the At this time, the originally small band oftop of each fold (fig. 2E). At the anterior closely clustered germinal cells has ex-and posterior ends of the stomach, the panded into a long band of labeled matureraphe, bulb, and crests, which are all cov- cells intermingled with unlabeled matureered by ciliated mucous cells, are continu- cells.ous with each other and with the mucous After 20 days, the entire groove heightcells in the esophagus, Chief and zymogen is labeled, but no pseudostratified cells arecells (terminology, Burighel and Milanesi, labeled any longer. By 30 days, few cells in73) cover the stomach grooves. The chief the groove population are still labeled.cells, however, are much more numerous Thus, most of the DNA synthesizing cellsthan the zymogen cells, and are absorptive labeled at the time of injection as well asin function. Pseudostratified regions occur several generations of their progeny haveat the base of each groove, at the side of been lost from the epithelium, evidentlyeach crest, and at the side of the raphe through cell extrusion at the junction ofand bulb. Mitotic figures occur along the the groove population with the crest popu-lumenal edge of the pseudostratified re- lation on the tops of the folds.gions, but they are most numerous in the Crest population. At one hour aftergroove population. injection, cells on the sides of the stomach The folds on the dorsal surface of the crests are labeled (fig. 16). No mucousstomach are the longest, while the ones cells on the tops of the crests, however,directly adjacent to the ventral raphe are are labeled. Labeled nuclei do not occur inthe shortest. The dorsal folds are straight sections of every crest, since the population(fig. 2C); the ventral folds, however, twist is small. The crest cells also have fewer around the raphe and bulb. The anterior grains above their nuclei than the cells inends intersect the raphe while the poste- the groove population.rior ends intersect the bulb, suggesting By ten days after injection, the twothat the stomach folds generate hom the bands of labeled cells on each side of theventral surface. As the folds move away crests have migrated toward each otherfrom the ventral surface, they lengthen (fig. 17), and by 15 and 20 days, the two and become higher. bands have met each other on the tops of Groove population. At one hour after the crests. By 30 days, the crests are gen-injection, only nuclei at the base of each erally unlabeled. Evidently, the mucouspseudostratified region are labeled (fig. 12). cells are extruded into the gut lumen at By three days after injection, nuclei at all the tip of each stomach fold. Thus, twolevels in the pseudostratified region are separate renewing cell populations occurlabeled (fig. 13). No mature cells are yetlabeled, but several mitotic figures are la-beled. As in the esophagus, nuclei from the Fig. 8 An autoradiogram of two esophageal basal portion of each pseudostratified re- folds three days after injection. Only cells at the base of the folds are labeled. X 200.gion had migrated toward the lumenal Fig. 9 An autoradiogram of some esophageal edges of the cells in preparation for mito- folds 20 days after injection. Cells on the tops of sis. Two labeled nuclei immediately adja- the folds are now labeled. X 200. Fig. 10 An autoradiogram of a pseudostrati- cent to each other in the intermediate levels fied region (left) and some adjacent band cells of the pseudostratified region are some- (right) one hour after injection. Only pseudostrati- times observed, indicating that they are fied cells are labeled. X 420. the two daughter nuclei of a recently di- Fig. 11 An autoradiogram of a pseudostrati- fied region (left) and some adjacent band cells 60vided labeled cell. By three days, there has days after injection. Pseudostratified cells are no been a slight migration of labeled cells longer labeled but a few band cells are now la- towards the mature zone. beled. X 420.
458 THOMAS H. ERMAKon each fold, each renewed by its own ger- terns in the rectum are similar to thoseminal cells. observed in the intestine. The major dif- The stomach raphe and bulb are basic- ference is in the size of the germinal andally large crest populations which are re- mature zones, with fewer cells proliferat-newed on the same order of time. However, ing in the germinal zone of the rectumthese populations are much larger in size. than in the germinal zone of the intestine.Many cells are labeled on the bulb at onehour, and migration (in microns per day) Pyloric glandis rapid. By 15 to 20 days after injection, The pyloric gland is composed of numer-the two large bands of mucous cells have ous tubules which are exceedingly numer-met each other on the top of the raphe and ous at the level of the mid-intestine andbulb, as on the crests. are solidly packed close to the intestinal epithelium (fig. 2F). Although the tubules lntes tine are also fairly abundant in the stomach The intestine (fig. 2F) is a long tube wall, they are not packed close togetherwith a typhosole on one side and a deep next to the gut epithelium as in the intes-groove running along the length of the tine. A few pyloric tubules even occur inother side. The intestinal groove is lined the esophagus and intestine. Although theby pseudostratified cells while the rest of tubules occur associated with the maturethe intestinal wall is lined by absorptive cells of the digestive tract, they are notand secretory cells. closely associated with the germinal zones, At one hour and three days after injec- especially in the intestine. The fact thattion, labeling is much like in the groove the pyloric tubules occur directly adjacentpopulation of the stomach with only pseu- to the absorptive cells in the intestine butdostratified cells in the intestinal groove not the germinal cells suggests that thislabeled (fig. 18). Cells migrate at a differ- organ might be involved with the processent rate, however. After ten days, cells are of nutrient absorption and assimilation.labeled up to the top of the intestinal At all times after injection, few cells aregroove and frequently onto the side walls labeled in the pyloric tubules.of the intestine. Both absorptive and se- DISCUSSIONcretory cells are labeled. By 15 days, theleading edge of labeled cells has migrated The classification of cell populations (asaround the side walls almost to the begin- used by Messier and Leblond, 60; Camer-ning of the typhosole. on, 70) in the digestive tract of Styelu After 20 days, labeled cells have mi- c l a m is given in table 1. Static populations,grated up the sides of the typhosole (fig. which undergo no cell proliferation, do not19). The band of labeled cells is clearly occur in the digestive tract nor in the restdefined, unlike in the shorter mature zones of the body (Ermak, 75). Expanding popu-in the esophagus and stomach. In front of lations have a slow rate of cell prolifera-the band of labeled cells, no cells are la- tion and no detectable cell turnover withinbeled. Behind it, cells are usually only the life span of the animal (about 12-18lightly labeled. By 30 days, the leading Fig. 12 An autoradiogram of the stomachedges have met each other at the top of the grooves one hour after an exposure to tritiatedtyphosole in a few animals. In others, the thymidine. Cells are labeled at the base of eachleading edges have almost met each other. groove. x 150. Fig. 13 A n autoradiogram of a germinal zone Rec turn from a stomach groove three days after injection of tritiated thymidine. Nuclei at various levels The rectum is the terminal part of the within the pseudostratified region and a mitoticdigestive tract and is characterized by the figure (arrow) are labeled. X 600.lack of a groove of pseudostratified cells Fig. 14 An autoradiogram of some stomach grooves ten days after injection of tritiated thymi-and by a typhosole which has transformed dine. Labeled cells now occupy approximately halfinto a thin flap. A region of basophilic of the groove height. X 150.cells runs along the dorsal surface of the Fig. 15 A n autoradiogram of the groove pop- ulation 15 days after an exposure to tritiated thy-rectum in a position corresponding to the midine. Labeled cells now occur along the entirepseudostratified region of the intestine. groove height. Few cells at the base of the groove Cell proliferation and cell migration pat- remain labeled. X 150.
460 THOMAS H . ERMAKmonths, Johnson, 71). Although zone 3 of believed that the undifferentiated cells re-the endostyle appears to be an expanding place only the zymogen cells which arepopulation, longer termed studies would then lost from the epithelium. He consid-be required to decide whether i t is expand- ered that the chief cells represent a sepa-ing or only slowly renewed. rate cell-type not derived from the zymogen Renewing populations have a high rate cell population. My results clarify theseof cell proliferation, usually demonstrate points. Two separate cell populations (thecell migration, and have turnover times groove and crest populations) occur in thewithin the life span of the animal. Renew- stomach of Styela clava, each renewed bying populations are divided into two cate- its own germinal cells. The chief and zymo-gories based upon their turnover times. gen cells in the groove population, how-Type I populations have turnover times of ever, differentiate from the same germinalless than 60 days (usually between 1 and cells at the groove bases.5 weeks) whereas type I1 populations have In a typical renewing population in theturnover times of more than 60 days (prob- postbranchial digestive tract, DNA is syn-ably up to several months). The reason thesized in pseudostratified cells whosewhy type I1 cell populations have such long nuclei lie close to the basal portion of therenewal times in comparison to the other cell. As each cell enters mitosis the nucleusrenewing populations is not presently un- migrates toward the lumenal edge of thederstood. The renewal of the two popula- cell. Labeled mitotic figures appear as soontions in the esophagus is of particular in- as five hours after labeling (Ennak, unpub-terest since both mucous cells (type I) lished results). After cell division, the twoand band cells (type 11) originate from the daughter nuclei move back into the regionsame stem cell compartment. The patterns of interphase nuclei. The process of nu-of cell migration and loss in the major re- clear migration in preparation for mitosis,newing populations (dorsal lamina and interkmetic migration, is responsible forbranchial bars excepted) are shown sche- the dispersal of labeled nuclei throughoutmatically in figure 20. the various levels of the pseudostratified From the limited work on cell popula- region by three days after injection. Inter-tion kinetics of invertebrates, it seems kinetic migration of nuclei also occurs insafe to assume that renewing gut cell pop- many other epithelia such as the epitheliumulations are rarely found. Cell renewal of the neural tube (Watterson, 65), theoccurs in the ascidian branchial wall and mouse intestinal epithelium (Kataoka, 70),also in the gill of the freshwater mussel and the mammalian oviduct epithelium(Tomasovic and Mix, 74). In addition to (Wrenn and Wessells, 70). The exactthe ascidian gut, only part of the crayfish mechanism by which nuclear migrationgut (the hepatopancreas) is known to con- is achieved is not known, but it might in-tain renewing cell populations (Davis and volve microfilaments (Hinds and Ruffett,Burnett, 64). More commonly, the gut 71; Wessells et al., 71).cell populations of invertebrates are of the Cell division is responsible for the in-expanding type; such populations occur crease in the number of nuclei labeled within sea urchins (Holland, 64) and probably tritiated thymidine at increasing time in-sea stars (Anderson, 65) and oysters (Mix,71). Fig. 16 An autoradiogram of a stomach crest My results for Styela clava have con- three days after administration of tritiated thymi- dine. Cells are labeled on the sides of the crest.b e d earlier reports of stem cell popula- x 600.tions in the digestive tract of ascidians Fig. 17 An autoradiogram of a stomach crest(Fouque, 53; Relini-Orsi, 68; Thomas, ten days after administration of tritiated thymi-70; Aros and Viragh, 69). In Styela pli- dine. Columnar cells on the top of the crest are nowcata, Fouque (53) suggested from static labeled.18 600. autoradiogram of the intestinal Fig. X Anlight microscope evidence that the cells at groove one hour after injection of tritiated thymi-the base of the grooves in the stomach dine. Nuclei are labeled at the base of the groove.migrate up the folds, becoming at first X 160.chief and zymogen cells and then differ- intestine 20 An autoradiogram of one half of the Fig. 19 days after administration of tritiatedentiating into ciliated mucous cells on the thymidine. Cells are labeled up to the base of thecrests. In Ciona intestinalis, Thomas (70) typhosole. X 150.
462 THOMAS H . ERMAK A Fig. 20 Schematic diagrams of cell renewal in the digestive tract of Styela cluua showing directions of cell migration (small arrows) a n d regions of cell loss (large arrows). Black regions indicate germinal zones. Diagrams not to scale. A, lip epithelium (shaded) a n d funnel epithe- lium of the dorsal tubercle; B, zone I of the endostyle; C, stigmatic cells; D, ciliated band cells (shaded) a n d ciliated mucuous cells of the esophagus, E, crest population (shaded) a n d groove population of the stomach; F, intestinal epithelium.tervals after injection. After a DNA syn- eventually move out of the germinal zone,thesizing cell has divided, the daughter pass through the transitional zone, andcells may differentiate or divide again. then migrate through the mature zone inThose cells in the germinal zone which lie a linear fashion. Those cells initially la-close to the transitional zone are more beled and their immediate progeny movelikely to differentiate into mature cells through the epithelium as a band of heav-while those cells which lie far away from ily labeled cells. In front of the band, nothe transitional zone are more likely to di- cells are labeled. Behind it, cells are onlyvide a few more times. In any case, all lightly labeled or not labeled at all. Thosethose cells initially labeled with tritiated cells which are lightly labeled are descend-thymidine and/or their labeled progeny ants through many generations from those
CELL PROLIFERATION IN ASCIDIAN GUT 463 TABLE 1 Classification of the cell populutions in the digestive tract of Styela clava according to proliferative behuvior Type of cell population Example (with transit time) Expanding populations Branchial tentacles Unciliated pharyngeal cells Zones 2, 3,4, , 6 , 7, 8, and 9 of endostyle 5 Pyloric gland Renewing populations A. Type I Funnel epithelium of dorsal tubercle (2.5 weeks) (renewal in less than 60 days) Lip epithelium of dorsal tubercle (2.5 weeks) Dorsal lamina and branchial bars (several weeks) Stigmatic cells (1.5 weeks) Mucous cells of esophagus (2.5 weeks) Crest population of stomach (2.5 weeks) Groove population of stomach (2.5 weeks) Intestinal epithelium (5 weeks) Rectal epithelium (5 weeks) B. Type I1 Zone 1 of endostyle (several months) (renewal in more than 60 days) Band cells of esophagus (several months)cells which were initially labeled with and intestine, however, are dramaticallytritiated thymidine and have had their different. Cells in the intestine migrateDNA diluted with unlabeled precursors. faster yet are renewed slower than cells inAssuming a chromosomal number of 32 the stomach. It is reasonable to assume(Minouchi, 36) for Styela, the progeny of that the stomach is under more demandslabeled cells would have only one labeled for cell turnover than the intestine. Mortonchromosome after six cell divisions (Cron- (60) claims that the stomach is the pri-kite et al., Sl), if a labeled cell remained mary site of digestion in ascidians whilein the germinal zone long enough to divide Yonge (25) and van Wee1 (40) state thatsix times, that is. In any case, the band of the intestine is the primary site of absorp-labeled cells is finally lost from the cell tion. Presumably the stomach provides apopulation, presumably through extrusion more formidable environment of digestiveof cells into the lumen of the gut. enzymes. Although most of the cell proliferation The renewing populations in the ascidianin the germinal compartments is for the postbranchial digestive tract are quite sim-replacement of mature cells lost from the ilar to the renewing populations in thecell populations, some proliferation pro- stomach and intestine of mammals. Thevides for the growth of the digestive tract, renewal of the mucous cells in the esoph-especially in small individuals. As animals agus and stomach of Styela is similar togrow larger, the stigmata multiply, the the renewal of the surface epithelium infolds in the esophagus and stomach grow the mammalian stomach (Messier, 60).longer, and the intestine grows wider. The renewal of the band cells in the esoph- There also appears to be some mecha- agus of Styela is similar to the renewal ofnism of regulating cell proliferation and the parietal cells in the mammalian stom-maturation in the ascidian digestive tract ach (Raggins et al., 68; Kataoka, 70) andsuch as that proposed in the rat intestine the Paneth cells in the mammalian intes-(Galjaard et al., 72; Rijke et al., 74). tine (Cheng et al., 69; Cheng, 74b). TheTransit times are the same for the mucous band cells, parietal cells, and Paneth cellscells in the esophagus and stomach, al- are all renewed at a much slower rate thanthough the sizes of the cell populations most of the other cells lining the digestive(esophageal fold; stomach crest, raphe and tract. They also originate from germinalbulb) and the actual migration rates (in cells which also give rise to the rapidly re-microns per days) are different. Migration newed cells of the digestive tract.rates are proportional to the sizes of the The renewal of the groove populationgerminal and mature compartments. Tran- in the stomach and the renewal of the in-sit times for the chief cells in the stomach testinal epithelium of Styela is most like
464 THOMAS H. ERMAKthe renewal of the intestinal villi of mam- Cheng, H. 1974 Origin, differentiation and re-mals (Leblond and Messier, 58; Messier newal of the four main epithelial cell types in the mouse small intestine. 11. Mucous cells. Am.and Leblond, 60; Cheng and Leblond, J. Anat., 141 : 481-502.74a,b; Cheng, 74a). In mammals, cell 1974 Origin, differentiation and renewalproliferation occurs only in the crypts of of the four main epithelial cell types in the mouseLieberkiihn. Crypt cells migrate up the small intestine. IV. Paneth cells. Am. J. Anat., 1 4 1 : 521-536.villi, differentiate into chief and goblet Cheng, H., and C. P. Leblond 1974 Origin, dif-cells, and are finally extruded at the villus ferentiation and renewal of the four main epi-tips. Cell migration in mammals, however, thelial cell types in the mouse small intestine.is out of pits and up fingerlike projections. I. Columnar cell. Am. J. Anat., 141 : 461-480. 1974 Origin, differentiation and renew-Migration in Styela, on the other hand, is al of the four main epithelial cell types in thelinear and up longitudinal folds. Perhaps mouse small intestine. V. Unitarian theory ofthe most conspicuous difference between the origin of the four epithelial cell types. Am.ascidian and mammalian gut renewal is J. Anat., 141 ; 537-562. Cheng, H., J. Merzel and C. P. Leblond 1969in transit time. The time for a labeled cell Renewal of Paneth cells in the small intestine ofto reach the villus tip is about one and the mouse. Am. J. Anat., 126: 507-524.one-half days in the rat (Messier and Le- Cronkite, E. P., S. W. Greenhouse, G. Brecher,blond, SO), about three days in the mouse V. P. Bond 1961 Implication of chromosome structure and replication on hazard of tritiated(Leblond and Messier, 58), and about five thymidine and the interpretation of data on cellto six days in man (MacDonald et al., 64). proliferation. Nature, 189: 153-154.In Styela, transit times are about two and Davis, L. E., and A . L. Burnett 1964 A study ofa half weeks in the stomach and about growth and cell differentiation i n the hepato-five weeks in the intestine. Cell renewal pancreas of the crayfish. Devel. Biol., 10: 122- 153.times in the intestine of Styela, however, Ermak, T. H. 1975 Cell Proliferation in the As-are comparable to those in the newt intes- cidian Styela clava: An Autoradiographic andtine (OSteen and Walker, 60) where re- Electron Microscopic Investigation Emphasizingnewal is on the order of a month. Cell Renewal i n the Digestive Tract of This and Fourteen Other Species of Ascidians. Ph.D. dis- ACKNOWLEDGMENTS sertation, Univ. Calif., San Diego. Fouque, G. 1953 Contribution ~3 1Ctude de la I am indebted to Dr. Nicholas D. Holland glande pylorique des ascidiaces. Ann. Inst.for his advice, support, and criticism. I Oceanogr. (Paris), 28: 18CL317.thank Emily Reid for the final drawings Galjaard, H., W. Van der Meer-Fieggen and J.of figures 1 and 2. Giesen 1972 Feedback control by functional villus cells on cell proliferation and maturation LITERATURE CITED in intestinal epithelium. Exp. Cell Res., 73: 197- 207.Abbott, D. P. 1975 Phylum chordata: Introduc- Godeaux, J., and H. Firket 1966 Ultrastructure tion and Urochordata. In: Lights Manual. R. I. de lendostyle de Molgula manhattensis Kay (As- Smith and J. T. Carlton, eds. Univ. Calif. Press, cidie stolidobranche). Compt. Rend. Acad. Sci. Berkeley, pp. 6384355. (Paris), 262: 48EL490.Abbott, D. P., and J. V. Johnson 1972 The as- 1969 Etude au microscope electronique cidians Styela burnharti, s. plicatu, s. clava, de lendostyle dune ascidie stolidobranche M o l - and S. montereyensis in California waters. Bull. gula manhattensis Kay. Ann. Sci. Nat. Zool. S. Cal. Acad. Sci., 71 : 95-105. Biol. Animale, 10: 163-187.Anderson, J. M. 1965 Studies on visceral regen- eration in sea stars. 11. Regeneration of pyloric Hinds, J. W., and T. L. Ruffett 1971 Cell pro- caeca in Asteriidae with notes on the source of liferation in the neural tube: an electron micro- cells in regenerating organs. Biol. Bull., 128; scopic and Golgi analysis in the mouse cerebral 1-23. vesicle. Z. Zellfrsch. Mikr. Anat., 1 1 5 : 226-264.Aros, B., and S. Viragh 1969 Fine structure of Holland, N. D. 1964 Cell Proliferation in Post- the pharynx and endostyle of an ascidian (Ciona embryonic Specimens of the Purple Sea Urchin intestinalis). Acta. Biol. Acad. Sci. Hung., 20: (Strongylocentrotus purpuratus): an Autoradio- 281-297. graphic Study Employing Tritiated Thymidine.Berrill, N. J. 1950 The Tunicata. Ray Society, Ph.D. dissertation, Stanford Univ. London. Johnson, J. V. 1971 The annual growth andBurighel, P., and C. Milanesi 1973 Fine struc- reproductive cycle of Styela sp. in the Marina ture of the gastric epithelium of the ascidian del Rey, Venice, California. M. S. thesis, Dept., Botryllus schlosseri. Vacuolated and zymogen Zool., Univ. Nebraska. cells. Z. Zellforsch. Microscop. Anat., 1 4 5 : 541- Kataoka, K. 1970 The fine structure of the pro- 555. liferative cells of the mouse intestine a s revealedCameron, I. L. 1970 Cell renewal in the organs by electron microscopic autoradiography with and tissues of the non-growing adult mouse. H3-thymidine. Z. Zellforsch. Mikr. Anat., 103: Texas Rep. Biol. Med., 28: 203-248. 170-178.
CELL PROLIFERATION I N ASCIDIAN GUT 465Leblond, C. P., R. C. Greulich and J. P. M. Pereira OSteen, W. K., and B. E. Walker 1960 Radio- 1964 Relationship of cell formation and cell autographic studies of regeneration. Anat. Rec., migration in the renewal of stratified squamous 137: 501-509. epithelium. Adv. Biol. Skin, 5: 39-67. Ragins, H.,F. Winlze, S. M. Liv and M. Dittbren-Leblond, C. P., and B. Messier 1958 Renewal ner 1968 The origin and survival of gastric of chief cells and goblet cells in the small intes- parietal cells in the mouse. Anat. Rec., 162: tine as shown by radioautography after injection 99-1 10. of thymidine.Hs into mice. Anat. Rec., 132: Relini-Orsi, L. 1968 Prime osservazioni morfo- 247-259. logiche ed istochimiche sull apparato digerenteLeblond, C. P., B. Messier and B. Kopriwa 1959 di Styela plicuta. Les. Boll. Mus. Inst. Biol. Thymidine-Hs as a tool for the investigation of Univ. Genova, 36: 157-184. the renewal of cell populations. Lab. Invest., 8: Rijke, R. P. C., W. Van der Meer-Fieggen and H. 296308. Galjaard 1974 Effect of villus length on cellLevi, C., and A. Porte 1964 Ultrastructure de proliferation and migration i n small intestinal lendostyle de lascidie Microcosmus claudicans epithelium. Cell Tiss. Kinet., 7: 577-586. Savigny. 2. Zellforsch. Mikr. Anat., 62: 293- Sokolska, J. 1931 Contribution lhistologie de 309. lendostyle des Ascidies. Folia Morfol., 3: 1-34.MacDonald, W. C., J. S . Trier and N. B. Everett Thomas, N. W. 1970 Morphology of cell types 1964 Cell proliferation and migration in the from the gastric epithelium of Ciona intestinalis. stomach, duodenum, and rectum of man: radio- J. Mar. Biol. Ass. U. K., 50: 737-746. autographic studies. Gastroenterol., 46: 405- Tomasovic, S. P.,and M. C. Mix 1974 Cell re- 41 7. newal in the gill of the fresh-water mussel, Mar-McGinitie, G. E. 1939 The method of feeding garitifera margaritifera: an autoradiographic of tunicates. Biol. Bull., 77: 44-47. study using high specific activity tritiated thy-Messier, B. 1960 Radioautographic evidence for midine. J. Cell Sci., 14: 561-569. the renewal of the mucous cells in the gastric Watterson, R. L. 1965 Structure and mitotic be- mucosa of the rat. Anat. Rec., 136: 242. havior of the early neural tube. In: Organogene-Messier, B., and C. P. Leblond 1960 Cell pro- sis. R. L. Dehaan and H . Ursprung, eds. Holt, liferation and migration as revealed by radio- Rinehart, and Winston, New York, pp. 129-159. autography after injection of thymidine-Ha into Weel, P. B. van 1940 Beitrage zur Ernahrungs- male rats and mice. Am. J. Anat., 106: 247-265. biologie der Ascidien. Pub. Sta. Zool. Napoli,Minouchi, 0. 1936 Notiz iiber die chromosomen 18: 5 C 7 9 . von Tethyum plicatum Les. (Ascidia). Z . Zell- Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. forsch. Microscop. Anat., 23: 790-794. Bradley, M. A. Luduena, E. L. Taylor, J. T.Mix, M. C. 1971 Cell renewal systems in the Wrenn and K. M. Yamada 1971 Microfila- gut of the oyster Crassostrea gigus (Mollusca: ments in cellular and developmental processes. Bivalva). Veliger, 14: 202-203. Science, 171 ; 1 3 S 1 4 3 .Mochan, 0. T., N. L. Liebson and L. T. Frolova Wrenn, J. T., and N. K. Wessells 1970 Cyto- 1971 On localization of proliferative process in chalasin B: Effects upon microfllaments involved the guts of some species of invertebrates (In in morphogenesis of estrogen-induced glands of Russian). Sci. Comm. Inst. Mar. Biol. Far East. oviduct. Roc. Nat. Acad. Sci., 66: 904-908. Sci. Cent. Akad. Nauk. S. S. S. R., 2: 152-157. Yonge, C. M. 1925 Secretion, digestion, andMorton, J. E. 1960 The functions of the gut in assimilation in the gut of Ciona intestinalis. ciliary feeders. Biol. Rev., 35: 92-140. Brit. J. Exp. Biol., 2: 373-388.