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The uptake of tritiated thymidine by the dorsal epidermis of the fetal and newborn rat.

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The Uptake of Tritiated Thymidiine by the Dorsal
Epidermis of the Fetal and Newborn Rat'
University of W a s h i n g t o n Sch~oolof Dentistry, Seattle, W a s h i n g t o n 981 05
The dorsal epidermis of fetal and newborn rats was examined
to determine the difference in ability of the basal cells to bind tritiated thymidine
during different stages of epidermal morphogemesis. Five rats were examined for
each time period from the eleventh day of gestation to the fifth day after birth.
The number of labeled cells in 5000 basal ce'Lls was counted and expressed as
a percentage. The labeling index is + 10% from the eleventh to the fifteenth
fetal day. It increases to 30% by the eighteenth day, decreases to 10% from
the twenty-first day until the first postpartum day and drops to 5% or less from
the second to fifth day. These changes in labeling index are accompanied by and
apparently correlated with the normal differentiation of rat epidermis. The
growth of the epidermis is continuous during the course of the study. Keratohyalin granules begin to form on the eighteenth day and by the twentieth day
the first cornified cells appear. The s. corneum becomes progressively thicker each
day thereafter. The s. Malpighii, on the other hand, decreases somewhat in thickness after birth. The labeling index curve represents a relationship between basal
cell activity and control or influencing mechanisms inherent in the maturational
system of skin. The increase and decrease are not related to growth alone, but
appear to be related to differentiation.
Fraser ('28) and Hanson ('47) have
published comprehensive histologic descriptions of the development of fetal rat
epidermis. This subject is of interest since
it provides general comparative information concerning epidermal development in
a common laboratory animal. In addition,
keratinization of fetal r a t epidermis occurs
in a relatively short time and thus this
animal also serves as a n excellent subject
for the study of keratinization.
Both Fraser ('28) and Hanson ('47) described the number of cell layers, the histologic appearance of the cells and the
sequential alterations in cell layers and
epidermal thickness from the eleventh fetal
day to birth and beyond. Hanson ('47), in
addition, gave the percentage of cells in
the various strata; the data indicate that
there is a proportional decrease in the
number of cells in the s. germinativum and
a proportional increase in the number of
differentiating cells during the early days
of epidermal development.
Hanson ('47) also determined the
number of mitoses in the s. germinativum
ANAT. REC., 170: 225-234.
exprlessed as the percentage of mitotic cells
in the total population of nucleated cells.
There is a small and graded decrease in
this percentage with time until birth. However, when one extrapolates the data and
calculates the percentage of mitotic cells
in the s. germinativum alone, it can be
seen that there is some peaking of mitotic
actikity before birth.
Tritiated thymidine can be employed as
a n indicator of DNA synthesis and thus
indirectly, of mitosis. Wessells ('63) has
shown that frequency of incorporation
might vary with age in chick embryo
shank skin. Basal cell orientation and
mitosis as well as inductive interaction
with the dermis are necessary prerequisites
for epidermal histodifferentiation (Wessells., '62). At different times and in different parts of the embryo proliferating
cells are characterized by varying degrees
of columnarity and by varying mitotic
activities (Wessells, '64b). A specific com-
Received Aug. 14, '70. Accepted NOV.24, '70.
1 This investigation was supported by U.S.P.H.S.
grant DE 01925-07.
parison of DNA synthetic activity at different ages has not yet been reported for
fetal epidermis. There may be a transient
diminution or halt in 3H-thymidine binding activity in the basal cells overlying the
dermal condensations that form during the
development of embryonic chick feathers
(Wessells, '65) and developing mouse
vibrissae and pelage hairs (Wessells and
Roessner, '65). Is the development of the
s. corneum accompanied by a similar
diminution? Wessels ('63) suggests that
the next essential step in understanding
epithelial ontogeny would be the establishment of the role of differentiation jn
relation to mitotic rate. This study proposes to examine fetal rat epidermis with
reference to these questions using tritiated
thymidine as an autoradiographic label.
By limiting the counts to the basal cell
population we can compare the percentages
obtained for the different days of gestation
with more precision. Because there is some
difficulty in handling and labeling the
younger fetuses, various approaches have
been used to administer the tritiated thymidine. Comments will be made concerning the comparative effectiveness of these
In this paper labeling index is defined
as the percentage of basal cells labeled;
growth, as the overall increase in cellularity; differentiation, as the cytological
changes leading to and involved in the
formation of the various epidermal strata
including the s. corneum; and cornification, as the production of a microscopically
evident amorphous surf ace layer commonly
described as the s. corneum. Cornification
is considered by most authorities to be
synonymous with keratinization.
Female Sprague-Dawley, Berkeley strain
rats weighing 330-380 gm, determined to
be in proestrus by means of vaginal washings made in the morning, were placed
overnight with males and examined the
following morning either for vaginal plugs
or the presence of sperm in vaginal washings. If positive findings were made, this
day was considered day zero of gestation.
Specimens were obtained from the
eleventh fetal to the fifth postnatal day.
On appropriate days pregnant rats were
anaesthetized with Diabutal at apprsximately 2 :00 PM and Caesarian section was
performed. Five fetuses (1 or 2 per dam)
were obtained for each fetal age. The
fetuses were either left in the mother or
maintained in Hanks BSS during the labeling period. The specimens were administered 0.03 to 0.06 cm3 (according to age)
of tritiated thymidine; specific activity
40 pc/ml, for one-half or one hour. The
dorsal skin was then fixed, dehydrated,
embedded in paraffin - 3% piccolite and
sectioned at 3 p. Tissues were fixed in
10% neutral buffered formalin for 24
hours at
5°C for specimens 18 days or
younger; in Lillie's fixative for 24 hours
for specimens 19 days to birth and 48
hours for specimens one to five postnatal
New England Nuclear Corp., Boston, Massachu-
SB, s. basale
P, periderm
SI, s. intermedium
SG, s. granulosuin
SS, s. spinosum
HG, hair germ
PG, periderm granule
KG, keratohyalin granule
SC, s. corneum
LBC, labeled basal cell
CT, connective tissue
LF, labeled fibroblast
Figs. 1-7 Richardson's stain - Epon Sections;
fig. 8-10, paraffin, Mayer's H. & E.
Fig. 1 Eleven day fetal rat skin. T i e epidermis
is composed of a cuboidal basal layer and a
flattened outer layer. x 1380.
Fig. 2 Fourteen day fetal rat skin. The epidermis is composed of several layers, the outermost constituting the periderm and the inner, the
s . basale and s. intermedium. x 900. Oil.
Fig. 3 Seventeen day fetal rat skin. The epidermis is made up of three strata; the s. basale,
the s. intermedium, the periderm. The cells of the
hair germ are columnar. x 540.
Fig. 4 Eighteen day fetal rat skin. By the
eighteenth day the s. intermedium forms a n s.
spinosum and a n s. granulosum. The periderm
consists of two layers. The inner periderm layer
contains periderm granules. x 680.
Fig. 5 Nineteen day fetal rat skin. The epidermis now forms a more prominent s. graniilosum. Other features are unchanged. x 580.
Fig. 6 Twenty day rat skin. The epidermis
has begun to cornify. Note the changes in the
condensation of the connective tissue cells below
the hair germ from the seventeenth to the twentieth day. x 530.
Figures 1-6
days. Slides were dipped in Kodak NTB3
and exposed for 12 days. Developed slides
were stained with Mayer's H. & E. Some
specimens were also fixed in glutaraldehyde, postfixed in osmium tetroxide, embedded in Epon, sectioned at 1 to 2 p. and
stained with Richardson's stain.
Dosage and administration of the tritiated thymidine was as follows :
Age of specimen
Amount of
11-17 fetal day
18-19 fetal day
20-21 fetal day
Newborn and 1-3 day
4-5 day
0.03 cm?
0.04 cm3
0.05 cm3
0.05 cm3
0.06 cm3
The following methods were used to administer the 3H-thymidine. In all cases
skin from the dorsal scapular region was
examined. Methods 2 to 6 were not always successful and unlabeled specimens
were discarded.
1. Subcutaneous injection. The embryo was removed from the uterus and
embryonic membranes and placed in
Hank's BSS. 'H-T solution was injected in
the dorsal scapular area.
2. Subcutaneous sac injection.
above, except the embryo was left within
the sac which was placed in the Hank's
3 . Sac. 'H-T solution was injected into
the amniotic fluid in uteto.
4. Placental. 'H-T solution was injected into the placenta.
5. Drop. The embryo was placed in
Hank's BSS and the 'H-T solution placed
on the skin of the scapular region.
6. Heart. The embryo was placed in
Hanks BSS and the 'H-T solution was
placed over the heart.
Two separate counts were made of the
number of labeled cells in 2,500 basal
cells, each by a different worker who was
unaware of the results obtained by the
other. The total was expressed as a percentage (labeling index.) Cells in every
third section were counted (i,e., there were
intervals of 6 p, between the sections
counted). A cell was considered labeled
if there were four or more grains over the
nucleus. Cells in hair germs and follicles
were not counted. The mean and standard
deviation were calculated from the label-
ing indices of the five animals in each time
period. A statistical analysis was done to
determine whether the method of rtpplication of 'H-thymidine affected the counts
obtained. Rank tests were performed according to a method of Kruskal and Wallis
('52) on Xjk/xj. for specimens 11 to 17
fetal days old grouped according to labeling technique used (where Xjk equals
labeling index in specimen, k, of fetal age,
j , and xj. equals the mean labeling index
at age, j.). In addition the data obtained
for one-half and one hour of administration of 'H-thymidine were subjected to
similar rank tests for significance.
The epidermis is one or two cells thick
on the eleventh fetal day (fig. 1 ) . In the
medial dorsal area it may remain one cell
thick for several days. However, laterally
there are two cell layers by the twelfth or
thirteenth fetal day. Figure 2 shows 14day fetal skin. The outer layer forms the
periderm; the inner layer is the stratum
germinativum. The tissue is
4 cell
layers thick in some places. Hair germs
are present by the seventeenth fetal day
(fig. 3). The tissue is
5 cells thick.
The s. basale is composed of cuboidal or
low columnar cells except for the hair
germs where the cells are columnar. The
periderm dces not contain periderm granules. The tissue continues to increase in
number of cells each day and is about
eight cells thick by the eighteenth fetal
day, at which time the stratum spinosum
and stratum granulosum begin to form
(fig. 4). The periderm, which was at f m t
one cell layer thick, has become bilayered.
The inner layer forms the periderm granules which differ from keratohyalin gran-
Fig. 7 Newborn rat skin. The s . corneum is
expanding and the s. Malpighii is slightly constricted. The s . granulosum is also narrower and
the granules are larger. x 800.
Fig. 8 The labeling of 13 day fetal rat skin is
relatively heavy. x 1170.
Fig. 9 The labeling of 19 day fetal rat skin is
greater than the 13 day fetal rat skin. Note also
the labeling of hair germ cells. x 800.
Fig. 10 Five day old rat skin. The labeling has
become more sparse than the 19 day old specimen. x 1020.
Figures 7-10
ules on the basis of staining and also in
morphology and location. When stained
with hematoxylin and eosin the periderm
granules stain very faintly blue and the
keratohyalin granules stain a darker bluish
purple. In Epon sections the periderni
granules stain intensely with Richardson's
stain. By the nineteenth fetal day (fig. 5)
the tissue is thicker. By the twentieth
fetal day the first cornified cells have
appeared below the periderm layers. (fig.
6). The thickness of the nucleated layers
reaches a maximum shortly before birth
and decreases slightly in the neonatal and
alder rat skin (fig. 7). The thickness of
the s. corneum continues to increase from
the twentieth fetal through the fifth postnatal day. The periderm persists in some
cases for several days following birth.
Autoradiograp hy
Figures 8, 9 and 10 show the labeling
at the thirteenth and nineteenth fetal and
the fifth postnatal day respectively. The
mean labeling index (fig. 11) is about
10% from the eleventh to the fifteenth day
(fig. 8). There is a sharp increase to
about 21% on day 16, an apparent lag in
the seventeenth day, and a peak of about
30% from the eighteenth to the nineteenth day (fig. 9 ) . Thereafter the labeling
index drops to about 10% by the twentyfirst day and remains there to the first postpartum day, dropping to 5% and less from
the second to the fifth day (fig. 10).
Figure 9 shows labeled epidermis from a
19 day fetal rat. Epidermal labeling occurs
in the s. basale. Heavy labeling occurs in
the developing hair germs (follicles), but
cells in these areas were included in the
counts of neither labeled cells nor total
basal cells.
Rankings and calculations performed to
test whether the method of administration
of 3H-thymidine influenced labeling index,
figure 12, reveal that there is no significant
difference in labeling due to the method of
application used in these experiments. K
(fig. 12) has a x2 distribution with c - 1
(in this case, 5) degrees of freedom. For
K = 3.28, 0.70 > P > 0.50. Thus, more
than 50% of the time a K value as large
as observed here would occur due to chance
fluctuations. Similar rank tests indicate
that no significant differences exist between fetuses labeled for one-half hour and
those labeled for one hour ( K = 0.017, d.f.
= 1, 0.90 > P > 0.80).
x -
Mean value o f samples
from one-time-period
Standard deviation
0 Subcutaneous injection
A 11
through sac
0 - Injection into sac
0 Placental injection
A - Drop of H3T placed on fetus
m /I
11 fetal heort
,\" 5 -
Fig. 11
D a y s of g e s t a t i o n and postnatal period
Graph of labeling index from the eleventh fetal to the fifth postnatal day.
100 and rank (in parentheses)
Mean rank
1. 125.6(25), 123.8(23), 123.3(22), 132.4(26), 156.6(32),
99.9(18), 60.4(3), 135.1(28), 72.5(7)
2. 140.4(30), 50.0( l ) , 101.0( 19), 70.4(5), 149.7(31)
3. 74.2(9), 72.4(6), 55.8(2), 95.1(15), 134.6(27)
4. 125.5(24), 102.4(20), 80.6( l o ) , 82.9( l l ) , 95.4( IS), 93.7(14)
5. 72.9(8), 90.0(13), 137.5(29), 65.5(4)
6. 88.0(12), 96.0(17), 106.4(21)
N = total number of ranks = 32.
ni = number of ranks in method i.
c = number of methods = 6.
Figure 12
The observations made in this study,
more or less agree with those of Fraser
('28), Hanson ('47) and Bonneville ('68)
insofar as the sequence of events is concerned. In order to compare findings in
terms of time it should be noted that Hanson ('47) counted the first day postcoitum
as day 1, and also adjusted ages of specimens taking into account the approximate
age and stage of epidermal histogenesis,
since litters and even embryos within a
litter may vary in their rate of development
(Nicholas, '32). In the present study, the
morning postcoitum is counted as day 0.
Animals are sacrificed six to seven hours
past the day count. Fraser ('28) and Bonneville ('68) do not explain their calculation of fetal age.
Hanson ('47) reports that the s. granulosum forms on day 18 and the s. corneum
on day 19 ( 1 day should be deducted for
comparison). Fraser ('28), on the other
hand, reports the appearance of the s.
granulosum at 20 days and the s. corneum
at 21 days. Fraser's rats have a gestation
period of 21 days as do ours, and it can
be assumed that the chronologic notations
correspond. Bonneville ('68) reports the s.
granulosum as early as 12 days. This may
be an error in interpretation. Bonneville's
age and crown-rump length data appear
to differ from the table found in Shepard,
Lemire, Aksu and Mackler ('68), so that
there may be some question concerning the
observation of cornification at ca. 18-19
days. In addition to differences in methods
of determining age and the possible differ-
ences in rats of different litters, one must
take into account the different strains of
white rats used by Fraser, Bonneville and
in the present study. Hanson used the gray
rat from which the albino rat has been
developed. Although dorsal skin was
studied in each case there is also the possibility that the sites from which the tissue
samples were taken varied. With these differences in mind the findings of the studies
cited may be compared with our observation (of the formation of the s. granulosum
at the eighteenth-nineteenth day and the
formation of the s. corneum beginning at
the twentieth day.
Fraser ('28) reports the presence of periderml granules by the sixteenth day and
they occasionally are s t i l l seen by the
twenty-first day. Hanson and Bonneville
indicate that the periderm may still be
present at birth although Bonneville ('68)
states that it becomes detached from the
underlying epidermis two or three days before birth. As reported above, we have
found it to be present and apparently
attached in some cases for several days
after birth. One may conclude that so long
as the periderm is present the cells subjacent to it cannot desquamate.
The tissue thickness and the number of
cells of the epidermis increase from the
eleventh fetal day until birth. The s. corneuni thickens from the twenty-first fetal
day through the fifth post-natal day. Keratinization first occurs in the outermost
layer of the s. granulosum and then the
next most superficial layers sequentially so
that keratinization procedes in a basal
direction. The passage of daughter cells
upward following mitosis adds to the thick-
ness of the tissue and the cells that sequentially keratinize lead to the thickening of
the s. corneum. When the periderm Snally
desquamates, the cells of the s. corneum
can begin to desquamate. It has been
reported that the rate of formation of
keratinocytes is equal to the rate of desquamation (Bertalanffy, '63; Leblond, Greulich
and Pereira, '64; Ascheim, '68) so long as
the thickness of the epidermis is constant
(Storey and Leblond, '51). The thickness
of s. corneum of the two day old rat is far
less than that of the 150 day old rat
(Fraser, '28). After birth the thickness of
the nucleated zone diminishes somewhat
until the tenth day (Hanson, '47). Its cells
cornify and contribute to the thickness of
the s. corneum. However, with this exception, mitosis must be more frequent than
desquamation in order for the s. corneum
to thicken as stated. Until such time as the
s. corneum reaches a constant thickness, a
steady state cannot prevail and mitotic
and desquamative rates must differ.
One may reason that the increased surface area and thickness of the epidermis
would require an increase in the number
of labeled basal cells. However, the increase in labeling persists from the sixteenth to the twentieth fetal day and drops
to its lowest level after birth despite the
continued growth of the animal. Thus,
growth alone cannot account for the increase in labeling index. The basic questions still remain: What control mechanisms produce, first, the increase and, later,
the decrease in DNA synthesis? How
are mitosis and histodifferentiation interrelated?
Wessells ('67) has reviewed the role of
the dermis in the differentiation of epidermis and epidermal derivatives. Some
component of the dermis is an essential
prerequisite to the epithelium's ability to
differentiate, undergo mitosis or to incorporate 'H-thymidine, as has been demonstrated in the recombination experiments
with chick skin (McLaughlin, '61; Wessells,
'61, '63; Dodson, '67) and mouse skin
(Kollar, '66). Wessells ('67) points out
that mesenchymal cells appear to regulate
the rate and type of differentiation of the
epidermis. A common feature of this regu-
lation appears to be the control of mitosis.
It is possible that the information passed
between the mesenchyme and the epithelium during this regulation may act by
other means. Fetal mouse skin collagen
fibers mature on the eighteenth fetal day
coincident with the differentiation and increase in thickness of the epidermis (Rimmer, '68). One phenomenon may control
the other. Similarly, there may be a series
of reciprocal inductive interactions such
as occur during amelogenesis and odontogenesis (Koch, '67).
The dip noted at the seventeenth fetal
day (fig. 11) may reflect a reduction in
DNA synthesis relative to hair growth,
since hair germ formation occurs on this
day in the rat (Fraser, '28 and our own
observations). Such a decrease in basal
cell mitotic activity (Wessells, '64a) has
been noted during the initial stages of
chick feather and mouse hair formation.
When the dermal condensation of the chick
feather germ forms, the mesodermal cells
and in many cases the epidermal basal cells
above it enter a state of decreased proliferative activity for 20-30 hours (Wessells,
'65) or longer for mouse hair (Wessells
and Roessner, '65; Wessells, '67). It is
suggested (Wessells, '65) that specialized
synthesis occurs at this time which may be
related to the induction of morphogenesis.
The decrease in labeling of basal cells from
the eighteenth fetal to the third postnatal
day, with the onset of keratinization occurring on the twentieth fetal day may be
another example of decreased proliferation
relating to histodifferentiation.
When the rate of formation of cells in
adult animals exceeds the rate of gain in
body weight, it may be considered to serve
cell renewal (Bertalanffy and Lau, '62).
The steady state presumes that cell formation and cell loss are balanced. In the
present study mitotic rate and desquamation rate are not equal since the s. corneum
continues to increase in thickness, and the
periderm persists until birth and in some
cases for a few days after birth. As long
as the periderm persists the subjacent cells
do not desquamate. After the second postnatal day when the young rats are in a
period of rapid growth, the epidermal labeling index is at a level barely above that
described by Messier and Leblond ('60) for
mature rat epidermis. While the labeling
indices per se in the present study are indicative of renewing (> 3.0% ) and not expanding (0.4-1.0% ) populations (Messier
and Leblond, ’60),the cell populations are,
in fact, expanding populations involved in
the growth and differentiation of this
Wessells (’67) states, “To date, it has
been impossible to obtain differentiation
without prior division. Therefore, it is still
not possible to say whether dermis has a
direct effect upon differentiation or
whether the latter process results secondarily from homotypic interreactions within
an expanding epithelial cell population.”
Bell (’64) suuggests that induction means
essentially, a change in rate of cell proliferation; and further that initiation of the
differentiation process depends on it.
Thus, the significance of the increase in
labeling index in this case may be that it is
concerned with the initiation of differentiation and therefore it is basic to the morphogenesis of epidermis.
Once differentiation has been initiated,
what causes the diminution in labeling index? Bullough and Laurence (’64) and
Eullough, Laurence, Iverson, and Eljo (’67)
have presented data indicating that a
tissue-specific mitotic inhibitor produced
by the epidermis itself controls mitotic
rate. It is possible that initial production of
such an inhibitor when the process of
keratinization begins might explain the decrease in mitotic activity that we have observed from the eighteenth fetal day to the
third postnatal day. Wessells (’63) suggests that certain factors such as epidermal thickness, among others, influence
mitotic rate. Bertalanffy (’63) reports that
desquamation may be partly effectuated by
pressure from below due to cell division.
It is possible that the persistance of the
periderm in fetal epidermis causes cell
crowding below it, impeding cell migration
and cell division. It is also possible that
nutritional or energy requirements might
possibly act in a Malthusian sense to limit
cell division (Bullough, ’52; Ascheim, ’68).
In conclusion, speculations concerning
phenomena that may first stimulate and
may later restrict cell division have been
presented. The observation in the current
investigation that labeling index reaches a
peak immediately prior to differentiation
and decreases as the s. corneum forms,
provides support for the contention that
mitosis and histodifferentiation are interrelated in epidermal development. Our
data indicate that fetal rat epidermis, an
expanding cell population about to become
a renewing one, would be a good system
in which to study control factors for mitosis and histodifferentiation and the interrelationships of these processes. It is now
necessary to discover the biochemical and
(or) biomechanical factors that mediate
these inductive or homotypic interactions.
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thymidine, epidermis, dorsal, tritiated, rat, uptake, newborn, fetal
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