1. Cell Proliferation in the Digestive Tract of Styela clava
(Urochordata: Ascidiacea) as Revealed by
Au 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 was
and autoradiography has been confined followed by taking samples of the digestive
to 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 has
been done on relatively few non-mamma- General orientation
lian vertebrates and invertebrates (Davis The digestive tract of ascidians (shown
and Burnett, '64; Anderson; '65; Holland, schematically in fig. 1A) is composed of a
'64). The sparsity of such information on branchial basket, the food collecting and
ascidians is especially unfortunate because respiratory apparatus, and a postbranchial
of their vertebrate affinities. Previous work region, which functions in digestion and
on ascidian gut cell kinetics is limited to a absorption of nutrients. The postbranchial
cell 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 the
dence alone, the replacement of gut epi- main course of the digestive tract at the
thelial cells has been proposed in several junction of the stomach and intestine. Sea
ascidians (Aros and Viragh, '69; Fouque, water enters the branchial basket through
'53; 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. The
tion patterns in the digestive tract of the major portion of the branchial basket is
ascidian 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 branchial
coast. 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

2. 3
8
X V N X S ‘ H SVNOHL OSP

3. CELL PROLIFERATION I N ASCIDIAN GUT 451
walls and with the creation of water cur- the lumenal edges of the pseudostratified
rents to bring in plankton and suspended regions.
organic matter. The mucous net is se- For more complete descriptions of the
creted by the endostyle and moves along mechanism of feeding in ascidians and the
the 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), Fouque
ticulate matter is trapped by the mucous (5) and Relini-Orsi ('68).
'3,
net and moves toward the anterior part
MATERIALS A N D METHODS
of the basket. Here, the mucous passes
down the dorsal lamina to the esophageal Styela c l a m is a club-shaped ascidian
opening. Below the branchial siphon, the native to Korea and Japan and lives on
branchial 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 length
dorsal tubercle, which projects into the are relatively clean, showing the brown
branchial basket, plays a role in chemore- wrinkled test, but larger ones usually have
ception (Carlisle, '53). a coating of bryozoans, algae, and other
The postbranchial digestive tract lies ascidians. Seventy specimens of S. clava
on the left side of the branchial basket. were collected from the docks at Quivira
The 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 tritiated
the esophagus, the mucous net forms a thymidine (New England Nuclear Corp.)
food cord, passes through the stomach and per gram fresh weight. Individuals ranged
becomes compacted in the intestine. Mu- in weight from one to four grams. The
cous secretions are added in the esopha- tritiated thymidine (specific activity 6.7
gus; enzymes are secreted mainly in the curies per millimore) was diluted 1: 1 with
stomach; 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 time
of Styela is extensively folded, a feature of sacrifice. Ten individuals were sacrificed
characteristic of solitary ascidian species. by fixation in Bouin's fluid at each of the
Each 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 60
specialization) on the outside and lined by days after injection.
the digestive epithelium on the inside. In The digestive tract was dissected from
between the epithelia occur blood spaces the body after at least one day's fixation
and connective tissue. In the branchial and the various organs dehydrated in alco-
region, smooth muscles pass below the hol and embedded in paraffin. Sections
branchial bars, and in the postbranchial were cut at 7 microns and covered with
region, the pyloric tubules run below the Kodak Nuclear Track Emulsion type NBT-2
digestive epithelium. by the dipping method. Autoradiograms
Much of the digestive tract is lined by were exposed for periods from two weeks
simple columnar epithelia, the majority to two months and developed in Kodak D-19
of which are either ciliated, secretory, or developer (3 minutes) at 68°F. All slides
absorptive in nature. In several restricted were stained through the emulsion with
zones throughout the digestive tract, the hematoxylin.
epithelia are modified into basophilic zones The following structures or organs were
of pseudostratified columnar cells (fig. 2D). examined: branchial tentacles, dorsal tu-
These zones are most obvious at the bases bercle, endostyle, dorsal lamina, branchial
of 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 not
proliferation (Fouque, '53; Relini-Orsi, '68), studied: the penpharyngeal band, which
since mitotic figures frequently occur along is continuous with the dorsal lamina, and

4. 452 THOMAS H . ERMAK
the 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 of
esophagus. 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 days
cross sections of the tentacles, two or three after injections, no migration of cells is
nuclei being labeled per section. No changes detected. After 60 days, however, several
are 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, they
The exact shape of the groove, however, show no migration or loss of labeled cells
varies among individuals. The groove is at increasing time intervals. Zones 2 and
lined by the heavily ciliated funnel epithe- 4 are the lightest labeled populations while
lium while the surface is lined by the non- zone 3 is the heaviest labeled population
ciliated lip epithelium (fig. 1D). Pseudo- in the endostyle, including zone 1 (see
stratified regions occur at the base and at DI sc u s s ION).
the top of the groove. Beneath the funnel
epithelium is a band of acidophilic mate- Branchial bars and dorsal lamina
rial 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 while
dostratified cells are labeled, but no cells the transverse bars and the dorsal surface
on the sides of the grooves or on the sur- of each longitudinal bar are covered by a
face of the dorsal tubercle are labeled (fig. relatively unspecialized epithelium. The
3). By 20 days after injection, cells have transverse bars are an integral part of the
migrated 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 underlying
branchial basket (fig. 1B). Most early ac- row of stigmata; either end of the bridge
counts 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 the
ascidians (Godeaux and Firket, '66, '69) ventral surface of the longitudinal bars are
have shown that this zone consists of two Fig. 2 A. A cross section through the endostyle
separate 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 the
through 9 (fig. 2A). The nine cell popula- twisting longitudinal folds o n the ventral surface
tions in the endostyle of Styela clava are and the straight folds o n the dorsal surface of the
roughly 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 correct
cilia which reach the top of the endostylar position in relation to the stomach in E. cb, band
furrow. 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, transitional
and 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.

5. CELL PROLIFERATION I N ASCIDIAN GUT 453

6. 454 THOMAS H . ERMAK
labeled, 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 each
beled 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 those
no distinct region of cell proliferation oc- nuclei which occur in a basal position in
curs, 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 mucous
loss of labeled cells from both the longitud- cells and band cells on the sides of the
inal 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 are
no definite patterns of cell migration could labeled at all levels in the pseudostratified
be 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 band
the dorsal lamina and away from the at- cells are labeled. The labeling of mitotic
tached 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 basal
terns 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 mucous
scattered 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 the
cells which beat outward when producing same animal.
water currents. Small pseudostratified re- By 60 days, mucous cells are no longer
gions 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, the
A 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 originate
nuclei are not observed in sections of every from the pseudostratified region on the
pseudostratified 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 produces
the stigmatic walls are labeled. At ten days both mucous cells and band cells; mucous
after injection, cells on the side walls are cell production occurs on the opposite side
now labeled (fig. 7) indicating that cells of the pseudostratified region from band
from the anterior end of each stigma have cell production.
migrated posteriorly, and that the cells at
the posterior end have migrated anteriorly. Stomach
The two bands of labeled cells have met The stomach has 25 to 30 longitudinal
each other on the side wall, leaving behind folds on its dorsal and lateral surfaces.
lightly labeled cells in the pseudostratified
regions. A t 20 days after injection, the cells Fig. 3 An autoradiogram of the lip and funnel
lining 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 stigmata
by 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 stigmata
ventral fold where band cells occur. The ten days after injection showing labeled ciliated
band cells are restricted to one side of the cells on the side walls. X 600.

7. CELL PROLIFERATION IN ASCIDIAN GUT 4 55

8. 456 THOMAS H . ERMAK
The ventral surface is covered by a raphe By ten days after injection, mature cells
which 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 still
testine. The stomach folds are covered by labeled (fig. 14). Animals killed 15 days
two cell populations: the groove population after injection have several folds where the
which 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 of
top 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 mature
raphe, bulb, and crests, which are all cov- cells intermingled with unlabeled mature
ered by ciliated mucous cells, are continu- cells.
ous with each other and with the mucous After 20 days, the entire groove height
cells in the esophagus, Chief and zymogen is labeled, but no pseudostratified cells are
cells (terminology, Burighel and Milanesi, labeled any longer. By 30 days, few cells in
'73) cover the stomach grooves. The chief the groove population are still labeled.
cells, however, are much more numerous Thus, most of the DNA synthesizing cells
than the zymogen cells, and are absorptive labeled at the time of injection as well as
in function. Pseudostratified regions occur several generations of their progeny have
at the base of each groove, at the side of been lost from the epithelium, evidently
each crest, and at the side of the raphe through cell extrusion at the junction of
and 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 after
groove population. injection, cells on the sides of the stomach
The folds on the dorsal surface of the crests are labeled (fig. 16). No mucous
stomach 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 in
the 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 in
ends intersect the raphe while the poste- the groove population.
rior ends intersect the bulb, suggesting By ten days after injection, the two
that the stomach folds generate hom the bands of labeled cells on each side of the
ventral surface. As the folds move away crests have migrated toward each other
from 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 mucous
pseudostratified 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, two
levels in the pseudostratified region are separate renewing cell populations occur
labeled (fig. 13). No mature cells are yet
labeled, 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 60
vided 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.

9. CELL PROLIFERATION IN ASCIDIAN GUT 457

10. 458 THOMAS H. ERMAK
on each fold, each renewed by its own ger- terns in the rectum are similar to those
minal cells. observed in the intestine. The major dif-
The stomach raphe and bulb are basic- ference is in the size of the germinal and
ally 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 rectum
these populations are much larger in size. than in the germinal zone of the intestine.
Many cells are labeled on the bulb at one
hour, and migration (in microns per day) Pyloric gland
is 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 and
bulb, 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 together
with 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 in
other side. The intestinal groove is lined the esophagus and intestine. Although the
by pseudostratified cells while the rest of tubules occur associated with the mature
the intestinal wall is lined by absorptive cells of the digestive tract, they are not
and secretory cells. closely associated with the germinal zones,
At one hour and three days after injec- especially in the intestine. The fact that
tion, labeling is much like in the groove the pyloric tubules occur directly adjacent
population of the stomach with only pseu- to the absorptive cells in the intestine but
dostratified cells in the intestinal groove not the germinal cells suggests that this
labeled (fig. 18). Cells migrate at a differ- organ might be involved with the process
ent 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 are
groove and frequently onto the side walls labeled in the pyloric tubules.
of the intestine. Both absorptive and se- DISCUSSION
cretory cells are labeled. By 15 days, the
leading edge of labeled cells has migrated The classification of cell populations (as
around 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 not
19). The band of labeled cells is clearly occur in the digestive tract nor in the rest
defined, 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 within
beled. Behind it, cells are usually only the life span of the animal (about 12-18
lightly labeled. By 30 days, the leading Fig. 12 An autoradiogram of the stomach
edges have met each other at the top of the grooves one hour after an exposure to tritiated
typhosole in a few animals. In others, the thymidine. Cells are labeled at the base of each
leading 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 mitotic
digestive 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 half
into 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 entire
pseudostratified region of the intestine. groove height. Few cells at the base of the groove
Cell proliferation and cell migration pat- remain labeled. X 150.

11. CELL PROLIFERATION I N ASCIDIAN GUT 459

12. 460 THOMAS H . ERMAK
months, 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 are
population, 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 these
of cell proliferation, usually demonstrate points. Two separate cell populations (the
cell migration, and have turnover times groove and crest populations) occur in the
within the life span of the animal. Renew- stomach of Styela clava, each renewed by
ing 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 germinal
less 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 the
turnover times of more than 60 days (prob- postbranchial digestive tract, DNA is syn-
ably up to several months). The reason thesized in pseudostratified cells whose
why type I1 cell populations have such long nuclei lie close to the basal portion of the
renewal times in comparison to the other cell. As each cell enters mitosis the nucleus
renewing populations is not presently un- migrates toward the lumenal edge of the
derstood. The renewal of the two popula- cell. Labeled mitotic figures appear as soon
tions 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 two
and band cells (type 11) originate from the daughter nuclei move back into the region
same 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 for
branchial bars excepted) are shown sche- the dispersal of labeled nuclei throughout
matically 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 in
safe to assume that renewing gut cell pop- many other epithelia such as the epithelium
ulations are rarely found. Cell renewal of the neural tube (Watterson, '65), the
occurs 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 exact
the ascidian gut, only part of the crayfish mechanism by which nuclear migration
gut (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 with
in 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 now
cata, Fouque ('53) suggested from static labeled.18 600. autoradiogram of the intestinal
Fig.
X
An
light 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 tritiated
entiating into ciliated mucous cells on the thymidine. Cells are labeled up to the base of the
crests. In Ciona intestinalis, Thomas ('70) typhosole. X 150.

13. CELL PROLIFERATION IN ASCIDIAN GUT 461

14. 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, and
cells may differentiate or divide again. then migrate through the mature zone in
Those 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 move
likely 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, no
the transitional zone are more likely to di- cells are labeled. Behind it, cells are only
vide a few more times. In any case, all lightly labeled or not labeled at all. Those
those cells initially labeled with tritiated cells which are lightly labeled are descend-
thymidine and/or their labeled progeny ants through many generations from those

15. 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 dramatically
tritiated thymidine and have had their different. Cells in the intestine migrate
DNA diluted with unlabeled precursors. faster yet are renewed slower than cells in
Assuming 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 demands
labeled cells would have only one labeled for cell turnover than the intestine. Morton
chromosome 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 while
in the germinal zone long enough to divide Yonge ('25) and van Wee1 ('40) state that
six 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 a
population, presumably through extrusion more formidable environment of digestive
of cells into the lumen of the gut. enzymes.
Although most of the cell proliferation The renewing populations in the ascidian
in 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 the
cell populations, some proliferation pro- stomach and intestine of mammals. The
vides 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 to
grow larger, the stigmata multiply, the the renewal of the surface epithelium in
folds 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 of
nism 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) and
such 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). The
Transit times are the same for the mucous band cells, parietal cells, and Paneth cells
cells in the esophagus and stomach, al- are all renewed at a much slower rate than
though 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 germinal
bulb) 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 population
germinal 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

16. 464 THOMAS H. ERMAK
the 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 renewal
proliferation occurs only in the crypts of of the four main epithelial cell types in the mouse
Lieberkiihn. 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 the
linear and up longitudinal folds. Perhaps mouse small intestine. V. Unitarian theory of
the 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 1969
in transit time. The time for a labeled cell Renewal of Paneth cells in the small intestine of
to 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 cell
to 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 of
a 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 and
tine (O'Steen and Walker, '60) where re- Electron Microscopic Investigation Emphasizing
newal 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 1'Ctude de la
I am indebted to Dr. Nicholas D. Holland glande pylorique des ascidiaces. Ann. Inst.
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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
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