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Epithelial cell proliferation during organogenesis of rat colon.

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Epithelial Cell Proliferation during Organogenesis
of R a t Colon
GREGORY L. EASTWOOD AND JERRY S. TRIER
Department of Medicine, Boston University School of Medicine,
Boston, Massachusetts 021 18, and Departments of Medicine,
Harvard Medical School and Peter Bent Brigham Hospital,
721 Huntington Avenue, Boston, Massachusetts 021 15
Between the 16th to the 20th day of gestation, the mucosa of the
colon of fetal rats changes from a simple tube with a tiny lumen lined by stratified epithelium to a much more complex structure with a large lumen and welldeveloped crypts lined by a single layer of columnar epithelium. Autoradiographic
studies with 3H-thymidine show that cell proliferation is present throughout the
stratified epithelium but becomes confined to the lower half of colonic crypts
immediately on their formation. The number of epithelial cells shown to be proliferating after exposure to a single pulse of 3H-thymidine is high during this
period of organogenesis but decreases rapidly after the 20th day of gestation
once the adult mucosal pattern has formed.
ABSTRACT
Recent work has shown that the pattern vein of the pregnant rats. One to 1.5 hours
of epithelial cell renewal in the duodenum later, fetuses were removed from the
of fetal rats changes markedly during the mother immediately after induction of
last few days of gestation (Hermos et al., ether anesthesia. To label proliferating
'71). Proliferating epithelial cells may be cells in neonatal animals, 1.5 &i of 3Hfound along the entire length of villi as thymidine/gm body weight was injected
late as one to two days before birth. How- intraperitoneally and the animals were
ever, just prior to birth, the adult pattern killed 1.5 hours later. Segments of distal
of epithelial cell renewal becomes estab- colon were taken from each fetus or neolished and proliferating epithelial cells are nate and immediately immersed in chilled
confined to the crypt region (Leblond and chrome-osmium fixative (Dalton, '55).
Messier, '58; Quastler and Sherman, '59). After dehydration in graded alcohol soluEpithelial cell proliferation in the colon tions, these were embedded in epoxy resin
of adult rodents is also confined to a spe- (Luft, '61) and oriented so that cross seccific region of the mucosa, namely the tions of the entire gut lumen could be
lower half of the crypts of Lieberkuhn cut or, in specimens from older animals,
(Lipkin and Quastler, '62). Since the rat so that crypts could be longitudinally seccolon like the rat small intestine under- tioned.
Autoradiographs were prepared by coatgoes rapid growth and differentiation during the last week of gestation, we ex- ing 1 sections mounted on glass slides
amined the pattern of colonic epithelial with Ilford K-5 photographic emulsion
cell renewal during this period of organo- (Ilford, Ltd., Ilford, Essex, England). The
slides were kept refrigerated during expogenesis.
sure for 6 weeks, then developed, and
MATERIALS AND METHODS
stained with toluidine blue. To quantitate
Fetuses from timed-pregnant Sprague- epithelial proliferation, we counted total
Dawley albino rats were studied from the epithelial cells and labelled epithelial cells
16th to the 22nd day of gestation. Neo- to determine the epithelial cell labelling
nates aged 1, 10 and 20 days were also index in well-oriented sections for each
studied. To label proliferating cells in gestational or neonatal age. Cells were confetuses, a dose of 250 ,&i of 3H-thymidine sidered labelled if 3 or more exposed silver
(6.7 Ci/mM; New England Nuclear, grains overlay the nucleus. Different secBoston, Mass.) was injected into the tail
Received Oct. 30, '73. Accepted Dec. 6, '73.
ANAT.REC., 179: 303-310.
303
304
GREGORY L. EASTWOOD AND JERRY S. TRIER
tions from the same specimen were separated by at least 10 serial sections to
avoid scoring labelled cells more than once.
For electron microscopy, thin sections
were cut with a diamond knife, mounted
on uncoated copper mesh grids, stained
with uranyl acetate (Watson, '58) and
lead citrate (Reynolds, '63) and examined
with a Phillips 300 electron microscope.
On the 20th day of gestation, differentiated goblet cells which stain dark blue
with toluidine blue can first be identified.
Moreover, proliferating epithelial cells with
labelled nuclei are confined strictly to the
lower half of the crypts (fig. 3). When
examined with the electron microscope, the
colonic goblet cells of the 20-day old fetal
rat appear well-differentiated with numerous formed mucous granules, a wellRESULTS
developed Golgi region and abundant eleThe colon of 16 day gestational fetal ments of granular endoplasmic reticulum
rats is a simple tubular structure with a (fig. 4).
tiny lumen which is lined by a stratified
During the last 2 days of gestation and
layer of undifferentiated epithelium 2 to 3 the early post-partum period, the crypts
cells thick (fig, 1). Proliferating epithelial continue to lengthen, the colonic lumen
cells are abundant at this age and, as enlarges further and the number of goblet
shown in autoradiograms prepared from cells in the epithelium increases. By 20
animals sacrificed within 90 minutes after days after birth, the crypts are 30 to 40
3H-thymidine injection, they are scattered cells deep (fig. 5). Throughout this period
throughout the entire epithelium in a more of continued growth, proliferating epitheor less random fashion (fig. 1 ) . Moreover, lial cells remain c o n k e d to the lower half
many nuclei in the surrounding mesen- of the crypts (figs. 3 , 5).
chyme are also labelled (fig. 1).
The results of quantitation of proliferatThe epithelium is still stratified at 17 ing epithelial cells identified by pulse ladays of gestation, but its thickness has in- belling with 3H-thymidine during organocreased to 3 to 5 cells. By this time, pro- genesis of the rat colon are summarized
liferating cells are not as evenly distrib- in table 1. During the 16th and 17th days
uted; the nuclei of many basally-located of gestation, the nuclei of up to 35% of
cells are labelled whereas the nuclei of colonic epithelial cells are labelled after
relatively few cells in the inner layer near exposure to a single pulse of the 3H-thymithe lumen are labelled.
dine. Over the next 5 days, in concert with
By the 18th day of gestation, rudimen- maturation of the gut and segregation of
tary crypts up to 7 or 8 cells in height the proliferative zone of the lower half of
have formed, markedly increasing the size colonic crypts, the percentage of cells with
of the gut lumen (fig. 2 ) . The cells form- labelled nuclei following a single pulse of
ing the base of these crypt-like invagina- isotope gradually declines so that the
tions seem oriented with their long axis colonic epithelial cell labelling index is
perpendicular to the crypt lumen whereas only 11-12% by the day prior to birth.
the more superficial cells appear randomly Thereafter the percentage of epithelial cells
arranged (fig. 2 ) . Proliferating cells are with labelled nuclei appears to remain connow most abundant among those cells lin- stant until at least 20 days after birth
ing the lower half of the crypt-like in- (table 1).
vaginations and among the intervening
DISCUSSION
basally-located cells between invaginations.
However, a few cells with labelled nuclei
The colonic mucosa of the fetal rat
are seen among the randomly-arranged changes strikingly during the final week
more superficially-located cells (fig. 2).
of fetal life. Firstly, i t increases markedly
By the 19th day of gestation, aggregates in size, Secondly, i t changes in about 4
of mesenchyme begin to invaginate the days from a small tube with a tiny lumen
basal region of the epithelium between the lined by undifferentiated stratified epithecrypt-like clefts thus forming true colonic lium (fig. 1) to a highly organized mucosa
crypts with intervening lamina propria with well-developed though shallow crypts
separating the epithelium of adjacent separated by the mesenchymal lamina propria and lined by a single layer of differcrypts.
305
CELL PROLIFERATION DURING COLON ORGANOGENESIS
TABLE 1
Percent of epithelial cell nuclei labelled with thymidineJH (labelling index) in
fetuses aged 16 through 21 days and newborns aged 1 , 10 and 20 days
Age
Litter
No. of animals
Cells
counted
% labelled
(labelling index)
16 days gestation
A
B
A
B
A
B
A
B
A
B
A
4
2
5
3
4
3
4
3
3
3
3
3
3
3
3
3
3
3
1112
386
1151
1164
1494
1468
1700
1865
1894
2002
2730
4060
4659
2981
3000
3000
3000
3000
26.6
29.8
35.4
34.0
22.9
23.0
20.9
20.0
17.8
18.2
16.3
11.5
11.8
10.8
14.7
11.6
10.2
11.2
17 days gestation
18 days gestation
19 days gestation
20 days gestation
21 days gestation
B
22 days gestation
1day after birth
A
B
A
B
10 days after birth
20 days after birth
entiated columnar epithelial cells which
include differentiated goblet cells (fig. 3 ) .
Thirdly, whereas proliferating epithelial
cells are scattered throughout the stratified epithelium on the 16th day of gestation (1 week prior to birth), proliferating
cells are compartmentalized in a proliferative zone confined to the lower half of the
crypts by the 19th or 20th day of gestation in a manner similar to that seen in the
large intestine of normal adult rats. Indeed, some segregation of proliferation occurs as early as the 18th day of gestation
in primitive crypt-like structures which
form in the stratified epithelium before
mesenchyme penetrates the epithelium to
form discreet epithelial crypts separated
by lamina propria (fig. 2). Thus, the
transition from the fetal to the adult proliferative pattern occurs 2 days earlier in
the fetal rat colon than in fetal rat duodenum in which epithelial cell proliferation does not become confined to the crypt
region until the 22nd day of gestation,
just prior to birth (Hermos et al., '71).
Epithelial cell proliferation is extremely
active during the period of colonic morphogenesis in which the mucosa assumes its
characteristic adult configuration. Following a pulse of 3H-thymidine, which is available for new DNA synthesis for only a
few minutes (Rubini et al., '60), up to
35% of epithelial cell nuclei are labelled in
fetuses sacrificed during the 16th and 17th
day of gestation (table 1). As the gut matures and the epithelial cells differentiate
and the proliferative zone compartmentalizes, the epithelial cell labelling index
decreases sharply. Indeed, in fetuses killed
1 to 2 days before birth, the labelling index
is the same as that found in the colon of
rats 10 and 20 days after birth (table 1).
These studies indicate that between 6
days before and 2 days before birth, the
colonic mucosa changes from a simple
primitive stratified epithelium with very
active but randomly oriented cell proliferation to a highly organized mucosa whose
structure and epithelial cell proliferative
pattern closely resemble that of the adult
colon.
ACKNOWLEDGMENTS
This study was supported by U.S.P.H.S.
grants AM17537 and AM05005.
LITERATURE CITED
Dalton, A. J. 1955 A chrome-osmium fixative
for electron microscopy. Anat. Rec., 121: 281.
Hermos, J. A., M. Mathan and J. S. Trier 1971
DNA synthesis and proliferation by villous
epithelial cells in fetal rats. J. Cell Biol., 50:
255-258.
Leblond, C. P., and B. Messier 1958 Renewal
of chief and goblet cells in the small intestine
as shown by radioautography after injection of
thymidine-H3 into mice. Anat. Rec., 132: 247260.
Lipkin, M., and H. Quastler
1962 Cell popula-
306
GREGORY L. EASTWOOD AND JERRY S. TRIER
tion kinetics i n the colon of the mouse. J. CLin.
Invest., 41: 141-146.
Luft, J. H. 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem.
Cytol., 9: 409-414.
Quastler, H., and F. G. Sherman 1959 Cell population kinetics in the intestinal epithelium of
the mouse. Exp. Cell Res., 17: 420438.
Reynolds, E. S . 1963 The use of lead citrate at
high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212.
Rubini, J. R., E. P. Cronkite, V. P. Bond and
T. M. Fliedner 1960 The metabolism and fate
of tritiated thymidine i n man. J. Clin. Invest.,
39: 909-919.
Watson, M. L. 1959 Staining of tissue sections
for electron microscopy with heavy metals. J.
Biophys. Biochem. Cytol., 4 : 4 7 5 4 7 8 .
PLATE 1
EXPLANATION OF FIGURES
1 Light microautoradiograph of a cross section of the colon from a 16
day gestation fetal rat. The tubular gut consists of a tiny lumen surrounded by a 2-3 cell thick layer of stratified epithelium, which in
turn is surrounded by concentrically arranged mesenchyme. Labelled
nuclei (arrows) are more or less randomly scattered throughout the
epithelium and the mesenchyme. Toluidine blue. x 600.
2
Light microautoradiograph of a cross section of colon from a n 18 day
gestation fetal rat. Rudimentary crypt-like invaginations have formed
i n the epithelial layer and labelled nuclei are more basally oriented
(arrows). Toluidine blue. x 600.
3
Light microautoradiograph from a 20 day gestation fetus. Labelled
nuclei are confined to the lower portions of well-formed crypts separated by mesenchymal lamina propria and goblet cells (arrows) have
appeared. Toluidine blue. x 600.
CELL PROLIFERATION DURING COLON ORGANOGENESIS
Gregory L. Eastwood and Jerry S . Trier
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
308
4
Electron micrograph of a goblet cell and adjacent absorptive cells
from the upper crypt region of a 20 day gestation fetal rat. This
well-diff erentiated goblet cell contains closely-packed mucous granules ( M ) , abundant granular endoplasrnic reticulum (ER), Golgi material ( G ) and aggregates of glycogen (arrows). x 10,000.
5
Light microautoradiograph from a 20 day old neonatal rat. The proliferative zone at the crypt bases is defined by the labelled nuclei.
Many goblet cells (arrows) are now present in mid and upper crypt
regions. Toluidine blue. x 600.
CELL PROLIFERATION DURING COLON ORGANOGENESIS
Gregory L. Eastwood and Jerry S. Trier
PLATE 2
309
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