Cell Migration Kinetics in the Stomach of Styela clava(Urochordata: Asc id iacea) THOMAS H. ERMAK’ Scripps Institution of Oceanography, La Jolla, California 92037 ABSTRACT By autoradiography with tritiated thymidine, the rate of cell migration was studied for the groove population of epithelial cells lining the stomach of Styela. After one hour, the-tritiated thymidine is incorporated into undifferentiated, dividing cells. Labeled differentiated cells appear after three days, and the migration of labeled cells to the extrusion zone takes about 16 days ( T = 1 . ) Stomach growth is accomplished by an increase in both number 9C. and height of epithelial folds. The percentage rate of cell migration probably remains fairly constant throughout adult life. However, it is decreased by starva- tion and low temperatures. In an earlier report (Ermak, ’75), cell the presumed zone of cell extrusion waspopulations in the digestive tract of Styela taken as 100% (fig. 1). The leading edgeclava were described qualitatively with au- of labeled cells was considered to be thetoradiography. On a stomach fold, cells percentage of distance migrated from theproliferate in restricted zones of pseudo- time of injection until sacrifice. Ten ran-stratified cells, migrate into mature zones domly selected grooves were measuredof simple columnar cells, and are finally from the base of the groove to the junc-extruded into the gut lumen. Cell renewal tion with the crest population. The medianon an ascidian stomach fold is quite simi- values from each individual were thenlar to that on a mammalian intestinal averaged and the median percentage mi-villus (Leblond and Messier, ’58), which gration value for each time interval listedhas been extensively investigated. The in table 1. The median instead of the meanpresent study defines quantitatively the was used for all studies since percentagesrate of cell migration in the stomach do not fit “t” distributions. Average transitgroove population, a renewal system where time was determined from the 100% inter-absorptive and zymogen cells are replaced cept on the graph of percentage migrationby relatively undifferentiated cells. Growth as a function of time.of the stomach folds as well as the influ- For a study of stomach growth, 35 speci-ence of animal size, nutritional state, and mens 0.25 to 30 g in weight had the stom-temperature upon the rate of cell migra- ach prepared for routine histology. Thetion are considered. number of folds was counted, and the height of each fold was measured as in MATERIALS AND METHODS the foregoing paragraph. Experiments on All specimens of Styela clava were col- the influence of animal size, nutritionallected at the Quivira Basin of Mission Bay, state, and temperature were conducted forSan Diego, California. The tritiated thymi- a period of ten days. This time was chosendine was administered and autoradiograms as an average time between the entrancewere prepared as previously described of labeled cells into the mature compart-(Ermak , ’75). For the cell migration study, ment and the beginning of cell extrusion.ten individuals were held at 19 1°C and For the temperature study, the Gabriel testsacrificed at the following time intervals: was used to test the statistical difference 1 hour, 3, 10, 15, and 20 days after injec- in two or more medians. For the rest of thetion. Cell migration was measured as fol-lows: the base of the pseudostratified re- versity of California Department of Physiology, Uni- 1 Present address: Medical Center, San Francisco,gion was taken as 0% migration whereas California 94143.J. ExP. ZOOL., 1 9 7 : 339446 339
340 THOMAS H. ERMAK crest POP 100% groove POP Fig. 1studies, the Mann-Whitney U test was usedto test the statistical difference betweenmedian values (Dixon and Massey, 69). To study the influence of animal size oncell migration, ascidians were divided intotwo groups of ten individuals each: sexu-ally immature animals 1.0 to 2.5 g inweight and sexually mature animals 11 to - 50Oo / OO/O Method of measuring cell migration on a stomach fold. ature for one day before injection. The aerated sea water was changed daily and kept within 2 0.5"C of the experimental temperature. No additional food, other than that in the changes of water, was provided, RESULTS Table 1 shows the percentage of stomach20 g in weight. Both groups were injectedand then isolated for two days; for the re- TABLE 1maining eight days, they were returned to Extent of cell migration i n groove population asrunning sea water averaging 19°C. revealed by median (range, number of animals) To examine the influence of nutritional percentage migrationstate on cell migration, 20 animals 2.5 to ~ ~ Time interval % migration4.5 g were collected. Half were starved infiltered sea water for ten days prior to in- 1 hour 9%jection and then starved thereafter. These ( 6-11%, 6)ascidians lost an average of 0.5 g during 3 days 18%the 2Oday period. The other animals were (12-22%, 10)maintained in Mission Bay during the 10 days 45%same time interval and gained about 1 g. (25-72%, 8)All animals were kept at 13-15°C. 15 days 94% For the temperature study, 40 animals (76-loo%, 8)3 to 6 g in groups of ten were graduallybrought to 6, 13, 20, and 26°C over a one 20 days 100%day period and maintained at that temper- (2 - 9)
CELL MIGRATION KINETICS IN ASCIDIAN STOMACH 34 1 35 30 0u) 0D 25 00Lc 0YI0L 20Q)9E 15 0 I I I I I 1 5 10 15 20 25 30 body weight (gm) Fig. 2 Number of stomach folds as a function of body weight. Each point represents the number of folds i one animal. ngroove height labeled at increasing time of 30 pm per day, is slower in the germinalintervals after injection. About 9% of the than the mature compartment.groove is labeled before any migration be- Histological sections show that the num-gins and may be taken as the approximate ber of folds in the stomach lining increasessize of the germinal compartment. By three with increasing body size (fig. 2). Animalsdays, most label is still confined to the weighing less than one g have 15 to 20pseudostratified region (18%, 12-22% ). folds while those more than a gram haveSince this zone occupies about 17% (11- 20 to 30. Most folds appear to be added2 4 % ) of the groove height, cell differ- before individuals reach a weight of aboutentiation must be completed in the first 5 g; the number does not significantly in-migrating labeled cells shortly after three crease after 10 g, Indeed, limited data sug-days. At 10 days, the variability in cell gest a slight decrease in the largest ani-migration rate is large, the percentage mals. These sections also show that smallerlabeled ranging from 25 to 72%. By ascidians have shorter folds (fig. 3 ) . Thegraphic interpolation, cell migration is esti- average height of a fold varies from 0.58mated to be completed by 16 (13-18) days. mm for a 3 g specimen to 1.65 mm for aMigration, which occurs at an average rate 15 g animal. The height of a fold increases
342 THOMAS H. ERMAK 1.80EE 1.40 W 1.oo @ @&0c 0.60 0.20 5 10 15 20 25 30 body weight (gm) Fig. 3 Height of stomach folds as a function of body weight. Each point represents the average height of ten folds from one animal. until an animal weighs about 10 g; there- centage basis, remains fairly constant, the after, the height remains constant with absolute rate of migration must be greater growth. Johnson (71) found that Styela in larger animals (about 50 pm per day) clava reaches sexual maturity at about 8 than in smaller animals (about 30 pm per cm. In the present investigation, 8 cm cor- day). responds to a weight of about 8 grams. In starved and fed animals, cell migra- Those ascidians smaller than 5 g are gen- tion is significantly greater in fed than erally sexually immature. Thus, the most starved individuals (table 2). Ten days rapid increase in fold length occurs dur- after the administration of tritiated thymi- ing the period of growth before sexual dine, 60% and 36% of the groove heights maturity. are labeled respectively in fed and starved Autoradiograms of the stomach from ascidians ( p = 0,006). In addition, the groups of small and large sized animals height of the grooves (428 -F- 45 pm vs show no significant difference in the per- * 259 21 pm) and the height of the epithe- centage of cell migration (table 2). The * lial cells from base to apex (57 5 pm vs constancy in percentage rate of cell migra- 31 f 3 pm) is likewise smaller in the tion implies that the rate of cell renewal starved group, with both germinal and ma- remains fairly constant throughout adult ture compartments affected. It is estimated life. Although the migratory rate, on a per- that transit time through the mature com-
CELL MIGRATION KINETICS I N ASCIDIAN STOMACH 343 TABLE 2 phases. Before three days, cells are re- Influence of animal size, nutritional state, and stricted to the pseudostratified region and temperature upon cell migration giving the migration is slow. During this initial pe- median (range, number of animals) riod, cells are involved in cell division and percentage migration and significance growth. A DNA-synthesizing cell has its nucleus located in the basal portion of the Condition % migration cell; before mitosis, the nucleus migratesSmall sized 51% (28-92%, 9) toward the cell apex (Ermak, 75). There-Large sized 39% (29-62%,6) after, the nuclei of the daughter cells mi-Significance N.S. grate basally. After three days, germinalStarved 36% (21-70%, 9)Fed 60% (27-82%,9) cells have differentiated into mature (ab-Significance p = 0.006 sorptive and zymogen) cells. They are con-(by a 1 tailed test) sidered to be differentiated by morphologi- 6" 21% (11-51%,6) cal criteria; biochemical, behavioral, and13" 25% (19-33%, 8)20" 46% (28-77%, 7) developmental criteria (as defined by Grob-26" 77% (12-loo%, 6) stein, 59) were not applied. After differ-Significance + 6" 20",0.01<p<0.05 entiation, the cells grow in volume and(by a 2 tailed test) 6"+26",0.01<p<0.05 their nuclei come to lie next to each other 13"+20", 0.01<~<0.05 13"+26";0.01<p<0.05 forming a simple columnar epithelium. Mi- 6"+ 13", N.S. 1 gration through the mature compartment 20"+26", N.S. is more rapid; the change in rate is due, at 1 N.S., not significant. least in large part, to proliferation of ger- minal cells. Cell size and shape seem topartment is lengthened about a week by affect migration rate in much the samestarvation. Qualitatively, labeled cells are way as changing tube diameter affectsstill confined to the pseudostratified region water flow through a pipe. Water flow in-i n starved animals, whereas most of the creases as water in a pipe of large diameterlabeled cells have migrated into the mature passes to one of small diameter.zone in the fed condition. Starvation ap- Growth of the stomach is due to an in-parently lengthens the turnover time of crease in both the number and height ofgerminal cells. However, it is not presently epithelial folds. Most folds must be addedknown which parts of the cell cycle are between metamorphosis and sexual matur-most affected; moreover, it is possible that ity since the post-metamorphic ascidianstarvation could decrease the growth frac- juvenile has a smooth walled stomach, andtion (defined by Cleaver, 67) of the cell folding occurs after metamorphosis (Scott,population. 52; Trason, 57; Cloney, 61). In Styela, the In the temperature study, significant stomach folds grow in height until an ani-differences are observed between all me- mal reaches about 10 g; thereafter, theydian temperatures except between 6 and stay about the same height. Both germinal 13°C and 20 and 26°C (table 2). At both and mature compartments increase in size,low temperatures, labeling is almost iden- and adult ascidians exhibit no significanttical. Ten days after the injection of tri- differences in the percentage rates of celltiated thymidine many pseudostratified but renewal during growth. In these respects,few mature cells are labeled. Although Styela differs from the mouse, in whichthere is no quantitative difference between the percentage rate of cell renewal on an20 and 26"C, there is a distinct qualita- intestinal villus changes with animal sizetive difference. In most ascidians at 26"C, (Koldovsky et al., 66). OConnor (66)only a few lightly labeled cells remain in states that villus growth in the fetal mousethe pseudostratified region. At 20"C, how- intestine is not due to a higher mitoticever, many labeled cells are still confined rate but the result of an extrusion rateto this region. below that of the adult. Such might also be the case in Styela. The influence of body DISCUSSION size upon cell renewal is comparable to the Cell migration in the stomch grooves of influence of cell population size in theStyela clava may be divided into two same ascidian. Mucous cells in the esoph-
344 THOMAS H. ERMAKagus and stomach (crests, raphe, and indirectly by influencing the rate of filterbulb) are renewed at the same rate al- feeding and, thus, nutritional state. Northough cell population sizes differ in each did it consider the role of low temperatureregion (Ermak, 75). isoenzymes (Hochachka and Somero, 73) In Styela, starvation decreases the cell in the long term adaptation of ascidians topopulation size, the compartment size, and winter temperatures. In the laboratory,the rate of cell renewal. In mammals, star- however, small changes in temperature ap-vation decreases the intestinal villus cell parently have little effect upon cell re-population size (Hooper and Blair, 58; Deo newal.and Ramalingaswami, 65). In the crypts, ACKNOWLEDGMENTSthe length of the cell cycle is also in- I am indebted to Dr. Nicholas D. Hollandcreased, especially the time of DNA synthe- for his support, criticism, and advice. Isis (Hooper et al., 68; Rose et al., 71). The thank Shirley Philibosian for her statisticaldecrease in cell population size after star- assistance and Dr. David Epel for his sug-vation is presumed to be due to a drop in gestions.the rate of cell proliferation below the rate LITERATURE CITEDof cell loss (Hooper and Blair, 58). There- Berrill, N. J. 1929 Digestion in ascidians andafter, the rate of cell loss would probably the influence of temperaure. J. Exp. Biol., 6 :come into equilibrium again with cell divi- 275-292.sion. MacGinitie (39) reports that food Cleaver, J. E. 1967 Thymidine Metabolism andsupplies of ascidians in Southern Cali- Cell Kinetics. John Wiley and Sons, Inc., New York.fornia bays are enriched by dinoflagellates Cloney, R. A. 1961 Observations on the mech-in summer. Such fluctuations in water anism of tail resorption in ascidians. Amer.quality could possibly cause changes in ZOO^., 1 : 67-87.renewal rates under field conditions. In Deo, M. G., and V. Ramalingaswami 1965 Re- action of the small intestine to inducted pro-any case, cell renewal times for ascidians tein malnutrition in rhesus monkeys - a studyin the field are probably somewhat shorter of cell population kinetics in the jejunum.than for animals maintained in the labora- Gastroenterol., 49: 150-157.tory (compare table 1 and 2 ) where food Dixon, W. J., and F. J. Massey 1969 Introduc- tion to Statistical Analysis. McGraw-Hill, Newis in comparatively short supply. York. The difference in renewal rates at 13 Dybern, B. I. 1965 The life cycle of Ciona in-and 20°C suggests that cell turnover slows testinalis L. F. Typica in relation to environ-down in winter. In San Diego, sea surface mental temperature. Oikos, 26: 109-131.temperatures range from about 12°C in Ermak, T.H. 1975 Cell Proliferation in the Di- gestive Tract of Styela clava (Urochordatawinter to 22°C in summer (Scripps Pier, Ascidiacea) as revealed by autoradiography72, M. Robinson, personal communica- with tritiated thymidine. J. Exp. Zool., 194:tion). During the winter of 1974, surface 449466.temperatures in Mission Bay dropped to Grobstein, C. 1959 Differentiation of vertebrate 15°C (measurements by the author). It cells. In: The Cell, Vol. I. J. Brachet and A. E. Mirsky, eds. Academic Press, New York, pp.is also during winter that reproduction is 437-496.interrupted in Styela calua (Johnson, 71). Hochachka, P. W., and G. N. Somero 1973Dybern (65) cites temperature as an im- Strategies of Biochemical Adaptation. W. B.portant factor in influencing reproductive Saunders Co., Philadelphia. Hooper, C. S., and M. Blair 1958 The effect ofcycle, growth rate, and life span in ascid- starvation on epithelial renewal in the rat duo-ians. The same species, depending upon denum. Exp. Cell Res., 14: 175-181. annual temperature range, may have a dif- Hopper, A. F.,R. W. Wannemacher and P. A.ferent growth rate and life span at differ- McGovern 1968 Cell population changes i n the intestinal epithelium of the r a t followingent latitudes and different seasons of the starvation and protein depletion. Proc. SOC.Exp.year. In ascidians, heart rate (Redick, 69) Biol. Med., 128: 695-698. and the passage of food through the diges- Johnson, J. V. 1971 The annual growth and tive tract (Berrill, 29) also slow down at reproductive cycle of Styela sp. in the Marinalow temperatures. The present investiga- del Rey, Venice, California. M.S. thesis, Dept. Zool., University Nebraska. tion did not demonstrate whether temper- Koldovsky, O., P. Sunshine and N. Kretchmer ature affects cell renewal directly or only 1966 Cellular migration of intestinal epithelia
CELL MIGRATION KINETICS IN ASCIDIAN STOMACH 345 in suckling and weaned rats. Nature, 212: heart rate in Molgula manhattensis. Am. Zool., 1389-1390. 9: 589.Leblond, C. P., and B. Messier 1958 Renewal Rose, P. M., A. F. Hopper and R. W. Wanne- of chief cells and goblet cells in the small in- macher 1971 Cell population changes i the n testine as shown by radioautography after in- intestinal mucosa of protein depleted or starved jection of thymidine.Ha into mice. Anat. Rec., rats. I. Changes in mitotic cycle time. J. Cell 132: 247-259, Biol., 50: 887-892.McGinitie, G. E. 1939 The method of feeding Scott, F. M. 1952 The developmental history of tunicates. Biol. Bull., 77: 443-447. of Amaroucium constellatum. 111. Metamor-OConnor, T. H. 1966 Cell dynamics in the in- phosis. Biol. Bull., 103: 226-241. testine of the mouse from late fetal life to ma- Trason, W. 1957 Larval structure and devel- turity. Am. J. Anat., 118: 525-536. opment of the oozoid in the ascidian Euherd-Redick, T. 1969 The effect of temperature on mania claviformis. J. Morph., 100: 509-545.