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Development of the pancreas of the rat embryo in vitroIslets and acini.

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Development of the Pancreas of the R a t Embryo
in Vitro: Islets and Acini'
MICHAEL R. SCHWEISTHAL? MARIA P. CaAS AND LEMEN J. WELLS
Department of Anatomy, University of Minnesota, Minneapolis, Minnesota
ABSTRACT
Developing pancreases from embryos of thirteen and one-half to
seventeen and one-half days were placed on rayon grids and cultivated on 2 liquid
medium of cock serum and chick embryo extract for periods of two to ten days. The
cultures were incubated a t 37°C in air supplemented by controlled oxygen and carbon
dioxide.
Pancreatic islets develop well in organ culture. This process usually involves
increases i n the size of islets and in the number of cells per islet, and sometimes
involves a n increase in the number of islets. The rate of morphogenesis of islets is
variable: slower than that in normal controls during equal time in days (most cases)
or equal to it (several cases) or faster than it (few cases). The development of
islets includes the formation of granulated beta cells (many cases). The beta cells
may reach stages as advanced as those i n normal pancreases OF postnatal day 3 (few
cases). The existence of granulated beta cells in cultures from explants too young
to have them constitutes convincing morphologic evidence of the production of insulin
or pre-insulin during the periods of culture.
The development of acini in organ culture includes the fcrmation of them from
pancreatic primordia with no acini and the appearance of zymogen granules in the
acinar cytoplasm. The morphogenesis may reach stages as advanced a s those i n
normal pancreases of prenatal day nineteen and one-half (several cases).
Before beginning our study in '62, we
could not find in the literature any record
of pancreatic islets with granulated beta
cells in organ cultures of the developing
mammalian pancreas. We knew, of course,
that small islets had been reported in cultivated pancreases of rat (Chen, '54) and
mouse embryos (Wells and Borghese, '61 ;
Golosow and Grobstein, '62). The literature on this subject has been reviewed in
a recent paper (Wells and Borghese, ' 6 3 ) .
We have recorded some of our observations in two abstracts (Schweisthal, C6as
and Wells, '63a, b). We now present a
definitive account of our study.
MATERIALS AND METHODS
The Sprague-Dawley strain of rats was
used (Simonsen Laboratories, Inc. ). Each
pregnancy was timed from the moment
of witnessed mating. Either the entire
pancreatic primordium (with or without
duodenum) or only the dorsal portion of
the developing pancreas was explanted.
The watch glass method of organ culture
was used. The liquid medium consisted of
equal parts of cock serum and chick embryo extract. This extract was the supernatant fraction from one part of minced
13-day chick embryo and two parts of buffered Tyrode's solution (pH 7.4 0.2).
An explant was held on the surface of the
liquid medium (0.5 ml) by means of 3
rayon grid which was supported by a grid
of stainless steel. A culture was kept in
a closed belljar and incubated at 37°C.
During a period of one hour everv day.
the air in the belljar was supplemented by
a controlled amount of introduced gas, a
mixture of 95% oxygen and 5% carbon
dioxide. A culture was transferred to fresh
medium every second day.4
Control pancreases were of two kinds,
those from litter-mates of the donors of
*
1 Aided by Grant A-1244 from the National Institute
for Arthritis and Metabolic Disease, Public Health
Service.
2 Trainee i n Anatomy in a program supported by a
Training Grant from the National Institutes of Health
to the Department of Anatomy.
3 Research
Fellow, 1962-63.
Present address:
Istituto Superiore di Sanita, Viale Regina
Elena.
Rome, Italy.
4 Antibiotics were not used. This and several other
particulars of the technique are consequences of the
many courtesies of European colleagues in whose
laboratories Lemen J. Wells studied while on sabbatical leave i n 196C61: Prof. Eli0 Borghese and staff
(Frascati), Prof. Etienne Wolff and staff (No ent
sur-Marne) and Prof. Honor B. Fell and staff (Sam:
bridge). In Cambridge, Prof. Fell and her group
supervised a reliminary study which confirmed the
observation of Chen ('54) that when the developin
panqreas .of the rat embryo is cultivated on a li&
medium zn uitro it may undergo significant development.
149
MICHAEL R. SCHWEISTHAL, MARIA P. C$AS AND LEMEN J. WELLS
150
explants (beginning controls taken at the
time of the explantation) and those from
embryos of other litters (table 1).
Two combinations of fixatives and stains
were used in preparing stained serial sections of cultures (culture periods of two,
four, six, eight and ten days) and of control pancreases : Bouin’s solution and aldehyde fuchsin with Ponceau’s counterstain;
Zenker’s solution and hematoxylin and
eosin.
Sections stained with aldehyde fuchsin
were used in estimating the granulation
age of an islet in a culture (figs. 1 4 ) .
This involved selection - selection of two
islets for simultaneous comparison in the
field of a Bausch and Lomb microcomparator. We routinely selected the largest
and best-developed islet of a culture for
comparison with the largest and bestdeveloped islet of a control pancreas. I n
estimating the granulation age of an islet
in a culture, two criteria were used: relative size of islet and relative abundance
of granulated beta cells5
Islets in a control pancreas and those
in a culture were counted in serial sections
TABLE 1
Explants a n d normal conhols
Number of
pancreases
Litters
No.
Age
days
205 Explants
70
64
44
22
5
8
8
6
2
1
44 Beginning controls
25
67 Other prenatal controls
22
11
1
1
7
4
10
5
8
3
2
4
2
1
13
5
9 Postnatal controls
2
1
2
1
2
1
1
1
1
1
1
1
13l/z
14%
15%
16%
17%
131/2-171/2
13%
15%
16%
17%
18%
19%
20Y2
21v2
1
2
3
4
5
6
stained with
(tables 2, 3 ) .
hematoxylin
and
eosin
TABLE 2
Counted islets per normal control pancreas
Number of islets
Groups
Cases
Age
Ranae
Averace
~~
days
A
B
C
D
11
11
8
8
13%.
14%
15%
16%
4 to 14
7 to 18
12 to 35
14 to42
7.9
12.2
23.3
30.7
Sections stained with hematoxylin and
eosin were used in comparing the largest
and best-developed acinus of a culture
with the largest and best-developed acinus
of a control pancreas in the field of the
microcomparator. In estimating the developmental age of an acinus in a culture.
three criteria were used: relative size of
acinus, relative height of cytoplasm of the
acinar cell and relative granulation of cytoplasm (zymogen granules).
OBSERVATIONS
Controls. The normal pancreatic primordium of thirteen and one-half days
had two regional portions, namely, the
dorsal and ventral pancreatic outgrowths
of the duodenum. These portions were
continuous with each other in the mesoduodenum in the region of the developing
portal vein. The dorsal pancreas, larger
than the ventral, extended into the meso
gaster as far as the spleen. The pancreatic primordium as a whole consisted
5 Such provisional criteria as number of granules
per cell, size of granules and intensity of coloration of
granules were abandoned for two reasons: First. our
attempts to devise satisfactory methods for obtainme
reliable data on the number and size of granules were
not successful. Second the intensity of coloration of
varies with thk time of exposure to aldehyde
uchsin and/or with the time of destaining.
mules
~-
Figs. 1 4 The tops of bars are two, four, six,
eight and ten days from the baseline, i.e., from
“zero time” or from the age at the time of explantation. In each figure the horizontal interrupted line at 17.5 days refers exclusively to
control pancreases. This line means that contrals of 17.5 days had several granulated beta
cells per islet. Controls older than 17.5 days
(above line) had still more granulated beta cells
per islet. Controls younger than 17.5 days (below
line) had one or two granulated beta cells per
islet (16.5 days) or one per pancreas in an
exceptional case (15.5 days) or none per p a n
creas (14.5 and 13.5 days).
ZZ
2
Cull
(Con1
dev odv do
. dev. odv. &
'Oq'
3.2
4
809'
EXPECTED BIRTH
Developmenlal odvance. doys 1.6
101ol days of culture
W Q 3
50q'
30
6
69%
5.5
B
,
,
I
I
80%
BO
10
,
8
I
(
,--.,
BETA GRANULATION IN CULTURES FROM DONORS OF 16$ DAYS
Figures 1 to 4
[
pornolol
4
"OY*
2.0
95x
38
lK%I
4015
110021
2
Narnal, prenllld
Mrd.
4014
0
:::
6
5015
73%
44
IIOOXI
J-
)
67 Y.
Cull
dev. odv. do.
dev ad".: do.
6
3015
160 Yo1
4
0012
4.0
Oe!
2
Developmenrol odvance. doys
Cultures ullh granules
Tot01 doys of cullure
( Con1
81 %
65
61 h
55 5
55
I80 %i
4.9
174 %I
10
120115
170123
8
n i
DAYS
68 Ye
68
10
4 6 4
llOO%l
,
8
I
---; :
DAYS
4 6 4
11007.)
BETA GRANULATION IN CULTURES FROM DONORS OF 13;
odv da.
C M I . dev. oav do.
( Cull.: dev
Developmenlal advance. days
culrureswdh
Tolol doys of culture
"
BETA GRANULATION IN CULTURES FROM DONORS OF 15:
152
MICHAEL R. SCHWEISTHAL, MARIA P. CPAS AND LEMEN J. WELLS
TABLE 3
Counted islets per culture
Groups
Cases
c$fz'ed
Number of islets
Range
Explanted at day 13%
1
2
E
F
G
2
4
2
H
I
10
6
8
10
J
4
2
K
4
4
L
7
3
2
6
8
10
5
AveraRe
1 .o
3 to 4
2 to 6
3.5
4.0
2 to 10
4 to 12
6.0
9.4
Explanted at day 141h
M
N
0
P
Q
R
S
T
U
V
2to 7
2 to 8
2 to a
4 to 12
10 to 12
Explanted at day 15%
2
2
2 to 8
3
4
6 to 14
4
6
3 t O 14
3
8
19 to 26
3
10
21 to33
Explanted at day 16Vz
2
2
24 to 33
3
4
33 to 54
2
6
62 to 71
4.0
4.8
5.7
8.3
11.0
5.0
10.7
9.0
23.0
26.0
28.5
43.3
66.5
of epithelium, developing islets, mesenchyme and peritoneum (figs. 5, 6, 10;
tables 1, 2 ) . The epithelium was arborescent; the rami were simple, finger-like
tubules with small lumina. The developing islets were highly eosinophilic. They
were relatively undifferentiated in that
they did not have granulated beta cells.
The developing pancreas of fourteen
and one-half days had an epithelial tree
and several young islets. The tree had two
generations of branches. The islets were
primitive (fig. 7). In sections stained
with aldehyde fuchsin the cytoplasm of
the islet cells had a purplish hue, but
lacked purple granules. Since the cytoplasm of all islet cells tended to be purplish, this tinctorial reaction was not regarded as specific for beta cells.
The embryonic pancreas of fifteen and
one-half days had young islets and an
epithelial tree with two or three generations of rami. The developing islets (fig. 8)
showed more advanced stages of differentiation in certain specimens than in other
specimens from different litters of the
same age. In each of two specimens
stained with aldehyde fuchsin, an exhaus-
tive study of all islets with the aid of an
oil-immersion objective brought into view
one islet cell with several purple granules
in the cytoplasm; this cell probably was a
young beta cell. In each of two other
similar specimens, it was not possible to
find any granulated beta cell; the cytoplasm of the islet cells was purplish but
without discrete, purple secretion granules.
The developing pancreas of sixteen and
one-half days had three kinds of primitive
structures: ducts, acini with zymogen
granules and islets (fig. 9). Some of the
primitive islets were being invaded by
capillaries. Among eight specimens stained
with aldehyde fuchsin, seven were suitable
for a careful study of islets with the aid
of an oil-immersion objective. In one of
them, one of the islets had large cells with
light purple cytoplasm but without purple
secretion granules in the cytoplasm. In
each of the six remaining specimens in
which a thorough search for granulated
beta cells likewise was made, at least one
per specimen was found; the largest number per specimen was two. The purple
granules, several per granulated beta cell,
were diffuse, not closely packed (cf. closely
packed granules in an older specimen.
fig. 12).
The embryonic pancreas of seventeen
and one-half days had developing ducts,
acini and islets (fig. 18). In each of six
pancreases stained with aldehyde fuchsin,
the islets had several granulated beta cells
per islet. The granules were purple and
closely packed (cf. closely packed granules
in an older specimen, fig. 12).
Prenatal controls of more than seventeen and one-half days and postnatal controls were studied and considered (table 1).
Certain essentials of representative specimens are depicted in selected photographs
which are accompanied by explanations
(figs. 12-14, 16, 22).
Granulated beta cells in cultures. Three
cultures from explants of seventeen and
one-half days, all from embryos of one
litter, were cultivated: one for two days,
one for four and one for six. The islets
of these three cultures were alkie in having many granulated beta cells per islet
and in having an estimated granulation
age of postnatal day 2. The islets of each
culture were unlike those in controls of
PANCREATIC DEVELOPMENT I N VITRO
postnatal day 2 in lacking internal networks of capillaries.
In all cultures from explants of sixteen
and one-half days, several to many granulated beta cells per islet were present
(figs. 1, 2 1 ) . The estimated granulation
ages of islets ranged from prenatal (postcoital) day seventeen and one-half (two
of the five cultures of two days, bottom of
range, fig. 1) to postnatal day 3, i.e., postcoital day twenty-four and one half (the
two cultures of ten days, fig. 1 ) . The tops
of ranges in figure 1 bring out the fact
that the developmental advance in the islets of a culture (advance in granulation
age) was sometimes equal to that of a
control pancreas during equal time in days
(2-day, 4-day and 8-day groups). This
was actually true of six cultures: three
cases after two days of cultivation, two
cases after four days and one case after
eight.
The cultures from explants of fifteen
and one-half days had at least several
granulated beta cells per islet (fig. 2 0 ) ,
in all cases except one, the one in which
the period of culture was four days (fig.
2 ) . There were eight cultures in which
the estimated advances in granulation age
were equal to those in control pancreases
during equal times: four cases after two
days of culture, three cases after four
days and one case after six (cf. tops of
ranges in two-day, four-day and six-day
groups, fig. 2). As shown in the bottom
horizontal column of data in figure 2, the
average developmental advance in the estimated granulation age after two days of
cultivation was equal to that in the control pancreas (100% ). After four, six,
eight and ten days of culture, the average
developmental advances lagged behind
those in controls (95%, 7 3 % , 81% and
68% ).
The cultures from explants of fourteen
and one-half days were of special interest
because granulated beta cells were not
present in beginning controls which were
taken at the time of the explantation (donors of explants and donors of control
pancreases were litter-mate embryos) and
because several to many granulated beta
cells per islet were present in most of the
cultures which were cultivated for periods
of six, eight and ten days (fig. 3 ) . Two
153
cultures had undergone advances in estimated granulation age which were equal to
those of control pancreases during equal
times: one case after eight days of culture
and one after ten (tops of ranges, fig. 3 ) .
The cultures from explants of thirteen
and one-half days were of still greater interest because the developing islets in the
beginning controls taken at the time of
the explantation were still more primitive
(figs. 4, 10, 11, 15, 19). Figure 4 shows
that several to many granulated beta cells
per islet were present in most of the cultures of six, eight and ten days. Not shown
are the numbers of cases at the tops of
ranges of the eight-day and ten-day groups,
namely six and two cases, respectively.
Among those 29 presented cultures
which lacked granulated beta cells (figs.
1-4), 23 were viable (consisted of healthy
tissue) prior to the fixation; the other six
showed either severe necrosis (four cases)
or atypical staining reactions which suggest early stages of necrosis (two cases).
The four cases of severe necrosis are distributed as follows: one case, six-day
group, figure 3 ; two cases, ten-day group,
figure 3 ; one case, ten-day group, figure 4.
The two cases of atypical staining reaction
are members of the ten-day group in
figure 4.
The 126 cultures which were subjected
to aldehyde fuchsin fell into three groups
with respect to the rates of morphogenesis
of islets (rates of attaining estimated granulation age); these groups now are designated X, Y and 2. In group X, 107 cultures including the four cases of necrosis
and the two cases of atypical staining
reaction (85% of cultures), the rates were
slower than those in normal controls during equal times in days. In group Y,
16 cultures (13% ), the rates were equal
to those in controls: six cases among the
cultures presented in figure 1; eight cases,
figure 2; two cases, figure 3. In group Z,
two cultures (2% ), both from explants
of seventeen and one-half days (one culture of 2 days and one of four), the rates
were faster than those of controls.
Among the cultures with estimated granulation ages of twenty and one-half days
or more, an occasional culture had two
kinds of large islets : those with granulated
beta cells and those with beta-like cells
154
MICHAEL R. SCHWEISTHAL, MARIA P.
which lacked purple secretion granules.
The significance of the latter kind of islets
will be considered in the discussion.
Other changes in islets of cultures.
Other developmental changes in islets
were studied in sections stained with hematoxylin and eosin. Sections of cultures
were compared with those of beginning
controls and with those of older controls.
During periods of culture as long as
six to ten days, the changes in the islets
of cultures usually included two : increase
in size of islets and increase in number of
cells per islet (see figs. 30, 11, 15, 19).
A third change, definite increase in
number of islets, occurred in the four-day
and six-day cultures from explants of sixteen and one-half days (groups U and V,
table 3); the evidences of this increase
are tabulated data and data in a protocol
to be presented, both of which should be
prefaced by words about methodology.
In each of eight cultures distributed
among six groups (Q-V, table 3 ) , only the
dorsal component of the developing pancreas was explanted (versus usual method
in which both dorsal and ventral pancreases were explanted en bloc: one case,
group Q; one case, R; one, S ; two, T; two,
U; one, V. The counted numbers of islets,
without any correction, were combined
with those for cultures of entire pancreases
in reckoning the tabulated ranges and
averages.
Returning to the data, the average number of islets per culture in group U and
that in group V were larger than the
average number of islets per control pancreas in group D of table 2. That these
differences reflected genuine increases in
the number of islets during the process of
organ culture in uitro is supported by data
on pancreases from three litter-mate embryos of sixteen and one-half days, as presented in the following protocol: The beginning control pancreas from one embryo
(entire gland) had 24 islets. An explant
of dorsal pancreas from the second embryo
was cultivated for four days; the culture
had 54 islets (culture in group U, table 3 ) .
The explant of dorsal pancreas from the
third embryo was cultivated for six days;
the culture had 71 islets (culture in
group v>.
CgAS
AND LEMEN J. WELLS
Acini i n cultures. At present, we shall
record only a brief account of the development of acini in cultures. Later, we plan
to publish a fuller account of it.
Among the cultures presented in table 4,
all had acini except the following nine:
one, group 1; two, group 2; one, group 3;
two, group 6; three, group 7. Acini in one
culture of group 4 are shown in figure 17.
In those 30 cultures of groups 1-10 in
which acini were present, these structures
must have appeared during the periods of
organ culture; acini were absent in the
beginning controls of thirteen and one-half
days and in those of fourteen and one-half
days.
The acini reached an estimated developmental age of prenatal (postcoital) day
nineteen and one-half in each of nine cultures in table 4, as follows: one culture,
TABLE 4
Estimated developmental ages of acini
in cultures'
Days
Groups
1
2
3
4
5
6
7
8
9
10
Cases
cultured
Explanted at day 13%
1
2
2
4
2
6
4
6
8
10
~
Explanted at day 14%
4
2
5
4
9
6
4
8
2
10
Estimated
age days
( average )
14.5
15.5
16.0
17.8
18.2
16.0
16.1
18.0
18.5
19.0
Explanted at day 15%
11
12
13
14
15
2
3
3
2
2
2
4
6
8
10
17.5
18.1
18.5
19.5
17.5
20
Explanted at day 16%
2
2
4
4
2
6
1
8
2
10
17.5
18.7
18.5
19.5
18.5
21
22
23
Explanted at day 17%
2
2
1
4
1
6
18.5
18.5
19.5
16
17
18
19
1 Any estimated.age ,of less than 16.5 days is that of
the pancreatic epithelium (acinl not yet present).
PANCREATIC DEVELOPMENT I N VITRO
group 9; one, group 10; one, group 13;
two, group 14; one, group 17; one, group
19; two, group 23 (figs. 23, 24). There
was not any case of an acinus in culture
which advanced beyond the stage of day
nineteen and one-half.
DISCUSSION
Islets with granulated beta cells and
acini with zymogen granules were observed in many of our cultures.
Granulated beta cells in cultures from
explants too young to have them must
have appeared during the periods of organ
culture; their purple granules are regarded
as representing insulin or pre-insulin. We
conclude that these cells produced and
stored insulin.
It is reasonable to assume that the beta
cells in cultures also released insulin into
the culture medium, for four related reasons: First, beta granules represent only
stored insulin, thus reflecting the physiological state of the cell at the moment of
fixation. Second, an occasional culture of
an estimated granulation age of twenty
and one-half days or more had not only
large islets with granulated beta cells but
also large islets with beta-like cells which
did not have purple secretion granules (cf.
degranulated beta cells). Third, any tenday culture from an explant of thirteen
and one-half days must have had ample
time for the release of insulin because
three cultures of six days and 17 cultures
of eight days already had granulated beta
cells (fig. 4). Fourth, the mere absence
of an internal network of capillaries in an
islet of a culture should not militate
155
against the release of insulin into the culture medium.
Our views are in harmony with the report that the abundance of granulated beta
cells correlates well with the insulin content of the pancreas, both in the dog and
in man (Wrenshall, Hartroft and Best,
'54). They also are in harmony with the
report of insulin in the culture medium
of cultures of bits of pancreas from rat
embryos of 17 days (Murrell and Lazarow,
'63).
Our study is being continued and expanded to include the cultivation of pancreatic primordia which are younger than
thirteen and one-half days.
LITERATURE CITED
Chen, J. M.
1954 The cultivation in fluid
medium of organized liver, pancreas and other
tissues of foetal rats. Exp. Cell Res., 7: 518-529.
Golosow, N., and C. Grobstein 1962 Epitheliomesenchymal interaction in pancreatic morphogenesis. Dev. Biol., 4: 242-255.
Murrell, L. R., and A. Lazarow 1963 Organ
cultures of fetaI endocrine rat pancreas o n
liquid medium. Anat. Rec., 145: 264.
Schweisthal, M. R., M. P. C6as and L. J. Wells
1963a Pancreatic acini and islets of the rat
embryo: development in organ culture. Anat.
Rec., 145: 282-283.
196313 Pancreatic islets of the rat embryo: development in organ culture. Diabetes,
12: 373.
Wells, L. J., and E. Borghese 1961 Sviluppo
embrionale in uitro del pancreas di topo. Boll.
Zool., 28: 235-239.
1963 Development of the pancreas of
the mouse embryo in vitro: acini and islets.
Gen. Comp. Endocrinol., 3: 265-273.
Wrenshall, G. A., W. S. Hartroft and C. H. Best
1954 Insulin extractable from the pancreas
and islet cell histology. Diabetes, 3: 444452.
PLATE 1
EXPLANATION O F FIGURES
5
Sectioned pancreatic primordium of a normal embryo of thirteen and one-half days,
showing epithelium (left and right), mesenchyme and two primitive islets of Langerhans (arrows). The illustrated portion of primordium is only the embryonic dorsal
pancreas in the mesogaster. Not shown is the peritoneum of the greater curvature of
the mesogaster (beyond left margin of photograph) and that of the omental bursa
(beyond right margin of photo). A study of the series of sections showed that the
entire primordium (both dorsal and ventral pancreases) has a total of five islets (see
group A, table 2). For tinctorial evidence that granulated beta cells had not appeared
at the illustrated stage, see figure 10. Hematoxylin and eosin, x 540.
6
Primordium of another embryo of thirteen and one-half days, showing epithelium,
mesenchyme and one of the five islets (arrow). The epithelium consists of unbranched
cords (not illustrated) and of luminated tubules (two shown). The islet cells are
highly eosinophilic. Hematoxylin and eosin, x 540.
7 Developing pancreas of a normal embryo of fourteen and one-half days, showing one
of the epithelial rami (left) and one of the 18 islets (right). The developmental stage
before us is more advanced than that in any other member of group B in table 2.
Not shown is the fact that the epithelial component of the illustrated primordium is
arborescent, that it has two generations of rami and that the terminal rami have two
regional portions: a relatively thin portion (primitive duct) and a more bulbous portion
(terminal and probably the parent tissue of future ducts and acini). Hematoxylin and
eosin, X 540.
8
Developing pancreas of a normal embryo of fifteen and one-half days, showing a n
epithelial tubule (right) and one of the 35 islets (left). The stage now i n view is more
advanced than that in any other member of group C in table 2. The ramification of
the epithelial tree is only somewhat more advanced than that described in connection
with figure 7; but the lumina of the epithelial tubules are larger. Hematoxylin and
eosin, x 540.
9
Developing pancreas of a normal embryo of sixteen and one-half days, showing a
primitive islet which is attached to a primitive duct from which it seems to have originated. This islet is one of the total of 42 in the entire pancreas. Not illustrated is the
fact that the ducts communicate with a system of two or three generations of branches,
that the terminal branches have young acini with zymogen granules and that certain
primitive islets are being invaded by capillaries; below the illustrated islet, a t “7 o’clock,’’
is a capillary. Not shown is the fact that i n six specimens stained with aldehyde
fuchsin one or two granulated beta cells per pancreas could be found. Hematoxylin
and eosin, X 540.
10 Islet (arrow) in a beginning control pancreas from a n embryo of thirteen and one-half
days. This embryo and the donor of the culture presented in figure 11 are members of
the same litter (litter-mates). Aldehyde fuchsin, X 540.
11 Granulated beta cells i n a n islet of a n eight-day culture from a n explanted primordium
of thirteen and one-half days (center and also above head of arrow); the donor of this
explant and that of the beginning control pancreas shown i n figure 10 are embryos of
the Same litter. The estimated granulation age of the illustrated islet is nineteen and
one-half days (see fig. 4, 8-day group, top of range). At the periphery of the illustrated
islet are nongranulated islet cells. The islet is almost surrounded by pancreatic ducts.
Aldehyde fuchsin, X 540.
12 Granulated beta cells in a n islet of a normal embryo of nineteen and one-half days.
The photograph also shows acini with zymogen granules. Aldehyde fuchsin, X 540.
156
PANCREATIC DEVELOPMENT IN VITRO
Michael R. Schweisthal, Maria P. CCas and Lemen J. Wells
PLATE 1
157
PLATE 2
EXPLANATION O F FIGURES
13 Granulated beta cells in an islet of a normal embryo of twenty and one-half days
(black). The photograph suggests the familiar pattern of the definitive islet in the rat
(definitive pattern has central beta cells and peripheral alpha cells). It is probable,
however, that the nongranulated cells at the periphery of the illustrated islet are forerunners of future cells of two types, alpha and beta. The islet is vascularized. Aldehyde
fuchsin, X 540.
14 Higher magnification of part of foregoing photograph (inked square in fig. 13), showing
additional details of the granulated beta cells. Aldehyde fuchsin, x 1,000.
15
Granulated beta cells in an islet of a ten-day culture from an explanted primordium of
thirteen and one-half days. This islet has an estimated granulation age of twenty-one
and one-half days (cf. fig. 4, 10-day group, top of range), but differs from a normal
islet of twenty-one and one-half days in that it lacks an internal network of capillaries.
The photograph also shows nongranulated islet cells (left) and four pancreatic ducts
(one at top right, three at bottom right). Not shown is the fact that this culture has
acini with zymogen granules. Aldehyde fuchsin, X 540.
16 Granulated beta cells in an islet of a normal embryo of twenty-one and one-half days
(embryo taken shortly before expected birth). The photograph also shows nongranulated islet cells (left), one pancreatic duct (bottom) and part of an acinus with zymogen
granules (right, above the number "16"). Aldehyde fuchsin, X 540.
17 Pancreatic acini in an eight-day culture from an explanted primordium of thirteen and
one-half days (one of the cultures in group 4 of table 4). These acini must have
appeared during the period of culture because acini were not present in the beginningcontrol primordium which was taken from a litter-mate embryo at the time of the
explantation (cf. figs. 5, 6). Zymogen granules, present in the acini of the culture,
are not visible in the photograph. The estimated developmental age of acini is seventeen and one-half days. The thin cellular membrane (top left) is of unknown origin:
its origin from mesothelium (embryonic peritoneum) would seem more likely than from
epithelium. Hematoxylin and eosin, X 540.
18 Acini (top and left), two small ducts and one islet (bottom) in pancreas of a normal
embryo of seventeen and one-half days. The islet is attached to the ducts from which
it seems to have originated. Hematoxylin and eosin, X 540.
158
PANCREATIC DEVELOPMENT I N VlTRO
Michael R. Schweisthal, Maria P. C6as and Lemen J. Wells
PLATE 2
159
PLATE 3
EXPLANATION O F FIGURES
19 Eight islets with granulated beta cells (arrows) in a ten-day culture from a n explanted
primordium of thirteen and one-half days. The estimated granulation age of the largest
illustrated islet is twenty-one and one-half days (cf. fig. 4, 10-day group, top of range).
Also shown are the ghosts of two strands of the rayon grid (center and bottom). Aldehyde fuchsin, x 125.
20 Granulated beta cells in a n islet of a six-day culture from a n explant of fifteen and
one-half days. The estimated granulation age of this islet is twenty-one and one-half
days (cf. fig. 2, 6-day group, top of range). Nongranulated islet cells more or less surround the cluster of granulated beta cells. Three ducts and half of another may be
seen in the photograph: two a t top left, half duct at top center and one duct at bottom
center. Aldehyde fuchsin, X 540.
21
Granulated beta cells in eight islets of a ten-day culture from a n explanted embryonic
dorsal pancreas of sixteen and one-half days. The estimated granulation age of the
largest islet is postnatal day 3 or prenatal (postcoital) day 24.5 (cf. fig. 1, 10-day group).
The photograph also shows several pancreatic ducts and the ghost of one filament of
the rayon grid (ghost below center and left of center). Aldehyde fuchsin, X 125.
22 Granulated beta cells in four islets of a normal rat of postnatal day 3. The photograph
also shows many pancreatic acini. Aldehyde fuchsin, X 125.
23 Acini, ducts and islets (arrows) in an eight-day culture from an explanted embryonic
dorsal pancreas of sixteen and one-half days. Not shown is the fact that the acini
have zymogen granules. The estimated developmental age of the acini is prenatal day
nineteen and one-half (see table 4 and fig. 12). At top left, the photograph shows in
part the upper (nongrid) surface of the culture. The photo shows the ghost of one
filament of the rayon grid (bottom right). Hematoxylin and eosin, x 125.
24
160
Zymogen granules in three acini of a n eight-day culture from a n explant of fifteen and
one-half days (acini arranged in form of a cloverleaf). The estimated developmental
age of the acini is nineteen and one-half days (see table 4 and fig. 12). Hematoxylin
and eosin, X 800.
PANCREATIC DEVELOPMENT IN VITRO
Michael R. Schweisthal, Maria P. CBas and Lemen J. Wells
PLATE 3
161
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