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Development of islets and acini from the explanted primordium of the rat embryo.

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Development of Islets and Acini from the Explanted
Primordium of the Pancreas of the R a t Embryo
MICHAEL R. SCHWEISTHALP LEMEN J. WELLS AND MARIA P. C f A S 3
Department of A n a t o m y , University of Minnesota, Minneapolis, Minnesota
ABSTRACT
In a n embryo of eleven and one-half to twelve and one-half days, the
stomach, duodenum, liver and primordium of the dorsal pancreas were excised en bloc,
placed on a rayon grid supported by a grid of stainless steel and cultivated on a liquid
medium of cock serum and extract of chick embryo for 8 or 10 or 12 days. The culture was incubated at 37°C i n air supplemented by controlled oxygen and carbon
dioxide.
One-hundred-fifteen cultures were fixed in Bouin's solution and the sections
stained with aldehyde fuchsin. Forty-one cultures were fixed i n Zenker's solution and
the sections stained with hematoxylin and eosin. The 269 cuntrol pancreases were of
two kinds: beginning controls taken at the time of the explantation (61 specimens);
reference controls taken during the period from day eleven and one-half postcoitum to
day five postpartum (208 specimens).
From an explant of a pancreatic primordium too primitive to have either islet or
acinus, a culture could give rise to islets with granulated beta cells and acini with
zymogen granules. The best-developed islet in a ten-day culture of a n explant from a
donor of eleven and one-half days had a n estimated granulation age of twenty-one and
one-half days; thus the differentiation in vitro ha2 kept pace with that which occurred
in viva during equal time in days. The best-developed acinus in a 12-day culture of a
pancreatic primordium from an embryo of 12 and one-half days had a n estimated developmental age of twenty and one-half days.
The developing pancreas in a rat embryo days had an estimated developmental age
of thirteen and one-half days is primitive in of twenty and one-half days (Schweisthal,
that it has small forerunners of a few is- Wells and Ckas, '64).
lets of Langerhans and cord-like forerunMATERIALS AND METHODS
ners of the future pancreatic ducts and
The
animals
used in this study were
acini. When it is cultivated in vitro for a
rats of the Sprague-Dawley strain (Simonperiod of ten days, it may differentiate into large islets with granulated beta cells sen Laboratories, Inc.). The age of a litter
and distinct acini with zymogen granules of embryos was reckoned from the moment of a witnessed mating.
(Schweisthal, C6as and Wells, '63).
At the beginning of an experiment, a litThe question arose as to whether a panter of embryos constituted an experimental
creatic primordium so primitive that it has
neither islet nor acinus might undergo sig- unit. From one embryo the developing
nificant differentiation in vitro. This ques- pancreas was removed, fixed and used as
a beginning control. From the other emtion was answered in the affirmative by the
bryos, the developing pancreases were exobservations to be recorded in the present
paper. Indeed, when such primordium planted and cultivated as organ cultures.
A block of tissue for an explant included
from a rat embryo of eleven and one-half
not only the primordium of the dorsal pandays is grown in organ culture for a period
creas but also the primordia of the duodeof ten days, it may give rise to islets with
granulated beta cells and to acini with num, liver, stomach and spleen (figs. 4,
zymogen granules. The best-developed 11).
1Aided by Grant HD 412 from the National Instiislet in a ten-day culture had an estimated
of Health.
granulation age of twenty-one and one-half tutes
2 Trainee in Anatomy, U.S.P.H.S. Present address:
Department of Anatomy, Medical College of Virginia,
days. The best-developed acinus in a 12- Richmond,
Virginia.
3 Presently conducting research
at the Instituto
day culture of a pancreatic primordium
Superiore de Sanita, Viale Regina Elena 299, Rome,
from an embryo of twelve and one-half Italy.
ANAT.REC., 151: 93-106.
93
94
MICHAEL R. SCHWEISTHAL, LEMEN J. WELLS AND MARIA P . CkAS
The watch-glass method of organ culture was used, and the essentials of it were
as follows. The medium was made from
equal parts of cock serum and chick-embryo extract. This extract was the supernatant fraction from one part of minced
embryo (12-day chick) and two parts of a
buffered Tyrode's solution (pH 7.4 2 0.2).
An explant was held on the surface of the
Acinar Granules in Pancreoses of 57 Normal Embryos
Poncreotic Islets in 114 Normol Embryos
Age of embryo i n doyr-plus h o u r i
Dot obove line. ~ r l e l i r lpresent
D o t below line islets ubsenl
. . . . .. . .
r!i"':..".....
I I doys
...
..
::;;;:
:
..j:i
: ' ' :* *
.
13doys
'
.. :::...
,
1
,
,
!
2
,
l
,
,
6
4
,
,
.!.:.i.
l
l
I
l
l
l
l
I
I
,
I
/
I
I
10 12 14 16 I8 2 0 2 2
8
Hours
Pancreatic Acini i n 59 Normol Embryos
Age O f embryo 8n doys-plui-hours
Do1 above line. ocini present
Dot below Ihne,ocinl obrenf
....
"!
13 days
:
f
I 5 days
I , I
2
0
2
4
6
,
,
8
,
,
:11:.
,
,
,
,
2
4
6
8
10 12 14 16 18 2 0 22
Fig. 3 Data showing that only six of the 31
embryos of about fourteen and one-half days had
large granules in the cytoplasm of the pancreatic
acini. Twenty-five of the 26 embryos of 15 days
and ten hours or older had such granules.
12days
0
0
Hours
: I
.. ..
.;I;
l6doys
1
,
1
,
,
,
1
/
,
10 12 14 16 18 20 2 2
Hours
Fig. 1 Data showing that in normal embryos
younger than 12 days the developing islets of
Langerhans had not appeared. In an embryo
of an age ranging from 12 days and 1 hr to 13
days and 0 hr, the pancreatic primordium had
either no primitive islet or no more than one
islet (see text).
Fig. 2 Data showing that in nine of ten embryos of about thirteen and one-half days the
developing pancreases did not have primitive
acini. In 48 of the 49 older embryos, the pancreases did have them.
liquid medium (0.5 ml) by a grid of rayon
acetate (upper platform) which was supported by a grid of stainless steel (lower
platform). Such culture-in-watch-glass was
kept in a covered Petri dish provided with
moistened cotton. The Petri dish was kept
in a closed desiccator and incubated at
37°C. Every day, for a period of one hour,
the air in the desiccator was supplemented
by a controlled amount of gas, a mixture
of 95% oxygen and 5% carbon dioxide.
Every second day, a culture was transferred to freshly-prepared medium. Antibiotics were not used at any time.4
Normal pancreases from litters of embryos not used as donors of explants were
used as reference controls. Those not
shown in figures 1 to 3 are recorded in
table 1.
In the preparation of sets of stained
serial sections of specimens, two combinations of fixatives and stains were used.
The first was Bouin's solution and aldehyde
fuchsin and Ponceau's counterstain. The
second was Zenker's solution and hematoxylin and eosin.
4This and certain other particulars of the techniques are consequences of courtesies of European
colleagues in whose laboratories Lemen J. Wells
studied i n 1960-61: Prof. Elio Borghese and staff
(Frascati), Prof. Etienne Wolff and staff (Nogent-surMarne) and Prof. Honor B. Fell and staff (Cambridge). In Cambridge, Prof. Fell and her group
supervised a preliminary study which confirmed the
report of Chen ('54) that the pancreatic primordium
of the rat embryo can undergo significant development on a liquid medium in vitro.
PANCREATIC ISLETS AND ACINI
TABLE 1
Control pancreases not recorded in figures 1 to 3
Pancreas
Aee of
r L in
days and
hours
Stain 1
AF
Hand
E
Embryos of days 171/z-21% postcoitum
17d and 12h
228A
X
17d and 12h
228B
X
17d and 12h
228c
X
17d and 12h
228D
X
17d and 12h
281
X
17d and 12h
282
X
17d and 12h
283
X
17d and 12h
284
X
17d and 16h
258
X
17d and 16h
259
X
246
18d and l l h
X
18d and l l h
247
X
18d and l l h
248
X
18d and l l h
249
X
19d and 13h
250
X
19d and 13h
251
X
252
19d and 13h
X
19d and 13h
253
X
254
20d and 12h
X
255
20d and 12h
X
256
20d and 13h
X
21d and 12h
680
X
21d and 12h
681
X
682
21d and 12h
X
21d and 12h
715
X
716
21d and 12h
X
717
21d and 15h
X
221c
21d and 15h
X
221E
21d and 15h
X
221F
21d and 15h
X
244
21d and 15h
X
245
21d and 15h
X
800
801
a02
803
823
899
895
Young rats of days 1-5 postpartum
X
Id
Id
X
2d
X
2d
X
3d
X
4d
X
5d
X
1.AF, aldehyde fuchsin; H and E, hematoxylin and
eosin.
The sections stained with aldehyde
fuchsin were used in estimating the granulation ages of islets in cultures, as follows.
The largest and best-developed islet of a
culture was selected for comparison with
the largest and best-developed islet of a
control pancreas. These selected islets
were compared with each other simultaneously in the field of a Bausch and Lomb
Microcomparator. If the islet of the control pancreas were more advanced or less
advanced than that of the culture, a new
islet of a control pancreas from an older
95
embryo or from a younger embryo was
brought into the field of the microcomparator for comparison. Thus, by trial and
error, the islet of a culture was suitably
matched with that of a control pancreas.
In the estimation of the granulation age of
a culture, two criteria were used: relative
size of islet and relative abundance of
granulated beta cells.
The sections stained with hematoxylin
and eosin were used in estimating the
acinar ages of cultures, as follows. The
largest and best-developed acinus of a culture was selected for comparison with the
largest and best-developed acinus of a control pancreas. These selected acini were
compared with each other simultaneously
in the field of the microcomparator. By a
method of trial and error, like that described in the foregoing paragraph devoted to islets, the acinus of a culture was
suitably matched with that of a control
pancreas. In the estimation of the acinar
age of 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).
Certain cultures were discarded because
they were not good enough to yield satisfactory data. Three of these had become
infected with bacteria, as demonstrated by
culturing samples of the media on slants
of nutrient agar. Thirtyeight cultures had
survived in part, but were discarded because they lacked islets with granulated
beta cells. Many other cultures had degenerated completely, some of them having crumbled and fallen through the
meshes of the grids. The degeneration not
attributed to bacterial infection seemed to
be largely a consequence of toxicity from a
film of precipitated soap on the glassware
used; the film originated from a preliminary wash with soapy tap water, and was
virtually impossible to remove. This temporary shortcoming of the technique eventually was rectified by preventing the formation of any film of precipitated soap.
In the perfected technique which yielded
the cultures to be considered, the glassware
was washed in soapy distilled water, and
all the soap was removed by means of several changes of boiling, glass-distilled
water.
96
MICHAEL R. SCHWEISTHAL, LEMEN J. WELLS AND MARIA P . C6AS
OBSERVATIONS
Controls. Two beginning control pancreases, numbers 1291 and 1301, were
chosen as type specimens for the purpose
of illustrating pancreatic primordia which
are too primitive to have islets and acini.
Pancreatic primordium 1291, from an embryo of eleven and one-half days (11 days
and 13 hours postcoitum), is illustrated in
figures 4 and 5. This primordium consisted
exclusively of the dorsal pancreas and had
three elements : epithelium, mesenchyme
and mesothelium (embryonic peritoneum).
Pancreatic primordium 1301, from an embryo of twelve and one-half days (12 days
and 15 hours), is illustrated in figures 11
and 12. It likewise was exclusively the
dorsal pancreas. It did not have any islet
(fig. 1). Its epithelial element had four
or five rows of cells, this being a consequence of an active proliferation of cells
(note mitotic figure in fig. 12).
Among 29 control pancreases taken
from embryos of ages varying from 12
days and 1 hr to 13 days and 0 hour, six
(including type specimen 1301) did not
have primitive islets of Langerhans; the
other 23 had from 1 to 3 primitive islets
per primordium of dorsal pancreas (fig. 1).
Each islet consisted of a few cells, about 2
to 10. In these cells, the cytoplasm was
highly eosinophilic.
Controls from embryos of thirteen and
one-half days had two regional portions per
pancreas, dorsal and ventral. They had
primitive islets (fig. l), the average number
per pancreas being about eight, as recorded
by Schweisthal, C6as and Wells ('63).
Nine of the ten pancreases from embryos
of thirteen and one-half days (fig. 2 of
present paper) did not have primitive
acini; the exceptional pancreas had only a
few bulbous swellings which had tiny lumina and which were regarded as primitive acini or as the forerunners of them.
Thirty-one controls from embryos of
fourteen and one-half days had primitive
islets, and 30 of these 31 had primitive
acini (fig. 2 ) . The islets did not have
granulated beta cells. Six of the 31 controls recorded in figure 3 showed large secretion granules in the cytoplasm of the
acinar cells.
Among 22 controls from embryos of
fifteen and one-half days, four were
stained with aldehyde fuchsin and 18 with
hematoxylin and eosin. In the four stained
with aldehyde fuchsin, each of two had a
single islet cell with purple secretion granules (single granulated beta cell) ; in each
of the other two, the cytoplasm was purplish but not granular. Eighteen of 18 controls stained with hematoxylin and eosin
had acini (fig. 2 ) . Fifteen of these 18 had
acinar granules, and one did not (fig. 3 ) ;
two of them had technical imperfections
which militated against the determination of presence or absence of granules,
and are not recorded in figure 3.
Six of seven controls from embryos of
sixteen and one-half days had one or two
granulated beta cells per specimen (aldehyde fuchsin); ten of ten controls had
granules in the cytoplasm of the acinar
cells (hematoxylin and eosin, fig. 3).
Among ten controls from embryos of
seventeen and one-half days, five were
stained with aldehyde fuchsin and five
with hamatoxylin and eosin (table 1).
Each control stained with aldehyde fuchsin had several granulated beta cells per
islet. Each stained with hematoxylin and
eosin had acini with secretion granules in
the cytoplasm (zymogen granules),
Among the 29 controls from rats killed
during the period from day seventeen and
one-half postcoitum to day five postpartum
(table l ) , four were photographed and
used for the purpose of recording the progressive development of the islets and
acini, as follows: control from embryo of
eighteen and one-half days, figure 8; control from embryo of twenty-one and onehalf days, figure 7; control from embryo
of nineteen and one-half days, Schweisthal, C6as and Wells' figure 12 ('63); control from young rat of day three postpartum, Schweisthal, C6as and Wells' figure
22 ('63).
Granulated beta cells in islets of cultures. The granulated beta cells in islets
were studied in two sets of cultures in
which the sections were stained with aldehyde fuchsin: set one, 84 cultures of explants from donors of about eleven and
one-half days; set two, 31 cultures of explants from donors of about twelve and
one-half days.
The 84 cultures of set one were obtained
from embryos of 25 litters, and each of the
97
PANCREATIC ISLETS AND ACINI
25 beginning control pancreases taken at
the time of the explantation - one control per litter - was too primitive to have
islets (figs. 4, 5; table 2). Not only the
granulated beta cells in the islets of cultures but the islets as well must have originated in vitro. The average granulation
ages of islets in cultures of 8, 10 and 12
days were, respectively 16.5, 18.2 and 16.8
days (fig. 16). The culture with the best-developed islets was one of the 75 cultures
of ten days, and its islets had an estimated
granulation age of 21.5 days (figs. 6, 7;
cf. printed explanation of fig. 16). The
data on the 74 other cultures of ten days
were as follows : two cultures, granulation age of 20.5 days; 12 cultures, 19.5
days; 22 cultures, 18.5 days; 34 cultures,
17.5 days; four cultures, 16.5 days (cf.
middle bar of fig. 16).
BETA GRANULATION IN 84 CULTURES FROM DONORS OF I l f DAYS
Tolol doyi of Culture
8
Corer with prDnUlel
2
Developmenla1 Odvonce. days 5 0
(Cult dev odY,do.
Canl.deuadv,da.)
63%
16
10
75
67
12
7
5.3
67x
44%
BETA GRANULATION I N 31 CULTURES FROM DONORS OF 12i DAYS
,
24.5 r
ToIoI days of cuIIur8
Cases With gmnular
17
8
12
Dcvelopmenlol~dvonee.doyr
5.7
10
20
62
4
6.0
(Cull dew. OdV do.
Con1:deu odv.;do)
71K
62%
50%
7
TABLE 2
Data regarding the 84 cultures shown in figure 16
No. of
cultures
1
1
Age of
donors of
explants
in days
and hours
Islet(s) in
beginningcontrol
pancreas
Two cultures of 8 days
l l d and 13h
l l d and 14h
7
14
16
19
2
2
12
2
Seventy-fivecultures of 10 days
l l d a n d 9h
l l d and 10h
l l d and l l h
l l d and 12h
l l d and 13h
l l d and 14h
l l d and 15h
l l d and 16h
l l d and 17h
4
3
Seven cultures of 12 days
l l d and 14h
l l d and 16h
1
0
0
0
0
0
0
0
0
0
0
0
0
0
Fig. 16 Combined bar graph and table, recording the estimated granulation ages of islets
in 84 cultures from donors of eleven and onehalf days. The tops of bars are 8, 10 and 12 days
from the baseline, i.e., from the age of donors
(11.5 days postcoitum). Concentrating on the
middle bar, the summit of this bar records the
granulation age of islets in control pancreases
from normal embryos of 21.5 days postcoitum.
Only one of the 75 cultures of ten days had
attained an estimated granulation age of 21.5
days (top of range); in this culture, the differentiation in vitro had kept pace with that in vivo
during equal time in days (explant of 11.5 days
plus ten-day period of culture equals 21.5 days).
Differentiation in most of the 75 cultures had
lagged behind that of control pancreases, the
average granulation age being 18.2 days, which,
in turn, represents a developmental advance of
6.7 days in ten days of culture. In other words,
this developmental advance in vitro was 67% of
that in vivo, both advances having occurred in
equal time, ten days. Turning to all three bars,
only one of the three average ages (18.2 days,
middle bar) is above the horizontal line of
dashes at 17.5 days postcoitum, the age at which
all control pancreases have islets with granulated
beta cells. The upper portion of the right bar
depicted by three lines of dashes extends from
21.5 to 23.5 days postcoitum, this period representing postnatal days 1 to 2 in normal embryos.
Fig. 17 Combined bar graph and table, recording the estimated granulation ages of islets
i n 31 cultures from donors of twelve and onehalf days. Note that all of the three average
ages (18.2, 18.7 and 18.5 days) are above the
horizontal line of dashes at 17.5 days postcoitum
(for significance of this line, see explanation of
fig. 16).
98
MICHAEL R. SCHWEISTHAL, LEMEN J. WELLS AND MARIA P. C f A S
The 31 cultures of set two were from
embryos of nine litters; four of the nine
beginning control pancreases taken at the
time of the explanatation - one control
per litter - had no islets (figs. 11, 12;
table 3); the other five beginning controls
had from 1 to 3 islets per pancreas - islets so primitive that granulated beta cells
had not appeared (see paragraph two in
the section devoted to controls). The granulated beta cells in the islets of cultures
must have originated in vitro. The average
granulation ages of islets in cultures of 8,
10 and 12 days were, respectively, 18.2,
18.7 and 18.5 days (fig. 17). The culture
with the best-developed islets was one of
the 20 cultures of ten days, and its islets
had an estimated granulation age of 21.5
days (figs. 13, 7; cf. figs. 11, 12; see footnote of table 3). The data on the 19 other
cultures of ten days were as follows: seven
cultures, granulation age of 19.5 days;
seven cultures, 18.5 days; four cultures,
17.5 days; one culture, 16.5 days (cf. middle bar in fig. 17). In 13 of 31 cultures
recorded in figure 17, not only the granulated beta cells but also the islets themselves must have originated in vitro be-
cause the beginning controls for these 13
cultures did not have any islets (table 3).
Nongranulated cells in islets of cultures.
Nongranulated cells were observed in the
islets of cultures stained with aldehyde
fuchsin. They were noted both in islets
which had granulated beta cells (figs. 6,
13) and in those which did not. Some of
them were beta-like cells in all respects
except the fact that they lacked purple seACINAR AGES IN 20CULTURES FROM DONORS
OF ll;
DAYS
( Cult
dev adv . d a
Con, dev adv.do)
55q'
59%
530/.
TABLE 3
Data regarding the 31 cultures shown in figure 17
ACINAR AGES IN 21 CULTURES FROM DONORS OF
No. of
cultures
Age of
donors of
explants
in days
and hours
Islet(s) in
beginningcontrol
pancreas
245
12i DAYS
r
~
2
2
1
1
1
Seven cultures of 8 days
12dand 6h
12dand 9h
12d and 13h
12d and 14h
12d and 15h
2
1
1
6l
2
2
6
Twenty cultures of 10 days
12dand 6h
12d and 12h
12d and 14h
12d and 15h
12d and 15h
12d and 16h
12d and 16h
1
1
2
Four cultures of 12 days
12d and 14h
12d and 15h
12d and 16h
+0
0
+
+
++
+0
+0
+
+
+
0
'One of these six cultures attained an estimated
granulation age of 21V2 days (fig. 17, top of range),
and is illustrated in figure 13; the beginning-control
pancreas (number 1301, see text) is illustrated in
figures 11 and 12.
Told days of ~ u l l u i e
8
Number of culluref
7
Developmenf~ladvance. days 5 4
19
(
Cult
Con,
dev o d v . d o
dw odv,do)
"%
10
I2
7
7
59
64
59q'
53q.
Fig. 18 Combined bar graph and table, recording the estimated developmental ages of
acini in 20 cultures from donors of eleven and
one-half days: The estimates were made in sections stained with hematoxylin and eosin.
Fig. 19 Combined bar graph and table, recording the estimated developmental ages of
acini in 21 cultures from donors of twelve and
one-half days. Estimates made in sections
stained with hematoxylin and eosin.
PANCREATIC ISLETS AND ACINI
99
cretion granules, hence were regarded as lease of insulin; granulated beta cells albeta cells which had released their secre- ready were present in the younger cultion granules into the culture medium (de- tures of eight days. Also, this view is in
granulated beta cells - see Di~cussion).~harmony with the reports that the relative
Acini in cultures. The acini were stud- abundance of granulated beta cells coried in two sets of cultures in which the relates well with the insulin content of the
sections were stained with hematoxylin pancreas (Wrenshall, Hartroft and Best,
and eosin: set one, 20 cultures from ’54); that pancreases of fetuses from diadonors of about eleven and one-half days; betic rats have enlarged islets of Langerset two, 21 cultures from donors of about hans with relatively few granulated beta
cells (Kim, Runge, Wells and Lazarow,
twelve and one-half days.
The 20 cultures of set one were ob- ’60); that the subjection of a guinea pig
tained from embryos of 12 litters in which to a prolonged, intravenous injection of
the 12 beginning control pancreases were glucose decreases the number of granutoo primitive to have acini; hence the acini les in the beta cell (Woerner, ’38); that
in cultures must have originated in vitro. the perfusion of glucose through the vesThe average developmental ages of acini sels of an isolated pancreas of the rat
in cultures of 8, 10 and 12 days were, causes the emerging perfusate to be rich
respectively, 15.9, 17.4 and 17.8 days (fig. in insulin (Anderson and Long, ’47); that
18). The data on the 11 cultures of ten cultivated pieces of pancreases from fetal
days were : three cultures, developmental rats of 17 days release insulin into the culage of 18.5 days - one specimen illus- ture medium (Murrell and Lazarow, ’63;
trated in figures 9 and 10; four cultures, cf. Coalson’s report, ’56).
17.5 days; four cultures, 16.5 days.
The large, eosinophilic granules in the
The 21 cultures of set two were obtained cytoplasm of acini of cultures are regarded
from embryos of nine litters in which the as granules of zymogen. This view is
nine beginning control pancreases were based exclusively upon morphological obtoo primitive to have acini; hence the servations which were not supplemented
acini in cultures must have originated in by chemical studies.
The growth of a culture, though slight,
vitro. The average developmental ages of
acini in cultures of 8, 10 and 12 days were, and the morphologic differentiation of the
respectively, 17.9, 18.4 and 18.9 days (fig. insular and acinar cells must have in19). The data on the seven cultures of 12 volved both the synthesis of proteins and
days were: one culture, developmental age the appearance of attendant, physicochemof 20.5 days - specimen illustrated in fig- ical changes. The synthesis of proteins reures 14 and 15; two cultures, 19.5 days; quired food which was furnished by the
three cultures, 18.5 days; one culture, culture medium. The morphologic differentiation of the insular and acinar cells
17.5 days.
might have required the action of embryDISCUSSION
onic organizers or inductors. If so, it is
It would seem that the purple secretion reasonable to assume that both the mesgranules in the beta cells of islets of cul- enchyme of the explant and the “juice”
tures represent stored insulin or pre-in- from the mesenchyme of the chick embryo
sulin, that they reflect the physiological served as embryonic organizers. It has
state of these cells at the moment of fixa- been reported that in a culture of dissocition and that certain beta-like cells without ated epithelium from the pancreas of a
purple granules represent genuine beta mouse embryo a dissociated mesenchyme
cells which had released their granules (in- which is separated from the epithelium by
sulin) into the culture medium (degranu- a micropore filter can act as embryonic orlated beta cells). Although this view was ganizer and induce the formation of pannot tested by assays of the medium for creatic acini (Golosow and Grobstein, ’62).
insulin, it is supported by the fact that It also has been reported that in a similar
the insular cells of 10-day and 12-day culThe islets of cultures were not subjected to stains
tures had Iived in vitro for considerable for alpha cells. alpha cells have been reported in the
pancreas of a’ normal rat of 48 hours after birth
time - presumably ample time for the re- (Hard, 44).
J
100
MICHAEL R. SCHWEISTHAL, LEMEN J. W E L L S AND MARIA P. C i A S
culture which lacks the dissociated mesenchyme but which is grown on a medium
rich in extract of chick embryo - rich in
juice from mesenchyme of the chick - the
formation of acini can occur (Grobstein,
'64).
The morphologic differentiation in our
cultures must have been preceded by physicochemical changes - changes which
have been called chemical differentiation
(Grobstein, '64) and which have been explored experimentally by means of radioautographs and metabolic inhibitors
(Wessells, '64). In cultures of epithelium
and mesenchyme from the pancreatic primordia of mouse embryos, tritiated thymidine was used to label the nuclei of the
developing acinar cells in radioautographs
(Wessells, '64). Two gradients were reported: a gradient of synthesis of desoxyribonucleic acid (DNA), the greatest number of labeled nuclei being at the periphery
of the culture; a gradient of formation of
acini, the active center of formation being
subperipheral (central). When epithelium
of a culture was cultivated with mesenchyme and the mesenchymal portion removed at the end of a period of 36 hours,
a continued cultivation of the epithelial
portion yielded acini with zymogen granules; thus, during the initial period of cultivation for 36 hours, a chemical differentiation of the epithelium had occurred.
When a metabolic inhibitor (5-bromodesoxyuridine) was added to the culture medium which was used during the initial
period of 36 hours, this inhibitor blocked
the Occurrence of chemodifferentiation as judged by the fact that a removal of
the mesenchymal portion of the culture
and a continued incubation of the epithelial portion on a medium without inhibitor
did not lead to the formation of acini with
zymogen granules. The manner in which
the metabolic inhibitor brought about this
blockade is not clear. Also, the manner in
which the synthesis of DNA was followed
by the formation of zymogen granules is
not clear. It is reasonable to assume, however, that messenger ribonucleic acid (messenger RNA) and ribosomes were involved
(cf. review by Hurwitz and Furth, '62).
The morphologic differentiation in our
cultures probably was preceded by morphologic changes which might have been
evident in cultures incubated for fewer
than eight days. Also, electronmicrographs of such cultures might have been
useful. Thus, in the pancreatic primordium of a normal mouse embryo of 11
days, electronmicrographs of sections seem
to confirm the existence of a few, primitive islet cells (Kallman and Grobstein,
'64). In the pancreas of a normal mouse
embryo of 13 to 14 days, electronmicrographs of the islet cells seem to demonstrate beta granules, despite the fact that
these cells were said to be too primitive to
show purple granules in sections stained
with aldehyde fuchsin and examined with
a light microscope (Munger, '58b). In the
pancreas of a normal mouse embryo of 15
days, electronmicrographs of the developing acini seem to exhibit prozymogen granules which could not be seen with a light
microscope (Munger, '58a).
Our experiments could be modified for
the study of a variety of important problems. Indeed, two new experiments already are in progress in the Department of
Anatomy.
LITERATURE CITED
Anderson, E., and J. A. Long 1947 Effects of
hyperglycemia on insulin secretion as determined by the isolated rat pancreas in a perfusion apparatus. Endocrinology, 40: 92-97.
Chen, J. M. 1954 The cultivation in fluid medium of organized liver, pancreas and other
tissues of foetal rats. Exp. Cell Res., 7: 518529.
Coalson, R. E. 1956 Functional and histological observations on islet tissue grown in vitro.
Anat. Rec., 24: 488 (abstract).
Golosow, N., and C. Grobstein 1962 Epitheliomesenchymal interaction in pancreatic morphogenesis. Develop. Biol., 4: 242-255.
Grobstein, C. 1964 Cytodifferentiation and its
controls. Science, 143: 643-650.
Hard, W. L. 1944 The origin and differentiation of the alpha and beta cells in the pancreatic islets of the rat. Am. J. Anat., 75; 369403.
Hunvitz, J., and J. J. Furth 1962 Messenger
RNA. Scientific American, 206: 41-49.
Kallman, F.,and C. Grobstein 1964 Fine structure of differentiationg pancreatic exocrine
cells in transfilter culture. J. Cell Biol., 20;
399413.
Kim, J. N.,W. Runge, L. J. Wells and A. Lazarow
1960 Pancreatic islets and blood sugars in
prenatal and postnatal offspring from diabetic
rats: beta granulation and glycogen infiltration. Anat. Rec., 138: 239-259.
Munger, B. L. 1958a A phase and electron microscopic study of cellular differentiation in
PANCREATIC ISLETS AND ACINI
pancreatic acinar cells of the mouse. Am. J.
Anat., 103: 1-33.
1958b A light and electron microscopic
study of cellular differentiation in the pancreatic islets of the mouse. Am. J. Anat., 103:
275-3 1 1.
Munell, L. R., and A. Lazarow 1963 Organ
cultures of fetal endocrine rat pancreas on liquid medium. Anat. Rec., 145: 264 (abstract).
Schweisthal, M. R., M. P. Cdas and L. J. Wells
1963 Development of the pancreas of the rat
embryo in vitro: islets and acini. Anat. Rec.,
147: 149-161.
101
Schweisthal, M. R., L. J. Wells and M. P. Cdas
1964 Organ culture of the pancreatic primordium of the rat embryo. Anat. Rec., 148: 333
( abstract).
Wessells, N. K. 1964 DNA synthesis, mitosis,
and differentiation in pancreatic acinar cells
in vitro. J. Cell Biol., 20: 415-433.
Woerner, C. A. 1938 Studies of the islands of
Langerhans after continuous intravenous injection of dextrose. Anat. Rec., 71: 33-57.
Wrenshall, G. A,, W. S. Hartroft and C. H. Best
1954 Insulin extractable from the pancreas
and islet cell histology. Diabetes, 3: 444-452.
PLATE 1
EXPLANATION OF FIGURES
4 Transverse section of a normal embryo of 11 days and 13 hours,
showing in part what usually constituted an explant from a donor of
this age, namely, foregut and outgrowths (rectangle in ink). Inside
the rectangle are three illustrated structures: developing duodenum
(middle), primordium of the dorsal pancreas (top) and hepatic diverticulum (bottom). Hematoxylin and eosin, X 40.
5
Higher magnification of the primordium of the dorsal pancreas shown
in figure 4, illustrating the three elements of this primordium, as
follows: (1 ) epithelium (horseshoe-shaped structure in the center of
the photograph), (2) mesothelium or embryonic peritoneum (right
and left portions of the photograph) and (3) mesenchyme (thick
layer between epithelium and mesothelium). x 540.
6
Section of a ten-day culture of an explant from a donor of 11 days
and 13 hours, showing an islet with granulated beta cells. The estimated granulation age of this islet is twenty-one and one-half days;
hence the differentiation in vitro had kept pace with that in vivo
during equal time in days (explant of 11% days plus ten-day period
of culture equals 21% days). At least two pancreatic ducts can be
seen, one in the middle of the left margin of the photograph, the
other to the observer’s left of the number “6.” The culture shown in
figure 6 and the pancreatic primordium shown in figures 4 and 5
were from two embryos of the same litter. Aldehyde fuchsin, x 540.
7 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 (bottom left) and a
portion of an acinus with zymogen granules (bottom right). Aldehyde fuchsin, x 540.
8
Section of developing pancreas from a normal embryo of eighteen
and one-half days, showing an islet (top left) which is adjacent to
three other structures: two ducts (top right) and one acinus (middle
right). Other acini can be seen in the bottom half of the photograph.
Hematoxylin and eosin, x 250.
9 Acini in a section of a ten-day culture of an explant from a donor
of 11 days and 11 hours. The estimated developmental age of the
acini is eighteen and one-half days (cf. acini in fig. 8). Hematoxylin
and eosin, X 250.
10 Higher magnification of the largest acinus in figure 9 (top left of
fig. 9 ) , showing zymogen granules in the cytoplasm of the acinar
cells. X 900.
102
PANCREATIC ISLETS A N D ACINI
Michael R. Schweisthal, Lemen J. Wells a n d Maria P. CBas
PLATE 1
103
PLATE 2
EXPLANATION O F FIGURES
11 Transverse section of a normal embryo of 12 days and 15 hours,
showing i n part what usually constituted a n explant from a donor
of this age, namely, duodenum, primordium of dorsal pancreas and
developing liver (rectangle i n ink). The right and left portions of
the primordium of the dorsal pancreas are not perfect mirror images
of each other because the illustrated section is somewhat oblique
(on observer’s left, note lateral bulge of this primordium). Hematoxylin and eosin, X 40.
12 Higher magnification of the primordium of the dorsal pancreas shown
in figure 11, illustrating the three elements of this primordium:
epithelium (much thicker on observer’s left than on right), mesothelium (right margin of photograph) and mesenchyme (between
epithelium and mesothelium). A small portion of liver also can be
seen (bottom right). x 540.
104
13
Section of a ten-day culture of a n explant from a donor of 12 days
and 15 hours, showing a n islet with granulated beta cells. The estimated granulation age of this islet is twenty-one and one-half days
(cf. fig. 7 ) ; hence the differentiation in vitro had lagged a bit behind
that in vivo during equal time in days (explant of 12% days plus
ten-day period of culture equals 22% days). Many nongranulated
islet cells also can be seen. The culture shown in figure 13 and the
pancreatic primordium shown in figures 11 and 12 were from two
embryos of the same litter. Aldehyde fuchsin, x 540.
14
Acini in a 12-day culture of a n explant from a donor of 12 days and
13 hours, showing zymogen granules in the cytoplasm of the acinar
cells. The estimated developmental age of the acini is twenty and
one-half days. Hematoxylin and eosin, x 400.
15
Higher magnification of two acini shown in figure 14, illustrating
still better the zymogen granules. x 900.
PANCREATIC ISLETS AND ACINI
Michael R. Schweisthal, Lemen J. Wells and Maria P. CBas
PLATE 2
105
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