J. Embryol. exp. Morph. Vol. 24, 2, pp. 335-355, 1970
Printed in Great Britain
335
The dependence of duodenal differentiation in
chick embryos on pars distalis hormones
By F. T. BELLWARE 1 AND T. W. BETZ1
From the Department of Biology, Carleton University
SUMMARY
Duodenal differentiation in normal chick embryos between 15-75 and 20-5 days of incubation was characterized by the following changes:
1. The dry weight increases from 4-1 to 12-4 mg.
2. The alkaline phosphatase activity increases from less than 12 to 426 units.
3. The length of the villi increases sixfold.
4. The height of the epithelial cells at the villous tips increases from 12-9 to 25-9 fi.
5. The shape of the mucosal cells changes from low columnar, to cuboidal, to high columnar.
6. The shape of the nuclei progresses from round to ovoid.
7. At first mitotic figures are distributed throughout the epithelium but become restricted
to the crypts of Lieberkiihn.
8. Cytoplasmic glycogen appears by day 15-75 and is mobilized by 20-5 days.
9. A mucopolysaccharide at the brush border of the mucosal cells progressively appears at
16-75 days and increases in amount.
10. Alkaline phosphatase activity (Gomori technique) at the brush border appears in low
levels at 16-75 days and becomes more intense.
11. Fresh body weights and third toe lengths at 19-75 and 20-5 days of incubation were
recorded as indices of body growth.
In 'hypophysectomized' embryos at 19-75-21-5 days of age:
12. The level of duodenal differentiation approximated that of normal .16-75—17-75 day
embryos.
13. In 'hypophysectomized' embryos which received a pars distalis chorioallantoic homograft at 9-5 days of incubation the duodena were normal.
14. In 'hypophysectomized' embryos with grafts which became atrophic the level of duodenal differentiation was not different from that of untreated 'hypophysectomized' embryos.
15. In chick embryos duodenal differentiation depends on pars distalis hormones.
INTRODUCTION
Several investigators, especially Moog and her co-workers in a series of
elegant studies, have described the major biochemical and morphological
changes which occur in the development of the duodenum of the chick embryo.
Apparently these changes are necessary for the functional differentiation of the
duodenum and include: (1) increases in duodenal length and diameter, (2)
delimitation of the villi from the pre-villous ridges and subsequent elongation,
1
Authors' address: Department of Biology, Carleton University, Colonel By Drive,
Ottawa 1, Canada.
336
F. T. BELLWARE AND T. W. BETZ
and (3) a transient accumulation of erythrocytes in the villous stroma. The epithelial cells clothing the villi change in size, shape, cytoplasmic density and basophilia. Their nuclei undergo changes in location and morphology. The distribution of mitotic figures and goblet cells as well as the appearance, distribution
and mobilization of cytoplasmic glycogen also change in characteristic ways. In
addition, the appearance and maturation of the microvilli of the brush border of
the mucosa cells parallel the appearance and increase in the amount of mucopolysaccharide and activity of alkaline phosphatase. These changes occur between
the 15th and 20th (last) day of incubation, presumably in preparation for the
ingestion of food after hatching (Hilton, 1902; Argeseanu & May, 1938; Moog,
1950, 1961; Moog & Wenger, 1952; Moog & Richardson, 1955; Kato, 1959;
Romanoff, 1960; Hinni & Watterson, 1963; Stocum, 1966; Penttila & Gripenberg, 1969).
Corticoids cause normal differentiation of the duodenum to occur precociously in vivo and in vitro (Moog & Richardson, 1955; Moog & Thomas, 1957;
Moog, 1958, 1959a, b, 1962; Hayes, 1965a, b; Overton, 1965) and ACTH has
the same effect in vivo (Moog & Ford, 1957). Thyroxine is apparently permissive
in conditioning the sensitivity of the duodenum to adrenocorticoids (Moog, 1961)
and encourages duodenal growth and villous morphogenesis (Stocum, 1966).
Accordingly, * hypophysectomy' (by partial decapitation) arrests duodenal
epithelial cell differentiation at a 16- to 17-day-old normal level (Hinni & Watterson, 1963) but only retards the rate of duodenal growth and villous morphogenesis. Also the activity of the major electrophoretic component of duodenal
alkaline phosphatase in partially decapitated chick embryos is reduced by 90%
(Manwell & Betz, 1966). The subnormal duodenal development in partially
decapitated embryos may be due to the absence of factors other than adenohypophyseal hormones (Hinni & Watterson, 1963). However, in other respects,
the effects of partial decapitation have been shown to be the same as if the embryos
had been hypophysectomized in the classical manner (Betz, 1967, 1968, 1970).
Hinni & Watterson (1963) reported that cortisone and thyroxine alone or
together apparently do not alleviate the arrested duodenal differentiation and
suggested since STH (somatotrophic hormone) is essential to maintain the
normal turnover rate of the duodenal mucosa of rats (Leblond & Carriere,
1955), it may also be necessary for normal duodenal differentiation in the chick
embryo. Hinni & Watterson (1963) review the important literature concerning
normal duodenal development, its hormonal modification and discussions of
the theoretical functional significance of these changes.
The gross duodenal morphology appeared normal in 'hypophysectomized'
chick embryos with a chorioallantoic graft of embryonic pars distal is gland
(Betz, 1967) but the details of the correction were not analysed. The results of
this study demonstrate that the pars distalis is essential for normal duodenal
growth and differentiation in chick embryos.
Duodenal differentiation and pars distalis
337
MATERIAL AND METHODS
Fertile eggs (Hyline strain 934 F) were selected at random and incubated without turning at 38 ± 0-5 °C and 60 ± 5 % relative humidity. There were four groups
in the experimental design: (1) normal embryos, (2) 'hypophysectomized'
embryos, (3) 'hypophysectomized' embryos which received a pars distalis
homograft, and (4) 'hypophysectomized' embryos with grafts which apparently
had not been vascularized and had atrophied. The embryos in group 1 received
no further treatment. Those for groups 2 and 3 were handled and 'hypophysectomized' by surgical partial decapitation at stage 11 as described previously
(Betz, 1967) except that in group 3 the hosts were 9-5 days old when they
received chorioallantoic grafts of partes distales from donor embryos which
ranged in age from 14 to 21 days. The experimental design, number of specimens
in each group and age of the embryos at sacrifice are summarized in Table 1.
At sacrifice the grafts were fixed in 3 % glutaraldehyde in 0-1 M phosphate
buffer, pH 70 for 12 h at 0 °C. To ensure that the condition of the embryos
in group 3 was due to hormones from the grafts and not from remnants of the
host's pars distalis, the heads were examined as before (Betz, 1965). The extraembryonic membranes and yolk sacs were removed and the embryos rinsed in
cold water and blotted. All heads were removed (Betz, 1967, 1968) to make the
bodies comparable (parts of the head are missing in operated embryos). The
headless bodies were weighed to the nearest 0 1 g and the length of the third
right toe (the two distal segments of the phalanyx including the claw tip) was
estimated to the nearest 0-5 mm. The duodenum was excised; the pancreas and
adherent fat removed and the apex of the loop was cut into several pieces
approximately 5 mm in length. Some pieces were fixed in glutaraldehyde as
above. Representative gut samples from 18-75-day-old normal embryos were
fixed in Bouin's fluid for 24 h at 22 °C. Samples from all embryos were fixed in
80% ethanol for 12 h at 4 °C for the localization of alkaline phosphatase
activity (Gomori, 1941). The tissues fixed in glutaraldehyde were dehydrated and
embedded in glycol methacrylate according to McCully (1966) and Feder &
O'Brien (1968). Thin sections, 0-5-2/* thick, were cut with glass knives on a
Porter-Blum, MT-1 Ultramicrotome and affixed to glass slides as described by
Feder & O'Brien (1968). The tissues fixed in Bouin's fluid and in 80 % ethanol
were processed for paraffin techniques and sectioned on a rotary microtome set
at 5//. The unfixed proximal and distal limbs of the duodena were immediately
frozen at - 1 5 °C, dehydrated in vacuo at room temperature for 12 h and stored
at - 15 °C no longer than 3 weeks. The storage time was kept constant within
and between experimental groups. The dried duodena were weighed to the
nearest 0-1 mg and homogenized in a glass system in glass-distilled water at 4 °C.
Alkaline phosphatase activity of the homogenates adjusted to a concentration
of 01 % was estimated by the method of Manning, Steinetz, Babson & Butler
(1966). Histological and histochemical observations were made after treating
338
F. T. BELLWARE AND T. W. BETZ
the sections with one of the following procedures or stains. (I) Acid fuchsin
and counterstained with toluidine blue as a general stain asdescribed by McCully
(1966). (2) Periodic acid-Schiff's reagent (PAS) for localization of glycogen and
mucopolysaccharides. Sections were placed in 1 % chlorous acid 20 min
(Rappay &Van Duijn, 1965); washed in tap water, 5 min; oxidized with 1 %
periodic acid, 10 min; placed in Schiff's reagent, 30 min; bleached in three
sulfite washes, 10 sec each; counterstained with 1 % fast green, pH 2-0, 2 min;
washed in tap water, dehydrated, cleared and mounted. Control sections were
pretreated for 30 min to 2 h with salivary amylase to remove glycogen. (3)
Gomori (1941) technique for localization of alkaline phosphatase activity.
Pieces of duodena fixed in 80 % ethanol at 4 °C were dehydrated in two changes of
absolute ethanol at 4 °C for 12 h each; cleared at room temperature in benzene
three changes, 1 h each; infiltrated and embedded with histowax (m.p. 52 °C).
Sections (5 /*) were mounted on slides, dried at 40 °C for 10 min, deparaffinized, cleared, hydrated and incubated 20 min in magnesium-free medium at
37 °C. (Hinni & Watterson, 1963). The sections were washed in cold tap water,
treated with 2 % cobalt nitrate, 5 min, washed in tap water; treated with dilute
ammonium sulfate (Gomori, 1952); washed in tap water; mounted in permount
and photographed 2 days later.
Representative sections were photographed. The lengths of the villi and the
heights of fourteen epithelial cells only at the tips of non-dilated villi were
estimated with an ocular micrometer from thin sections stained with acid
fuchsin-toluidine blue. The means, standard errors of the means of body weights,
third toe lengths, dried duodenal weights, units of phosphatase activity and
heights of the epithelial cells were calculated. References to similarities and
differences within and between experimental groups in the text are based on the
results of Student's t test, which was used to estimate significant differences
between the means, t values were chosen a t P = 005.
RESULTS
The gross morphology of the embryos in group 2 was defective but appeared
normal in group 3 except for the lack of the derivatives of the head parts resected
in the operation as described before (Betz, 1967, 1968). The embryos in group 4
were indistinguishable from untreated, partially decapitated embryos (group 2).
In all parameters (Tables 1-5) development is arrested at the 16th or 17th day
of incubation by ' hypophysectomy' and is restored to normal by one pars
distalis graft. In group 4 the developmental pattern is not different from that in
untreated ' hypophysectomized' embryos, indicating the effect exerted by the
grafts occurs only if the cells are viable. The histology of the duodena from
group 1 incubated 16-75, 17-75 and 19-75 to 21-5 days; group 2 incubated
19-75 to 21-5 days; groups 3 and 4 incubated 19-75 to 20-5 days are illustrated
by Figs. 1-27. The cytology of a pars distalis graft is shown in Fig. 8.
6
9
4
10
4
10
4
2
10
4
16-75
17-75
18-75
19-75
20-5
(hatching)
21-5
19-75
20-5
21-5
19-75
20-5
19-75
20-5
20-5
6
1
2
5
15-75*
14-2±l-5
120 ±0-9
ll-9±0-3
2-53, S
14-2 ±2-6
1-78, NS
13-6±0-3
14-8 ±0-4
10-9 + 0-5
= 0-48, NS
11-3 ±2-2
2-58, S
13-6±0-6
016, NS
13-8 ±1-2
1-5, S
160 ±0-7
5-97, S
230 ±0-6
Body
wt. (g)
12-5 ±0-2
13-6±O-9
13-7 ±0-6
11-9 ±0-6
12-1 ±1-0
t = 1 26, NS
10-5 ±0-5
603, S
13-7 + 0-3
1-89, NS
130±0-5
1-73, NS
14-4 + 0-4
Third
toe (mm)
12-1 ±1-3
4-6 ±0-9
4-3±l-3
6-5 ±0-3
40±l-4
3-8 ±0-3
4-1 ±1-0
/ = 0-42, NS
3-8+ 1-2
2-4, S
5-2 ±0-3
1-11, NS
5-7 ±0-4
1-14, NS
7-6 ±0-8
3-45, S
12-4±l-0
Dried
duodena
wt. (mg)
201 ± 3 1
1-61, NS
26-1 ±2-1
10-7±l-l
12-9 ±0-8
t = 1-15, NS
U-2±l-2
0-178, NS
ll-5±l-2
1-86, NS
15-6±l-6
3-23, S
21-3±0-8
1-24, NS
25-9 ±2-8
007, NS
26-2 ±1-8
12-1 ±0-9
316, S
15-2 ±0-4
villous tips (ji)
Height of
cells at
* 15-75 days of incubation = 15-75 x 24 h plus 3 h for thermal equilibration of the eggs.
t = values of t for means between successive days.
S = significant. NS = not significant. . = data not available.
'Hypophysectomized' plus
one atrophic graft (group 4)
'Hypophysectomized' plus
one graft (group 3)
' Hypophysectomized'
(group 2)
Normal (group 1)
Treatment
Days of
No. of
incubation specimens
(Mean values are given ± standard errors of the mean, followed by the /-test values in some cases.)
20
480 ±54
22±3
20±2
215 ±30
20±l
2400 ±200
21 ±2
21 ±2
2-4, S
29 ±2
4-2, S
74±18
2-5, S
188 ±28
4-5, S
426 ±53
< 12
Units of
activity/0-1%
homogenate
Table 1. Experimental design, numbers oj embryos, average weights oj the headless Jresh bodies, length oj the third toe, weight
oj dried duodena, height oj the epithelial cells at villous tips, and units oj alkaline phosphatase activity in the duodena oj the
chick embryos in groups 1, 2, 3 and 4
?^
u>
a
a
a
ss
a
f^
f^
^ ^
^•i
a
NJ
340
F. T. BELLWARE AND T. W. BETZ
In addition to the tabulated observations at 19-75 days the villi were apparently
more numerous than at 18-75 days and the stage of differentiation was more
variable than before or later. At 17-75 days of incubation amylase treatment of
Table 2. Comparison of experimental values in Table 1 (groups 2 and 3) with
normal mean values (group 1) by Student's t test1
Equivalent stage of normal development (group 1)
(days of incubation)
Parameter
Body weight
Toe length
Duodenal weight
Mucosal cell height
16-75
.17-75
G2-19*
G2-19
G2-19
G2-19
G2-20
G2-19
18-75
19-75
20-5
G2-20
G3-19
G3-19
G3-19
G3-19
G3-20
G3-20
1
All means were not significantly different from the normal mean at P = 005.
* G2-19: the average weight of the embryos in group 2 on day 19-75 of incubation was not
significantly greater than the normal body weight on day 16-75 of development.
Table 3. A summary of the changes in the characteristics of the
duodenal villi of the embryos in groups 1, 2, 3 and 4
Average
incuba- length Dilated
tion
(ji)
tips
Days
of
Treatment
Normal (group I)
15-75
16-75
17-75
18-75
19-75
20-5
(hatching)
21-5
'Hypophysectomized' (group 2) 19-75
20-5
21-5
19-75
'Hypophysectomized' plus
20-5
one graft (group 3)
'Hypophysectomized' plus one 19-75
atrophic graft (group 4)
100
250
250
300
420
+
++
+
+
600
0
600
250
250
250
400
600
250
0
0
++
++
++
+
+
_l_ _l_
RBC aggregations
Stroma
A few, small
More, larger
Same as 16-75 days
A few, smaller than before
Fewer, smaller than before
(now in vessels)
None
Compact
Compact
Compact
Loose
Loose
None
Same as 16-75+ normal
Same as before
Loose
Same as 15-75+ normal
Same as 19-75 normal
Same as 20-5 normal
Same as 16-75+ normal
Loose
Compact
Loose
Compact
Loose
Loose
Loose
thin sections was required for 2 h to remove the PAS-positive granules from the
normal mucosa cells but they were removed from thick, ethanol-fixed sections
after only 30 min. At 18-75 days of incubation perinuclear vacuolations were
common in the normal mucosa cells at the villous tips. There were low-intensity
Gomori reactions which did not change from 16-75 to 19-75 days of develop-
'Hypophysectomized' plus one
atrophic graft (group 4)
'Hypophysectomized' plus one
graft (group 3)
'Hypophysectomized' (group 2)
Normal (group 1)
Treatment
1+
6
4-6
Same
Crypts + bases
Same
Not villous tips
6
20-5
19-75
Ovoid
Round
Round
Round
Spheroovoid
Ovoid
Round
6
20-5
(hatching)
21-5
19-75
20-5
21-5
19-75
1+
1+
1+
Same as before
4-6
19-75
Same as before
Crypts + bases
Crypts + bases
Same
All over
Same as before
Only crypts
Spheroovoid
Ovoid
2-3
Same as before
Not villous tips
Not villous tips
Same
Only crypts
Crypts
Crypts + bases
Same as before
All but villous
tips
All over
All over
Not villous tips
Bases and crypts
Same as before
Goblet
cells
Round
Round
Round
Round
Mitotic
figures
1+
1+
1+
Nuclear
shape
15-75
16-75
17-75
18-75
Days of
Height/
incubation width
Distribution of
Mucosa cells
Present
Present
Present
Present
Present
Present
Present
Present
Present
Absent
Present
Present
Present
Brush
border
0
+ ++ +
+++
0
0
0
+++ +
+++ +
:
OJ
S-
^3
5!
<o
Si
<*•*.
S"
2S
+++
r^
+
^s
^^
si-
8-
s:
o
b
++
0
0
Gomori
reaction
Table 4. Summary of the changes in the characteristics of the duodenal mucosa cells of the embryos in groups 1, 2, 3 and 4
342
F. T. BELLWARE AND T. W. BETZ
ment in parts of normal duodena other than the free border. At 20-5 days there
were PAS-positive, amylase-fast vesicles in the brush border of many normal
mucosa cells.
Table 5. Summary of the amount, distribution and changes in the PAS reaction of
cytoplasmic granules and brush border of the mucosal cells of the embryos in
groups 1, 2, 3 and 4
PAS Reaction
Treatment
Normal (group 1)
' Hypophysectomized'
(group 2)
'Hypophysectomized' plus
one graft (group 3)
'Hypophysectomized' plus
one atrophic graft (group 4)
Granules (glycogen)
Brush border
A
A
Days
of
incubation
Amount
15-75
16-75
17-75
+
++
+++
18-75
+
19-75
Rare
0
20-5
(Hatching)
0
21-5
+
19-75
20-5
+
21-5
+
19-75
Rare
20-5
0
19-75
+
Distribution
Diffuse
Diffuse
Distal to
nucleus
Apical
Apical
Amylase sensitivity
Amylase
sensitivity
f
(thin
Thin
Thick
sections) Amount sections sections
Fast
i- labile
i- labile
0
+
+
Labile
Fast
Fast
Fast
Labile
Labile
0*
++
Labile
Fast
Fast
Fast
Fast
Fast
+++
+ + + + Fast
Diffuse
Diffuse
Diffuse
Apical
Diffuse
4 labile
4 labile
\ labile
+
0*
+
Labile
++
i labile
+++
+
Fast
Labile
Fast
Fast
Fast
Fast
Fast
m
Fast
Fast
Fast
Fast
* The brush border was 13 AS+ in the thicker Bouin's fixed sections.
In 'hypophysectomized' embryos at 19-75 and 20-5 days the stage of development did not differ from 16-75- and 17-75-day normal embryos except at 19-75
days there were perinuclear vacuolations and the frequency of villi distended with
red blood cells seemed greater. At 20-5 days the brush border was faintly PASpositive in ethanol-fixed sections (5 fi) but not in thin sections. By 21-5 days the
level of development was intermediate between the 15-75- and 16-75-day normal
levels. The cells in the established pars distalis grafts were indistinguishable
from normal (Fig. 8). Atrophic grafts were not sectioned.
DISCUSSION
This investigation determined that normal duodenal development occurred
only in the presence of pars distalis tissue, whether in situ or ectopically as a
Duodenal differentiation and pars distalis
343
graft. Most embryos were examined at 19-75 and 20-5 days of incubation (normally most landmarks of duodenal differentiation have occurred by this time)
because the differences between the normal and 'hypophysectomized' embryos
are so great correction would be obvious in group 3. In groups 2, 3 and 4 only a
few embryos were examined after 19-75 days due to the characteristic high
mortality in groups 2 (Betz, 1967, 1968) and 4. Also in group 3 the chorioallantoic circulation shuts off (day 20; before hatching), killing the grafts. Therefore the experiment is terminated because the source of hormones is lost. The
normal embryos hatched at 20-5 days of incubation. Thus 21-5 and 22-5 days of
'incubation' are the first and second post-hatching days in group 1. However,
in group 2 the embryos remain in the eggs (Betz, 1967). We used 14- to 21-dayold donors for pars distalis grafts as a source of hormones and did not detect
differences in duodenal development within groups in group 3. This may be
commensurate with the fact that the electrophoretic species of soluble proteins
in the pars distalis at hatching are almost the same as in the adult gland (Manwell & Betz, 1966). In addition partes distales, from even younger, 10-day-old
embryos correct or alleviate the defective development of partially decapitated
embryos (Betz, 1965, 1967, 1968), indicating secretory differentiation of the
pars distalis has occurred by that time or is synchronous with host development.
The pars distalis grafts effected normal duodenal differentiation in partially
decapitated embryos in the absence of the hypothalamus, epiphysis, pars nervosa and the brain parts of prosencephalic and anterior mesencephalic derivation. This demonstrates that these organs do not have important influences on
duodenal development.
The villi usually bend during growth; therefore the villous lengths were
approximated from the unbent villi. The extent to which fixation, embedding
and other procedural factors may affect the measurement of villus lengths was
not determined. Body weights and toe lengths were recorded as indices of body
growth which partially depends on pars distalis hormones (Betz, 1968, 1970).
In our results the absolute reduction and increase in body weight in groups
2 and 3 differ from the results of Betz (1967, 1968) for unknown reasons but the
difference between groups is almost equal. Our results indicate pars distalis
grafts in 'hypophysectomized' embryos can, but do not always, restore body
growth to normal. However, the between-group changes in toe length are in
accord with the results of Betz (1968). The slight subnormality of the dry duodenal weights in group 3 may be due to reduced albumen ingestion by the
operated embryos (Betz, 1968). The duodenal contents were not removed prior
to determining dry weights. However, the data of Coulombre & Coulombre
(1958), Hinni & Watterson (1963) and Stocum (1966) on the rate of duodenal
growth (increases in length and diameter) agree with our data. We did not
measure duodenal lengths and diameters because the peristaltic contractions
which occur before and during fixation change the length and diameter of the
intestine.
344
F. T. BELLWARE AND T. W. BETZ
Von Pap (1933) proposed the erythrocyte aggregations in the lamina propria
of the villi at 16-75 and 17-75 days of incubation arise de novo by erythropoiesis. He and Moog (1961) feel that they dilate the pre-villous ridges. Initially
EXPLANATION OF FIGURES 1-14
These photomicrographs are of thin cross-sections of the duodena of chick
embryos at various stages of development, except that Fig. 8 is a photomicrograph of
a section of a pars distalis homograft in the chorioallantois of a partially decapitated
chick embryo. All were stained with acid fuschin-toluidine blue except Figs. 9-13
which were treated with PAS and counterstained with fast green. All are magnified
x 380 except Figs. 8, 9, 13-14 which are x 1000, x 240 and x 100 respectively.
Fig. 1. 19-75-day-old normal embryo. The epithelial cells are columnar; the brush
border is well defined and the stroma is dense. 220 units of phosphatase activity.
Fig. 2. 19-75-day-old normal embryo. This section represents the least well developed duodenum examined at this stage of incubation. Note the apical vacuolation
of the cytoplasm (arrow). 150 units of phosphatase activity.
Fig. 3. 19-75-day-old 'hypophysectomized' embryo. The cells at the villous tips are
low columnar and the stroma is loose. Perinuclear vacuolations (arrow) are common. 18 units of phosphatase activity. The level of differentiation does not exceed day
16-75 normal (Fig. 7).
Fig. 4. 19-75-day 'hypophysectomized' embryo with a pars distalis graft. 220 units
of phosphatase activity.
Fig. 5. 19-75-day 'hypophysectomized' embryo with a pars distalis graft. The epithelial cells are columnar and the stroma is compact. 245 units of phosphatase
activity.
Fig. 6. 19-75-day 'hypophysectomized' embryo with an atrophic pars distalis graft.
Compare with the untreated'hypophysectomized'embryo (Fig. 3). 24 units of
phosphatase activity.
Fig. 7.16-75-day-old normal embryo. The epithelial cells (arrow) are cuboidal and the
stroma contains well developed erythrocyte aggregations. The cuboidal cells
average 11-2 /i in height. 21 units of phosphatase activity.
Fig. 8. Pars distalis homograft in the chorioallantois of a 20-5-day ' hypophysectomized' chick embryo. The cytology of the cells appears normal. Note the granular
cytoplasm (arrow).
Fig. 9. 19-75-day normal embryo. Goblet cells are near the tips of the villi (arrow)
and the brush border is intensely PAS-positive. 220 units of phosphatase activity.
Fig. 10. 19-75-day 'hypophysectomized' embryo. Note the erythrocyte aggregations
in the stroma and the PAS-positive granules in the cytoplasm of the epithelial cells
(arrow). 20 units of phosphatase activity.
Fig. 11. 19-75-day 'hypophysectomized' embryo with a pars distalis graft. The stage
of differentiation is not different from normal. 210 units of phosphatase activity.
Fig. 12. 16-75-day normal embryo. Note the localization of aggregates of PASpositive granules especially in the apical cytoplasm of the cell at the tips of the villi
(arrow). The brush border is faintly PAS-positive. 19 units of phosphatase activity.
Fig. 13. 19-75-day 'hypophysectomized' embryo. Note the distribution of PASpositive granules in the cytoplasm of the epithelial cells. Note the frequency and size
of erythrocyte aggregations (arrow). 20 units of phosphatase activity.
Fig. 14. 19-75-day 'hypophysectomized' embryo with a pars distalis graft. Note the
lack of large erythrocyte aggregations (arrow) and length of villi. 175 units of
phosphatase activity.
Duodenal differentiation and pars distalis
345
14
346
F. T. BELLWARE AND T. W. BETZ
Duodenal differentiation and pars distalis
347
the cells at the tips of the pre-villous ridges are low columnar but become cuboidal
or squamous after the red blood cell (RBC) aggregations develop. It is tempting
to ascribe the changes in shape of these cells to an increase in the mechanical
force supplied by the erythrocytes in accord with Moog (1961). The RBC
EXPLANATION OF FIGURES
15-27
These photomicrographs are of thin cross-sections of the duodena of chick
embryos in various stages of development. All were treated with PAS and counterstained with fast green except Figs. 18 and 26 which were stained with acid fuchsintoluidine blue and Figs. 21 and 22 which were treated with the Gomori technique.
The magnification is x 380 except Figs. 23 and 24 which are x 1000.
Fig. 15. 20-5-day normal embryo. Note the PAS-positive brush border, the absence
of cytoplasmic granules and the apical goblet cell (arrow). 350 units of phosphatase
activity.
Fig. .16. 20-5-day 'hypophysectomized' embryo. The pattern of distribution of PASpositive granules in the cytoplasm of epithelial cells at the tips of the villi (arrow) is
similar to 16-75-day normals, but the brush border is PAS-negative. 21 units of
phosphatase activity.
Fig. 17. 20-5-day 'hypophysectomized' embryo with an atrophic graft. Note
similarity to the 17-75-day normal (Fig. 19) and the 20-5-day hypophysectomized
embryo (Fig. 16). 20 units of phosphatase activity.
Fig. 18. 20-5-day 'hypophysectomized' embryo with a pars distalis graft. The stage
of cytological differentiation is more advanced than in 19-75-day normals. 400 units
of phosphatase activity.
Fig. 19.17-75-day normal embryo. Compare theamountand location of PAS-positive
granules (arrow) with Fig. 16. 26 units of phosphatase activity.
Fig. 20. 17-75-day normal embryo. The section was treated with salivary amylase
for 2h prior to PAS treatment. Note reduction of PAS-positive cytoplasmic
granules (arrow) compared to Fig. 19. Note the absence of a PAS-positive brush
border. 28 units of phosphatase activity.
Fig. 21. 20-5-day normal embryo. Note the heavy deposition of Gomori precipitate
showing alkaline phosphatase activity localization at the brush border of the epithelial cells (arrow) and its relative absence in other parts. Green filter in the light path.
395 units of phosphatase activity.
Fig. 22.20-5-day'hypophysectomized'embryo with a graft. Note similarity to Fig. 21.
(Compare area at arrow.) 420 units of phosphatase activity.
Fig. 23. 20-5-day normal embryo. Note the PAS-positive brush border and the
absence of cytoplasmic granules. Note also the goblet cell (arrow). 350 units of
phosphatase activity.
Fig. 24. 20-5-day 'hypophysectomized' embryo with a graft. The stage of differentiation is normal (cf. Fig. 23). 400 units of phosphatase activity.
Fig. 25. 21-5-day (hatched) normal chick. Note the number of goblet cells and the
intense PAS-positive brush border (arrow).
Fig. 26. 21-5-day 'hypophysectomized' embryo. Note the PAS-positive granules in
the cytoplasm of the cuboidal epithelial cells on the tips of the villi (arrow). 25 units
of phosphatase activity.
Fig. 27. 21-5-day 'hypophysectomized' embryo. The epithelial cells are cuboidal and
the stroma is filled with erythrocytes. These villi are less well developed than those
of the 17-75-day-old embryo (Fig. 19). 22 units of phosphatase activity.
348
F. T. BELLWARE AND T. W. BETZ
aggregations normally disappear and the villi become vascularized on the 17th
day of incubation (Von Pap, 1933). Thus, the increased frequency of villi with
RBC aggregations in the duodena of the 'hypophysectomized' embryos may
indicate the vascular development of the villi is subnormal as occurs in other
organs of 'hypophysectomized' embryos (Betz, 1967) or perhaps the period of
erythropoiesis is prolonged in these embryos.
The significance of the perinuclear vacuolations in the mucosal cells at 18-75
days is not apparent. However, they seem to be part of normal development
(Hinni & Watterson, 1963). In 'hypophysectomized' embryos at 19-75 and 20-5
days the epithelial cells have perinuclear vacuolations (a characteristic of
18-75-day normal cells) but in other respects they are like 16-75-day normal
epithelial cells, implying the cells are not as severely retarded. However, the
phosphatase activity does not exceed the 15-75-day level. The development of
the cytological and biochemical changes are not synchronous. This probably
indicates that they are not developmental prerequisites for each other nor
functionally interdependent but that individually they are affected differently
by the endocrine environment; this is in agreement with the results of others
(Moog & Nehari, 1954; Kato, 1959; Hayes, \965b). The PAS-positive amylasefast vesicles at the brush border on 20-5 days of incubation may be the lightmicroscopic equivalent of the vesiculations of microvilli which appear in vitro
if cortisone is added to the medium (Hayes, 1965Z?). If so they may be adrenocorticoid dependent in vivo. The heights of the mucosal cells reported by Hinni &
Watterson (1963) are lower than ours, perhaps due to differences in fixation. In
other respects, our results are in accord with those of others (Moog & Richardson, 1955; Moog, 1961; Hinni & Watterson, 1963) except for the location of
glycogen in the mucosal cells. It is impossible to correlate our results with their
reports since they did not observe the precautions of Pearse (1968) about glycogen fixation and we do not know how glutaraldehyde fixation affects glycogen.
Crude salivary amylase removed the PAS-positive granules from thick but not
thin sections unless incubation was extended to 2 h, perhaps contrary to expectation since there is less material in thin sections. Glutaraldehyde fixation may
retard enzymic hydrolysis or hold glycogen more strongly than other fixatives
especially since it causes proteins to cross-link (Swigart, Wagner & Atkinson,
1960). Also glycol methacrylate perhaps retards glycogen digestion. Further
studies may clarify this problem. Presumably glycogen is an energy source for the
synthesis of structural and enzymic proteins and is apparently mobilized by
20 days of development, perhaps because demand exceeds deposition (Moog,
1961; Hinni & Watterson, 1963).
In our slides the first consistent appearance of a PAS-positive, amylase-fast
material is at 19-75 days of incubation. Perhaps the difference between our
results and those above is due to the small amount of polysaccharide which
reacts with the PAS reagent in thin sections. The PAS-positive, amylase-fast
material at the border could not be detected on 15-75 and 18-75 days, perhaps
Duodenal differentiation and pars distalis
349
because the border contains another amylase-labile carbohydrate. In thick
sections there was enough mucopolysaccharide after the labile component was
removed to give a visible PAS reaction, but not in thin sections. Perhaps there is
less of this material on days 15-75 and 18-75. Moog & Wenger (1952) suggest
that the non-acidic mucopolysaccharide provides a cyto-skeletal framework
which orients the phosphatase molecules, thus providing a suitable environment
for activity and a finite number of enzyme accepting sites which limits enzyme
accumulation. A mucopolysaccharide is an integral part of the surface of the
microvilli (Ito, 1969; Millington, Critchley, Tovell & Pearson, 1969) and in
addition phosphatase molecules are apparently glyco-proteins (Portmann,
Rossier & Chardonnens, 1960).
We did not determine specific activities of alkaline phosphatase. Nevertheless
our results are in accord with those of Moog & Richardson, (1955) and Moog
(1961), who did determine specific activities. Activity expressed as units per mg
total duodenal protein is apparently not a more valid assessment of true activity
than units per mg dry duodenal weight, probably because the phosphatase is
mostly confined to the brush border (Overton, Eichholz & Crane, 1965; Eichholz, 1969) which is a small fraction of the total dry weight or protein. The duodenum does not contain adipose tissue which would contribute to the weight,
and the albumen ingested by chick embryos (Betz, 1968) would contribute to
the protein content and dry weight. Our pilot experiments demonstrated that
albumen has no significant alkaline phosphatase activity in accord with the
results of Manning et al. (1966).
We deleted magnesium ions from the medium in the Gomori method (1941,
1952) and incubated for 20 min at 37 °C in accordance with Hinni & Watterson
(1963), who reported the free border to be Gomori-negative prior to 17 days of
incubation. Moog's results (1961) concur even though she did not study embryos
younger than 17 days. Also she added magnesium to the medium and varied the
incubation time. With magnesium in the medium (Kabat & Furth, 1941)
phosphatase activity is demonstrable at the free border as early as 14 days
(Hancox & Hyslop, 1953). In our study duodena with less than 30 units of activity
would not yield enough Gomori-positive precipitate to be seen, which indicates
the resolving power of this modification of the Gomori technique with thin
sections is not very good. In spite of the controversy (Pearse, 1968) concerning
the validity of the Gomori technique for localizing alkaline phosphatase activity,
apparently this enzyme is localized in the outer membrane of the microvilli
(Clark, 1961; Holt & Miller, 1962; Goldfischer, Essner & Novikofif, 1964;
Overton & Shoup, 1964; Eichholz & Crane, 1965; Overton et al. 1965; Hugon &
Borgers, 1966; Mayahara, Hirano, Saito & Ogawa, 1967; Toner, 1968; Eichholz,
1969). The problem remains to show that the same localization occurs in vivo.
It has long been accepted that the microvilli are involved with absorption
because they increase the surface area of the cells (Toner, 1968). Clark (1961),
Overton & Shoup (1963, 1964) and Penttila & Gripenberg (1969) showed that
350
F. T. BELLWARE AND T. W. BETZ
microvillous maturation parallels alkaline phosphatase accumulation in the
brush border. The problem of correlating increases in enzymic activity and in
absorptive capacity has not been resolved although Hudson & Levin (1968) have
described the changes in electrical potential across the intestine of the chick
embryo during development in correlation with the transport of hexoses and
amino acids. Also Bogner (1961) demonstrated the absorption coefficient for
several sugars increases significantly in the chick intestine within three days after
hatching. The role played by alkaline phosphatase has not been elaborated,
although Tosteson, Blaustein & Moulton (1961) have suggested that it may act
as a 'sodium pump' coupled with the active uptake of sugars. However, the
physiological significance of the structural and biochemical changes which occur
during duodenal development remains obscure (Crane, 1965; Ugolev, 1965;
Toner, 1968; Korn, 1969).
Moog (1962) and Toner (1968) consider the progressive accumulation of
enzymes during development as evidence of adaptation to absorptive function.
Moog (1962) and Penttila & Gripenberg (1969) propose that the small intestine
passes suddenly from a prolonged non-functional state into active function and
point to the suggestive evidence of the upsurge in alkaline phosphatase activity
which always occurs shortly before function begins. Presumably in the chick this
means the duodenum begins to function at or shortly after the time of hatching.
The age at which the duodenum of the chick becomes functional is not known
but between 11 and 15 days of incubation albumen is ingested, digested and
utilized (Witschi, 1949; Carinci & Manzoli-Guidotti, 1968; Hinsch, 1967;
Betz, 1970) and if chick embryos do not ingest albumen they are dwarfed (Betz,
1968). The proventricular glands begin to secrete acid on day 13 and show
histochemical signs of heightened secretory activity on days 14 and 15 (Toner,
1965; Hinsch, 1967). This in addition to the results of Hudson & Levin (1968)
indicates the intestine functions, at least to some extent, before the changes which
have been associated with 'functional' differentiation of the duodenum by Moog
and her co-workers Hinni & Watterson (1963) and Baxter-Grillo (\969a, b)
have progressed very far. Thus, the developmental changes in the duodenum are
probably not prerequisites for absorption, digestion or transport but they may
be necessary to support the increased rates of function after hatching. It seems
unlikely but the ingested albumen may be stored in the gut until functional
onset or the digestion products may be absorbed in a more posterior part of the
small intestine.
This study demonstrates the indispensability of pars distalis hormones in
normal duodenal differentiation in the chick embryo during the last 4 days of
development. The developmental rate of the duodenum in 'hypophysectomized'
embryos apparently does not simply slow down because differentiation is
arrested at the 16-75- or 17-75-day level, even if incubation is prolonged for
2 or as many as 6 days beyond the time of hatching (Hinni & Watterson, 1963).
One or more pars distalis hormones probably affect duodenal differentiation
Duodenal differentiation and pars distalis
351
directly (perhaps STH, prolactin and/or ACTH) or ACTH and TSH may
stimulate the synthesis of adrenocorticoids and thyroxine (Betz, 1970). Betz
(1965, 1967, 1968) demonstrated that pars distalis grafts improve the subnormal
growth of' hypophysectomized' embryos, perhaps indicating that the embryonic
pars distalis secretes STH or prolactin or both. Also Bates, Miller & Garrison
(1962) have demonstrated that bovine growth hormone and ovine prolactin
have a splanchnotropic effect in hypophysectomized pigeons.
The possibility that STH is essential for duodenal differentiation in the chick
embryo is consistent with the results of Leblond & Carriere (1955), and Turner
(1966) points out that STH and thyroxine are complementary in many species
and may be essential for the full expression of other hormones. Also a growthpromoting activity (bio-assayed in Rana temporaria larvae) which appears on the
15th day of incubation is confined to the caudal region of the pars distalis in the
chick embryo (Enemar, 1967). Presumably, STH rather than prolactin is responsible for this growth-promoting activity. See review of Betz (1970). However,
the time at which STH may be released in vivo in amounts which may stimulate
duodenal development is not known. It is tempting to suggest the upsurge in the
rate of duodenal development at 18 days of development coincides with the
release of STH and perhaps other hormones. Other interpretations are possible.
Testosterone (Carriere, 1966) and estrogen (Bullough, 1946) can stimulate
mucosal mitogenesis. The effects of sex steroids on duodenal differentiation
have not been reported but may not be inconsiderable since the pars distalis—
gonadal axis probably becomes established at least by 13-5 days of incubation
(Woods & Weeks, 1969). However, there was no apparent sexual dimorphism
in duodenal development in normal embryos. A more precise analysis of the
hormones involved in duodenal differentiation in chick embryos is in progress.
RESUME
Influence des hormones de la 'pars distalis' sur la differenciation duodenale,
chez Vembryon de poulet
La differenciation duodenale chez les embryons de poulet normaux entre 15,75 et 20,5 jours
d'incubation est caracterisee par les changements suivants:
1. Le poids sec augmente de 4,1 a 12,4 mg.
2. L'activite de la phosphatase alcaline croit de moins de 12 a 426 unites.
3. La longueur des villosites augmente de six fois.
4. La hauteur de cellules epitheliales aux extremites des villosites augmente de 12,9 a 25,9 ju,.
5. Les cellules muqueuses sont d'abord cylindriques et courtes, puis cubiques, puis cylindriques et elevees.
6. Les noyaux, d'abord spheriques, deviennent ovoides.
7. Les figures mitotiques, initialement distributes sur tout Pepithelium, s'observent par la
suite uniquement sur les cryptes de Lieberkiihn.
8. Le glycogene cytoplasmique, apparait apres 15,75 jours et est mobilise apres 20,5 jours.
9. Un mucopolysaccharide apparait a la bordure en brosse des cellules muqueuses apres
16,75 jours et augmente progressivement.
352
F. T. BELLWARE AND T. W. BETZ
10. L'activite de la phosphatase alcaline, mise en evidence par la technique de Gomori,
appratt faiblement au niveau de la bordure en brosse, apres 16,75 jours et devient ensuite plus
intense.
11. Le poids frais et la longueur du troisieme orteil apres 19,75 et 20,5 jours d'incubation
ont ete pris comme indices de la croissance.
Dans les embryons 'hypophysectomises', apres 19,75 et 21,5 jours:
12. Le niveau de la differenciation duodenale est le meme que celui d'embryons normaux
ages de 16,75 a 17,75 jours.
13. Les embryons 'hypophysectomises', chez qui un greffon de la pars distalis chorioallantoidienne a ete implante, presentent un duodenum normal.
14. Chez les embryons' hypophysectomises' ou les greffons s'etaient atrophies, le niveau de
la differenciation duodenale est similaire a celui des embryons 'hypophysectomises,' non
traites.
15. Chez les embryons de poulet, la differenciation duodenale depend des hormones de la
'pars distalis'.
This work was supported by a grant to T. W. Betz from the National Research Council of
Canada.
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{Manuscript received 20 October 1969, revised 18 February 1970)
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