ermak comparative cell proliferation j exp zool 1981

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 217:325-339 (1981)

A Comparison of Cell Proliferation Patterns in the Digestive Tract of Ascidians

THOMAS H. ERMAK Scnpps Institution o f Oceanography, La Jolla, California 92037

ABSTRACT Cell proliferation patterns in the postbranchial digestive tract of a variety of ascidian species were investigated with autoradiography and tritiated thymidine. Based upon the distribution of radioactive nuclei, gut cell populations were classified as either static, expanding, or renewing. In most ascidians, renew. ing populations occur in the esophagus and stomach, but expanding populations sometimes occur in all or part of the intestine. From species to species, the number of cell renewal units (pairs of germinal and mature zones) in each portion of the digestive tract increases with increasing organ size, and this multiplication of renewal units follows the folding patterns in the gut. Cell turnover is fastest and the size of a cell renewal unit smallest a t the anterior end of the gut. The smallest CD.

lonial ascidians have a single renewal unit per cell population in the esophagus and stomach and an expanding population in the intestine. Epithelial folding and multiplication of cell renewal units occurs in solitary species of increasing body size. One cell population occurs on the stomach folds of more primitive solitary ascidians, two of those of advanced species, The digestive diverticulum, which only occurs in two families, is renewed much the same as the stomach, and probably evolved from that organ. In the intestine of primitive solitary ascidians, renewing populations only occur a t the anterior end; with evolutionary advancement, r e newing populations line the entire intestine.

In Styeh chva, a solitary stolidobranch asci- dim, most of the gut epithelia are renewing cell populations (Ermak, '75a, c, '76a). Cell proliferation occurs in restricted germinal zones of pseudostratified cells. With time, germinal cells migrate into mature zones of ciliated, secretory, or absorptive cells. Aging mature cells are presumably extruded into the gut lumen.

In other ascidian species, the postbranchial digestive tract (esophagus, stomach, and intestine) exhibits a great deal of variability in posi- tion, shape, and structure (Berrill, '50); it may be posterior or next to the branchial basket; on the right or left side of the body; U-shaped, S-shaped, or twisted into a variety of shapes. A particularly salient feature is the elaboration of the epithelial lining. This may be unfolded, folded, or modified into a system of canals and tubules. Ascidians with small bodies (most colonial ascidians) have unfolded or smooth gut linings, whereas ascidians with large bodies (a few colonial species and all solitary species) have highly folded epithelial linings.

This paper considers cell proliferation patterns in a variety of ascidian species in order to determine those features of gut cell renewal which are characteristic of the class Ascidiacea as a whole. Examples of each ascidian family (Berrill, '50) from California waters were injected with tritiated thymidine. In the esophagus, stomach, and intestine, sites of cell proliferation were detected with autoradiography in order to characterize each cell population. In some cases, individuals were sacrificed at increasing time intervals after injection in order to determine the fate of the DNA synthesizing cells. Cell proliferation patterns in different species were compared to each other and to those in S t y e h (Ermak, '75c).

MATERIALS AND METHODS

All ascidians were collected in California by picking them off the underside of docks, in the intertidal zone at low tide, or by diving with T.H. Ermak's present address is Department of Physiology,

University of California School of Medicine, San Francisco. CA 94143.

0022-104X/81/2173-0325$04.5001981 ALAN R. LISS. INC.

326 T.H. ERMAK

SCUBA (see Ermak, '75a, for localities). Those species included in this investigation are listed in Table 1 (for descriptions and additional illus- trations, see Ritter, '17, and van Name, '45). All families are represented except the family Dia- zonidae, of which there are no Pacific coast represent atives.

On the day of collection, specimens were exposed to tritiated thymidine for one hour (Er- mak, '752). In some cases, the solution of tritiated thymidine in sea water was again diluted with sea water. Solitary ascidians were injected with 5 pCi of tritiated thymidine per gm animal weight. At least three animals were injected for each time interval to be investigated. Two or three injections 24 hours apart were given to Molgula and Pyura in order to increase their low labeling index. Colonial ascidians would usually not take up the radioactive label if placed in a solution of tritiated thymidine in sea water and were, therefore, per- fused with the solution of tritiated thymidine by injecting approximately 15 pCi into the common tunic. Each sample of colonial ascidians injected contained numerous individuals. Polyclinum and Ascidia were sacrificed after one hour, 5 , 10, and 15 days. Ciona was sacrificed at one hour, 15, and 20 days. Pyura was sacrificed at one hour, 5, 10, 20, and 30 days. All other species were sacrificed after one hour only. Autoradiograms were prepared as previously described (Ermak, '75c) and exposed for two weeks, one month, or two months.

0.2mm H 0.3mm

H

L

RESULTS

General features Ascidians exhibit a large range in individual

body and organ size. Colonial species repro. duce rapidly through asexual reproduction, increasing the number of individuals in a colony, and are several millimeters in length, at most a few centimeters. Solitary species re- produce only sexually and spend much of their lives (usually only a year or two) increasing in size and complexity. They may reach 10 cm or more in length.

The postbranchial digestive tract, like in Styela (Ermak, '75c), is composed of anesopha- gus, stomach, and intestine (Fig. 1). The esophagus is a short tube in which the food cord is formed and is folded in certain solitary species. The stomach, the largest organ in which enzymes are secreted, has several folding patterns (listed in Table 1). In most colonial ascidians, the stomach has a smooth wall (Fig. 1A) and a raphe of mucous cells along one side. In some colonial species, e.g., Distaplia, the epithelium forms small longitudinal ridges (corrugated) which apparently do not represent multiple zones of proliferation, as in larger ascidians. The colonial species Euherdmania has a folded stomach (Fig. 1B). No colonial species examined had a folded esophageal or intestinal lining.

In solitary species, the stomach is always folded or has a digestive diverticulum off one

1 mm H

1Cm H

3mm H

Fig. I . Types of ascidian postbranchid digestive tracts. A) Colonial ascidian, smooth stomach. B) Colonial ascidian. folded stomach. C) Chelyosoma, pitted (areolated or waffle patterned) stomach. D) Ascidia, longitudinally folded stomach. E) Pyura, with digestive diverticulum.

CELL PROLIFERATION IN ASCIDIAN GUT 327

TABLE 1. List of ascidian species used in this investigation giving the foldingpattern and distribution of renewing (R) and expanding (E) cell populations in each region of the postbranchial digestive tract

SPECIES ESO STOM mr Aplousobranchia

Polyclinum planum (c) smooth (R) Archidistoma ritteri (c) smooth (R) Didemnum carnulentum (c) smooth (R) Distaplia occidentalis (c) smooth (R) Euherdmania claviformis (c) smooth (R)

Phlebobranchiata Perophora annectens (c) Ciona intestinalis (s) Chelyosoma productum (s) Ascidia ceratodes (s)

Stolidobranchiata Styela clava (s) Botrylloides diegense (c) Molgula verrucifera (s)

smooth (R) folded (RI smooth (R) smooth (R)

folded (R) smooth (ST?) folded (R)

smooth (R) smooth (R) smooth (R) corrugated (R) folded (R)

smooth (R) folded (R) pitted (R) folded (R)

smooth (El smooth (E) smooth (El smooth (E) smooth (E)

smooth (E) folded (R-E) smooth (R-E) smooth (R-E)

folded (R) folded (R) folded (ST?) smooth (ST?) diverticulum (R) smooth (R-E)

Pyuia haustor (s) folded (R) diverticulum (R) smooth (R) Names from Ahbott ('75). For comparison, Styela clava (from Ermak. '75c) is included in its appropriate place. 'R-E'indicates that renewing and expanding populations occur in different regions of that organ. c, colonial species; s. solitary species; ST, static population.

side. The folds may be pits (Fig. 1C) as in Chelysoma, or longitudinal folds, as in Ascidia (Fig. 1D). The digestive diverticulum may con- sist of folds or tubules (Fig. 1E). The molgulid diverticulum is actually a specialized region of the stomach, whereas the pyurid diverticulum (Fig. 1E) is a separate organ connected to the stomach by canals (Fouque, '59).

The intestine is a long tube in which nutri- ents are absorbed and faeces are compacted. In most ascidians, it is smooth walled in the largest species examined, however, a typhlosole may pass along one side.

The digestive tracts of most ascidian species share several histological features. Most or all of the esophageal wall is lined by mucous cells; in advanced species, a narrow strip of band cells (Ermak, '75c), whose function is as yet unknown, runs from the branchial basket into the esophagus. The stomach has a basic pattern re- gardless of the degree of folding. A narrow raphe of mucous cells like those in the esophagus passes along one side from the esophagus to the intestine. The rest of the stomach wall is covered by absorptive and zymogen (enzyme secreting) cells (germinal regions contain undifferentiated cells). This mixed population will here be referred to as the chief cell population. The intestine has a variable population of absorptive, zymogen, and mucous cells. Sever- al other types of cells, including endocrine cells, also occur in the ascidian gut (Burighel and Milanesi, '75; Fritsch, '76; Fritsch and Sprang, '77; Brevis and Thorndyke, '78; Thorn- dyke and Brevis, '78).

Colonial ascidians, smooth stomach In Polyclinum planum, the esophagus is el-

liptical in cross section; the stomach (about

250 pm wide) and intestine are approximately circular (Fig. 2A). A one-hour exposure to tritiated thymidine labeled germinal cells in the esophagus and stomach. Two germinal zones of mucous cells run opposite one another along the length of the esophagus (Fig. 3); in the stomach, germinal zones occurred on each side of the raphe and around the esophageal and intestinal openings. Both chief and mucous populations were labeled (Fig. 4); the chief germinal cells, however, were more heavily labeled than mucous germinal cells. Some mature cells were also labeled along the circumference of the stomach wall (Fig. 51, but most labeled cells were along the raphe. In the intestine, labeled cells were scattered throughout the epithelium.

At 5 and 10 days after injection, cells had migrated away from the germinal zones in the esophagus and stomach. Cells appeared to migrate away from the raphe and centrifugally away from the esophageal and intestinal openings (Fig. 2B). By 10 days, chief cells had migrated a third to all the way around the stomach wall. In the intestine, little change was observed.

In Archidistoma ritteri, Didemnum camu- lentum, and Perophora annectens, other colonial ascidians of approximately the same size as Polyclinum, labeling patterns after one hour were similar. However, in Archidistoma and Didemnum, only one germinal zone was observed in the esophagus.

Distaplia occidentalis In Distaplia, the esophagus and stomach

were smooth walled, but the stomach sometimes had several low, longitudinal ridges on its internal surface giving the lining a corruga-

328 T.H. ERMAK

C

Fig. 2. Cross sections and cell migration (B) in digestive tracts of aplousobranch (A-D), phlebobranch (E-H), and stolidobranch (I-L) ascidians. A, Polyclinum stomach B. Migration in Polyclinum stomach C, Distaplia stomach D, Euherdmania stomach E, Ciona stomach; F, Ciona foreintestine; G , Cionu hind-intestine; H. Ascidia stomach I, Mob

gula esophagus: J, Molguh stomach (st) and digestive diverticulum: K, Pyuru esophagus: L, Pyuru stomach (st) and digestive diverticulum. Stomach raphe is stipled. bp, Band population; ig, intestinal groove; mp. mucous population: mj, major fold mn, minor fold t, tubule of digestivediverti- culum: ty, typhlosole.

ted appearance (Fig. 2C). The esophagus also contained a band cell population.

In the esophagus, germinal zones occurred on each side of the band population. In the stomach, cells were labeled along the raphe. As in Polyclinum, several mature cells were labeled, but they were not distributed in any pattern related to folding on the stomach wall. As in other aplousobranchs, labeled cells were scattered in the intestine.

Euherdmania claviformis

This is one of the few colonial species with a folded stomach wall (Figs. l B , 2D). About six longitudinal folds each measuring about 200-225 pm high occurred on all sides except the side next to the intestine. At one hour after injection, localized regions of proliferation occurred in the esophagus and stomach, but not the intestine. Germinal cells were labeled


at the axial ends of an esophageal cross section and the base of each stomach fold (Fig. 6). Such labeled cells also occurred along the stomach raphe.

Ciona intestinalis This relatively large solitary species had

four folds in the esophagus, 30 to 40 alterna- tive major and minor folds in the stomach (Fig. 2E), and a typhlosole in the intestine (Fig. 2F, G). Each stomach fold measured from 375-500 pm in the animals examined. In the intestine, a deep groove ran opposite the typhlosole for a short distance past the stomach (Fig. 2F) and then disappeared, while the typhlosole continued throughout the course of the intestine (Fig. 2G). Pseudostratified regions of basophilic cells occurred at the base of each fold in the esophagus and stomach and in the intestinal groove, but not in the intestine after termina- tion of the groove.

One hour after injection, localized regions of cell proliferation occurred in the esophagus, stomach, and fore-intestine. In the esophagus, the pseudostratified cells but not the mucous or band cells were labeled. At 10 or 20 days after injection, mucous cells on the tops of folds but no band cells were labeled.

In the stomach, cells a t the base of each major and minor fold and on each side of the raphe were labeled after one hour. At increasing time intervals, mucous cells on the raphe and mature cells on the folds became labeled.

Three types of proliferative behavior occurred in the intestine. In the fore-intestine, pseudostratified cells were labeled in the intestinal groove. With time, cells migrated onto the side walls. In the mid-intestine, many columnar cells around the circumference of the intestine were labeled. Labeled cells were not evenly distributed throughout sections but were group- ed into bands of high and low labeling frequen- cies. In the hind-intestine, a small number of labeled cells was scattered throughout the sections. No cell migration was detected in the mid- or hind-intestine by 20 days after injection. However, the high percentage of labeled cells in the mid-intestine suggested that the epithelium might be transitional between the renewing population in the fore-intestine and expanding population in the hind-intestine.

Chelyosoma productum The esophagus and intestine of Chelyosoma

were smooth walled and oval in cross section. The stomach (Fig. lC), on the other hand, was pitted (waffle patterned) with about 10-15 pits

per cross section. The walls of the pits were about 200-250 pm high. An intestinal groove ran a short distance past the stomach before terminating.

At one hour after injection, germinal zones were labeled on each side of the esophageal band cells. In the stomach, proliferative zones were labeled at the base of each pit and on each side of the stomach raphe. In the fore-intestine, cells were laLCled in the intestinal groove; in the hind-intestine, they were scattered throughout the epithelium.

Ascidia ceratodes Both the esophagus, which had no band cell

population, and intestine of Ascidia were smooth walled and oval in cross section. The stomach, however, had 9-12 longitudinal folds on each side of a single raphe (Figs. ID, 2H); each fold was about 500-750 pm high.

At one hour, germinal zones opposite each other were labeled in the esophagus and intestine; in the stomach, germinal zones occurred at the base of each fold and on each side of the raphe (Fig. 7). In the hind-intestine, labeled nuclei were scattered throughout the epithe lium. At five days labeled cells had migrated towards each other in the esophagus and intestine. In the stomach, cells had migrated up the sides of the folds. By ten days, most or all the stomach folds were labeled (Fig. 8), and by 15 days most of the esophagus and intestine. No change was observed in the hind intestine.

Botrylloides diegense As in Styelu cluva (same family as Botry-

b ides) , the stomach wall was folded. However, the esophagus and intestine were smooth.

After one hour, no gut cells were labeled, al- though many blood cells, which are common to all zooids in the colony, were labeled. I t is possible that the radiochemical failed to reach the DNA synthesizing cells of the gut. The possibility that the gut cells cannot incorporate thymidine into the DNA is remote. Since electron micrographs of the gut do not reveal any unequivocal mitotic figures or germinal zones containing undifferentiated cells either in the esophagus or stomach (Burighel and Milanesi, '73; Ermak, unpublished results), it is conceiv- able that the gut populations in this species constitute non-dividing, static populations, and that proliferation only occurs during development, whether through sexual or asexual reproduction.

330 T.H. ERMAK


Molgula verrucifera

In Molgula, the esophagus had three folds with band cells lining one entire groove (Fig. 21) instead of just half a groove as in previous species (see also Ermak, '75c). The stomach and intestine were smooth walled, but a diverticulum composed of numerous epithelial folds ex- tended from the stomach maintaining an open connection with no collecting canals (Fig. 25).

At one hour, localized zones of proliferation occurred in the esophagus, stomach, diverticulum, and most of the intestine. In the esophagus, germinal regions occurred at the base of each fold and above the band cells. In the stomach, several germinal regions occurred along the circumference. In the diverticulum, labeled cells occurred at the bases of the folds (Fig. 9). Two germinal zones occurred in the fore-intestine; one germinal zone occurred in the mid-intestine (Fig. 10); and no germinal zone was present in the hind-intestine.

Pyura haustor Pyura had an esophagus with four folds,

a smooth stomach with a diverticulum, and a smooth intestine. In the esophagus, band cells lined the entire base of one groove as in Mol- gula (Fig. 2K). Three shallow grooves usually ran the length of the stomach. In the mid- intestine, however, the grooves were reduced t o two, and in the hind-intestine, only one groove remained. Each groove simply termin- ated while the others continued further along the length of the digestive tract.

The digestive diverticulum (Fig. 2L) extend- ed from the distal end of the stomach and con- sisted of numerous tubules and branching canals. The tubules were oval in cross section, about 100-150 pm in the long axis. Several tubules usually joined together a t an entrance to a canal. In living material, the tubules were bright orange with clear bands of cells running along each side and joining at the tips. These bands corresponded to small zones of basophilic cells in histological sections.

After a single injection of tritiated thymidine, only a small number of nuclei took up the

Figs. 3-8. Autoradiograms of digestive tracts from aplousobranch (Figs. 3-6) and phlebobranch (Figs. 7-8) ascidians.

Fig. 3. Esophagus of Polyclinum, one hour after injection of tritiated thymidine. showing two germinal zones (arrows). X 315.

Fig. 4. Stomach of Polyclinum, one hour after injection, showing labeled germinal cells (arrows) in chief population (cp) and mucous population (mp). X 400.

radioactive label. Three daily injections, however, greatly increased the number of labeled cells and clearly demarcated the zones of cell proliferation. Migration rates in Pyura haustor were much slower than in the other species examined (Polyclinum, Ciona, Ascidia, and Styelu). Even after 30 days, cells had migrated only a short distance. Only in a few tubules of the digestive diverticulum did the zones of labeled cells meet each other. The types of cell proliferation were as follows. In the esophagus, cells were labeled at the fold bases (Fig. 11) and above the groove lined by band cells (Fig. 12). Mucous cells migrated toward the crests of folds; band cells migrated toward the base of their groove. In the stomach, labeled cells were localized in three longitudinal grooves and at the entrance to the diverticulum. Thus, the stomach is roughly divided into quarters by germinal zones. In the canals of the diverticulum, the number of germinal zones depended upon the size of the canals, with several germinal zones in the large canals, and two opposite germinal zones in the small ones. The tubules of the diverticulum had two germinal zones, one at each axial end of a cross section (Fig. 13). Cell migration occurred along the sides of the tubule cross section. In the intestine, there were two germinal zones in the mid- and fore-intestine and one germinal zone in the hind-intestine (Fig. 14).

DISCUSSION

The cell populations lining the ascidian postbranchial digestive tract share several kinetic features (Table 1). Except in Botrylloides, which may well have nondividing populations throughout the adult digestive tract (Ermak, '75a; Burighel and Milanesi, '77; see also below), renewing populations consistently occurred in the esophagus and stomach of all ascidian species surveyed. Such renewing populations are characterized by a high rate of cell proliferation, migration of cells from germinal zones into mature zones, and loss of aging mature cells at secalled extrusion zones (Ermak, '7513. The populations turn over rapidly, and, at the steady state, the rate of cell production is carefully balanced by the rate of cell loss.

Fig. 5. Stomach of Polyclinum, 5 days after injection. Labeled mature cells (arrows) occur outside the region of labeled germinal cells. X 315.

Fig. 6. Stomach of Euherdmania one hour after injection, showing germinal cells at the base of each fold. X 150.

Fig. 7. Stomach fold of Ascidiu, one hour after injection. Only germinal cells at the base of each fold are labeled. X 100.

Fig. 8. Stomach fold of Ascidiu, 10 days after injection. Maturecells along entire height of fold arenow labeled. X 150.

332 T.H. ERMAK


Germinal cells have basophilic cytoplasm, are smaller in size than mature cells, and in many species form a pseudostratified epithelium. Mitotic figures frequently occur along the lu- menal edge of the epithelium. The germinal cells are relatively undifferentiated in comparison to the ciliated, secretory, or absorptive cells of mature zones (Thomas, '70; Ermak, '75a; Thorndyke, '77).

Both renewing and expanding populations occurred in the ascidian intestine. Expanding populations have a slower rate of cell proliferation, and cell division is not confined to a speci- fic region or group of cells (germinal cells). In this case, mature cells divide and maintain the cell population. Expanding populations line the entire intestine of all colonial ascidians and at least the posterior regions of most solitary ascidians. Only Styela (Ermak, '75c) and Pyura had renewing populations throughout the entire digestive tract.

Cell renewal units The renewing populations of the ascidian gut

are adapted to different degrees of body size, organization, and evolutionary advancement. The basic unit of cell renewal on a fold, pit, or tubule is a pair of germinal and mature zones, here defined as a cell renewal unit. With an increase in body size, the digestive organs undergo extensive folding and the number of cell renewal units increases, usually in proportion to the increase in folds. Only species with large- bodied individuals exhibit folding of the gut lining. Colonial ascidians, because of their small body size limitations, usually have smooth digestive tracts, whereas solitary species (phlebobranch and stolidobranch ascidians) have the greatest degree of folding.

In the esophagus, folding usually only occurs in solitary species, where they may reach at most three or four folds. The stomach is the most folded and largest of the postbranchial organs, and the number of folds usually increases with increasing body size. Large colonial ascidians have a few folds and cell renewal units. Euherdmania, a relatively large colonial ascidian, has about six folds. Clavelina, ano-

ther colonial species, has four folds which have mitotic figures at their bases (Ermak, '75a), indicating that cell renewal also occurs in this ascidian. The small folds of Distaplia forming a corrugated appearance do not appear to represent multiple regions of cell renewal, since only a single pair of germinal zones was observed for the chief population. A similar situation might also occur on the corrugated stomach of other colonial ascidians (Ermak, '75a), but this possibility needs further testing.

In solitary ascidians, the stomach has numerous folds. Ciona, one of the largest ascidian species, has 40 or more stomach folds. Surface area in the stomach is increased by longitudinal folding in Ciona, Ascidia, and Styela Both longitudinal and latitudinal infolding create the pits of Chelyosoma whereas folding in three dimensions produces the digestive diverticulum of Pyura, with hundreds of tubules. A pit differs from a tubule in its method of renewal. That is, the germinal zone for a pit only occurs a t the base and not along the side walls of the epithelium. The germinal zone for a tubule, however, is a strip which goes down one side, across the base, and up the opposite side. The number of folds and cell renewal units in the stomach does not necessarily correspond to absolute animal size. For example, Molgula and Pyura have the greatest number of cell renewal units but are not the largest species. However, they belong to the most evolutiona- rily advanced ascidian families (Berrill, '50).

In the intestine, folding of the gut lining may occur independently of cell renewal; thus, a large fold, the typhlosole, runs the entire intestine of Ciona However, the germinal zone ter- minates after the foreintestine and an expanding population continues through the rest of the intestine.

The size of a cell renewal unit is characteristic for each region of the gut, the largest ones occuring in the intestine. The smallest ascidian gut cell renewal units examined occurred in a tubule of the pyurid digestive diverticulum. Each region apparently has a size limitation for each population, for with an increase in organ size, the number of germinal and mature

Figs. 9-14. Autoradiograms of digestive tracts from stolidobranch ascidians.

Fig. 9. Digestive diverticulum of Molgula, one day after two consecutive daily injections of tritiated thymidine. Ger- minal cells at the base of each fold are labeled. X 150.

Fig. 10. Intestine of Molgula, one day after two daily injections, showing a single germinal zone. X 150.

Fig. 11. Mucous cells on an esophageal fold of Pyura. 30 days after three consecutive daily injections. Cells are still only labeled at the base of the fold. X 150.

Fig. 12. Esophageal groove of Pyuru, 30 days after three daily injections. Mucous cells (mc) above the groove are heavily labeled, but most band cells (bc) within the groove are still unlabeled. X 125.

Fig. 13. Tubules of digestive diverticulum of Pyura, 30 days after three injections, showing two opposite germinal regions for each tubule. X 220.

Fig. 14. Intestine of Pyuru, 30 days after three injections, showing one of two germinal regions extending along opposite walls. X 150.

334 T.H. ERMAK

compartments increased. Both the number and size of cell renewal units increased with on- togenic growth of a single species (Ermak, '76a). In Stye& most folds are apparently added between metamorphosis and sexual ma- turity, reaching 20-30 in large individuals. Those animals 1-30 gm in weight had folds ranging in height from about 0.60 to over 1.5 mm. Stomach folds in other solitary species averaged about 500 pm in height. In Ciona, the size of the cell renewal units alternated between major and minor folds. The correspon- ding length of a cell renewal unit in the smooth stomach of a colonial ascidian was about 250-300 pm.

Transit time In all the ascidian species surveyed, cell

turnover was faster in the esophagus and stomach than in the intestine, suggesting that the stresses placed upon the esophageal and stomach epithelia are greater than those upon the intestine (see Ermak, "75c). This is possibly related to the fact that digestive enzymes are secreted in the anterior portion of the gut, es- pecially the stomach and digestive diverticulum. The decrease in turnover in the intestine was accomplished by a decreased rate of cell renewal or by the transition to an expanding population.

Transit times for the renewing populations in Polyclinum, Ciona, and Ascidia appeared to be on the same order as in Styela, about 2.5 weeks in the esophagus and stomach and 2-5 weeks in the intestine. In Pyura, however, transit times were much longer, on the order of one or more months. The factors responsible for this differ- ence are as yet unknown. Transit times in ascidians are longer at lower temperatures (Er- mak, '76a). They are also significantly longer in poikilotherms (weeks to months) than in home- otherms (days) (Gas and Noaillac-Depeyre, '74; Garcia and Johnson, '72; Hyodo-Taguchi, '70; Hansen and Youson, '78; Messier and Leblond, '60; O'Steen and Walker, '60).

Phylogeny and ontogeny In addition to their amazing powers of regen-

eration and budding, the ascidians have a high number of renewing cell populations, compar- able to mammals. Renewal of gonads (Ermak, '76b) and blood cells (Ermak, '75b; '77) as it occurs in ascidians also occurs in many other phyla; renewal of the digestive tract, however, has been reported to occur mainly in the vertebrates. Among invertebrates, renewing gut epithelia have been found in the hepatopan-

creas of the crayfish (Davis and Burnett, '64) and, among lower vertebrates, in larval lam- preys (Hansen and Youson, '78), fish Wickers, '62; Hyodo-Taguchi, '70; Garcia and Johnson, '72; Gas and Noaillac-Depeyre, '74), amphi- bians (O'Steen and Walker, '60; Patten, '60; Martin, '71; McAvoy and Dixon, '77), and rep- tiles (Wurth and Mussachia, '64). The method of cell renewal in the lamprey is similar to that of ascidians in that it occurs on simple folded epithelia. Renewal of gut epithelia in the am- phibian and reptile, however, involves nests of germinal cells in stratified epithelia.

The similarity of renewing populations in the digestive tract of ascidians and mammals has been particularly noted (Ermak, '75c). Mucous cells in the ascidian esophagus and stomach are renewed much like similar mucous cells of the surface epithelium in the mammalian stomach (Messier, '60; Hunt and Hunt, '62; Mac- Donald et al, '64). and renewal of absorptive and zymogen cells of the chief population in ascidian stomach resembles renewal of absorptive and goblet cells on villi of the mammalian intestine (Leblond and Messier, '58; Messier and Leblond, '60; Cheng and Leblond, '74). As- cidian band cells were reminiscent of mammalian Paneth cells (Cheng et al., '69; Cheng, '74) in that they are both renewed slowly and ori- ginate from the same germinal cells which give rise to the rapidly renewed cells of the digestive tract. Unlike in most ascidians, however, renewal in mammals occurs in pits or on villi.

Several of the renewing populations in ascidians, notably the digestive folds and tubules, the stigmata (Ermak, "75c), and the dorsal tubercle (Ermak, '75c) undergo extensive mor- phological alterations during ontogeny. The precise role of the germinal and mature compartments during development or during the budding of colonial species has yet to be determined.

Evolutionary patterns The evolution of ascidians has proceeded

mainly in two directions: 1) Toward the elaboration of structures for the maintenance of large solitary animals, and 2) toward the elaboration of structures advantageous to the colonial habitat. In general, solitary forms have complex adult structures and simple or reduced larvae whereas colonial forms have simple adult structures and complex tadpoles. Ciona is gen- erally considered to be one of the most primitive ascidians, and the cionid juvenile is considered to most closely resemble the postulated ancestral ascidian (Berrill, '36; Millar, '66). Aplousobranch colonial ascidians apparently


evolved from these primitive ancestors where as the stolidobranch ascidians (Styela, Mol- gula, and Pyura) probably arose from advanced phlebobranch ancestors (Berrill, '36).

In colonial ascidians, adult structures usually become reduced and larval structures more complex; their zooids become fully func- tional units soon after metamorphosis. Berrill ('36) warns against using elaboration of an internal organ to explain evolutionary patterns in ascidians. Since structure is dependent upon size, the process of budding, which induces dwarfing, is likely to induce simplification of parts. The question arises whether a simple structure is primitive or the result of size r e duction. The primitive nature of the branchial basket in colonial ascidians has been question- ed by Berrill ('36) and such questioning may also be applied to the rest of the digestive tract. In colonial forms present today, a smooth gut lining might have resulted from the loss of epithelial folds. For example, Pero- phora, which has a smooth gut lining, is gener- ally believed to have evolved from groups which today have highly folded gut epithelia.

The colonial habitat might also result in the loss of cell renewal from the gut. In the Botryl- lidae, renewing populations on styelid type folds might have been lost from the gut in connection with the short lives of individual zooids in the colony. The botryllids represent an independent line of evolution stemming from rather advanced styelid stock. They have un- dergone size reduction and have lost complex structures such as folds in the branchial basket but have developed complex larvae and specialized budding patterns. Botryllus zooids live only about a week before they are absorbed into the colony to make room for the next gen- eration of zooids (Burighel and Milanesi, '73). In such a case, a renewing population would be obsolete. With a life span of only one week, none of the cells in the digestive tract need be replaced (see also Burighel and Milanesi, '77).

With an increase in body size, solitary ascidians developed greater demands for food, oxygen, and waste removal. These animals, thus, have greater feeding and respiratory sur- faces in the branchial basket, which can be very elaborate in advanced species. Likewise, these forms exhibit a greater degree of postbranchial gut folding. Large bodied ascidians exhibited several evolutionary trends: 1) Mul- tiplication of cell renewal units; 2) an increase in the number of cell populations per fold 3) formation of the digestive diverticulum; and 4) an increase in the renewal of the intestine.

The simplest condition for the renewal of a cell population is by a single germinal zone, as occurred in the esophagus of several colonial ascidians and in the intestine of many solitary ascidians (Fig. 15A). Duplication of germinal zones (Fig. 15B) was common in these organs and also defined the method of renewal on the stigmata of the branchial basket (Ermak, '75c). In the esophagus of more complex ascidians, further multiplication produced a folded epithelium with a germinal zone at the base of each fold (Fig. 15C).

In the esophagus of advanced stolidobranch ascidians, there was a duplication of the 'half cell renewal unit for the band population (Fig. 15D) as occurred in Styela (Ermak, '75c). In Molgula and Pyura, a pair of germinal and mature zones or a 'whole' cell renewal unit occu- pied the entire groove (Fig. 15E). Both germinal zones produced band cells from one side and mucous cells from the other side.

Multiplication of cell renewal units, an increase in the number of cell populations per fold, and the formation of the digestive diverticulum have all been part of stomach evolution. The most primitive condition in the stomach was most likely similar to the smooth stomach of colonial ascidians. A single mucous cell renewal unit occurred on the raphe and a single chief cell renewal unit occurred on the stomach wall (Fig. 16A). In response to epithelial folding and stomach growth, the chief cell population first underwent multiplication of renewal units (Fig. 16B) as in some aplousobranch and most phlebobranch ascidians. This was later followed in styelid ascidians by multiplication of the mucous cell population, apparently derived from the raphe, on the crests of each fold (Fig. 16C).

In the most advanced ascidian species, the folds of the stomach underwent infolding to form numerous epithelial sacs. The folded part of the stomach wall formed a new organ, the digestive diverticulum. In its simpler form (Fig. 16D), the diverticulum maintained an intimate connection with the stomach, thereby forming part of the stomach wall (see also Fouque, '59).

In this case, each sac of the diverticulum was lined only by a chief population and not a mucous population. The presence of only one cell population in this type of diverticulum sug- gests that the Molgulidae evolved from ascidians with only one cell population on each fold. Possibly, the molgulids evolved from a stolidobranch ancestor which had not yet evolved a styelid stomach. The pyurid diverticulum (Fig. l6E) probably evolved from the

T.H. ERMAK 336

molgulid condition by further separation of the folds from the stomach to form tubules and the formation of canals by mucous cells. The ar- rangement of germinal zones up and down each side of the pyurid tubules can be derived from the molgulid folds by folding the molgulid diverticulum grooves upon themselves to form pyurid tubules. Thus, the folds and tubules of the digestive diverticulum are apparently homologous to the stomach folds and not the pyloric caecum, as once suggested by Berrill (’50).

The intestine exhibits increasing degrees of renewal with evolutionary advancement. In the primitive condition, the entire intestinal epithelium was most likely an expanding population. Renewal was developed first in the anterior portion of the intestine with a single cell

renewal unit and, most likely, an expanding population toward the posterior region. Grad- ally, the entire intestine became renewed. However, even in this case, the number of cell renewal units decreased posteriorly.

The distribution of expanding and renewing cell populations in ascidians provides a clue to the possible origin and evolution of cell renewal in epithelial populations, i.e., that renewing populations might have evolved from expanding populations by increased proliferation and cell loss and by the separation of proliferative and mature compartments. In response to a greater need for turnover or epithelial growth, the number of cell renewal units might have multiplied. In light of the possibility that ascidians gave rise to the vertebrates (Berrill, ’55), it is possible that renewing populations in ver-

Fig. 15. Multiplication of cell renewal units. Black r e gions represent germinal zones, small arrows directions of cell migration, medium arrows sites of cell extrusion, and large arrows presumed evolutionary pathways. A) Single germinal zone in esophagus or intestine. B) Duplication of ger-

minal zone. C) Further multiplication in esophagus. D) Band population ‘half d renewal unit in esophagus. E) Band population ‘whole’ cell renewal unit in esophagus of molgulid and pyurid ascidians.

Fig. 16. Presumed evolutionary pathways (large arrows) of cell renewal in the stomach (and digestive diverticulum) of ascidians. Black regions represent germinal zones. Chief cell populations in white; mucous cell populations stipled. Small arrows represent directions of cell migration. Medium arrows represent main sites of cell extrusion. A) Smooth stomach wall with single chief and mucous cell population. as in most small colonial ascidians. B) Multiplication of chief cell re-

newal units on epithelial folds, as in larger colonial ascidians and phlebobranch solidary ascidians. C) Multiplication of mucous cell renewal units on top of epithelial folds. as in styelids. D) Formation of the molgulid digestive diverticu. lum lined by chief cell population. E) Formation of pyurid digestive diverticulum with mucous cell population in canals and chief cell population in tubules.


Figure 16

338 T.H. ERMAK

tebrates evolved through similar stages of cell proliferation patterns.

ACKNOWLEDGMENTS

I am indebted to Dr. Nicholas D. Holland for his support, guidance, and critical evaluation during the course of this study. I thank Dr. Donald P. Abbott for valuable discussion and comments.

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