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DEVELOPMENT AND HISTOGENESIS OF THE AVIAN
DIGESTIVE TRACT WITH SPECIAL REFERENCE
TO THE HISTOCHEMISTRY OF THE

CONNECTIVE TISSUE

By

Pierson J..!§n Alten

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Zoology
Year 1955

Approved /t¥/?“2%FV""é§f:‘

 

THE-251$

 

Pierson J. Van Alten

THESIS ABSTRACT

Specimens used in this study were obtained from single-
comb white leghorn hatching eggs which were incubated from
11 to 20 days. The digestive tract was removed from the
fetuses and the histogenesis of the alimentary canal and his-
tochemistry cf the connective tissues within it were studied.
Histogenesis was studied in tissues which were fixed in
Bouin‘s solution, sectioned at seven microns and stained in
one of the following: hematoxylin and eosin, Mallory's
triple stain, Masson's trichrome stain, Weigert's elastic
tissue stain, Dempsey's orcein procedure, and Van Giesen's
collagenous tissue stain. The histochemistry of connective
tissues was studied with the Schiff procedure of Hotchkiss
(1948) and with selective staining by toluidine blue at var-
ious pH levels. Basic proteins were also used to determine
what effect they had on selective staining of mucopoly-
saccharides by basic dye.

Comparison of the geometric growth in weight of the
fetus and alimentary canal indicated that the alimentary
canal was growing at a faster geometric rate than was the
fetus between the 11th and 20th day. This was based on

weights of approximately 270 fetuses and digestive tracts.

2

Pierson J. Van Alten

It was demonstrated that the epithelial layer was the
first distinct layer to differentiate. The circular muscle
fibers of the external muscular layer were the next tissue
to differentiate. The lamina propria and submucosa differen-
tiated after the muscle layer and the muscularis mucosae was
the last layer to differentiate. The epithelium of the
esophagus differentiated into stratified squamous epithelium
and the epithelium of the remainder of the tract differen-
tiated into simple columnar. The muscularis mucosae and the
lateral muscle fibers of the muscularis externa were absent
in the gizzard.

The Schiff procedure for soluble polysaccharides was
positive in all tissues of all ages of fetuses studied. The
staining reaction was diminished but not completely abolished
if tissues were treated with saliva before oxidation with
periodic acid. This positive reaction soluble in saliva was
attributed to glycogen. The connective tissues were found to
stain with the Schiff reagent for insoluble polysaccharides.
The staining of these tissues was not diminished by treatment
with saliva and it was concluded that they were "neutral"
polysaccharides.

The connective tissues of the gut stained pink (meta-
chromatic) with toluidine blue at pH 5.5 and 8.5 and blue
(basophilic) at pH 2.5 and 3.5. Basic proteins, albumin

and globulin, inhibited staining at pH 3.5. Protamine sulfate

3
Pierson J. Van Alten

inhibited staining with basic dye at pH 5.5. Metachromasia
persisted in connective tissues after treatment with
ribonuclease.

It was concluded that connective tissues of the diges-

tive tract of the fetus contained "neutral" polysaccharides

and acid mucopolysaccharides.

DEVELOPEENT AND HISTOGENESIS OF THE AVIAN
DIGESTIVE TRACT WITH SPECIAL REFERENCE
TO THE HISTOCHEMISTRY OF THE

CONNECTIVE TISSUE

By
Pierson J. Van Alten

A THESIS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Zoology
1955

ACKNOWLEDGEMENTS

The author wishes to express his sincere thanks to
Dr. R. A. Fennell for his suggestion of the research prob-
lem and also for his constant assistance, direction, and
interest during the course of this research.

Grateful thanks is also due Dr. K. A. Stiles, head of
zoology department, for approving the study and allocating
the funds necessary for carrying on this investigation.

The author is also indebted to Dr. D. W. Hayne,

Dr. T. W. Porter, and Mrs. B. McCarthy Henderson, who have
been helpful in their suggestions.

Thanks is also due Mr. Joseph G. Engemann, graduate
assistant, zoology department, for the photomicrographs.

The final acknowledgement is made to my wife, Lucille
Van Alten, without whose helpful advice and encouragement

this study could not have been conducted.

TABLE OF CONTENTS

Section

I
II

III

IV

VI

INTRODUCTION 0 o o o o o o o o o o o o o o o o
BCATERIALS AND IIETHODS . o o o o o o o o o o o 0
RESULTS,

1. Geometric growth of the fetus and alimentary

canal O O O O O O O O O O O O O O O O O O

2. Gross morphology of the alimentary canal in

the eleven-day fetLIS. o o o o o o o o o o

3. Gross morphology of the alimentary canal of

the twenty-day fetus. . . . . . . . . . .

4. The histogenesis of the digestive tract 0
the various ages of the fetuses . . . . .

5. Study of the metachromatic staining elements

in the digestive tract of chick fetuses b
the use of toluidine blue, "pH signatures
proteins, and ribonuclease. . . . . . . .

6. The periodic acid-Schiff test applied to
tissues of the digestive tract of chick
fetuses . . . . . . . . . . . . . . . . .

DISCUSSION

1. Growth and histogenesis of the digestive
traCt O 0 O O O O O O O O O O 0 O O O O O

2. Histochemical studies of the connective
tissues 0 0 O O O O O O O O O O O O O O O

S [IL/HUM Y O O O O O O O O O O O O O O O O O I O 0

LI TERATIIRE CI TED O O O O O 0 O O O O O O O 0 O

f

E!

l2

l2

19

2O

21

35

44

49

52

59

LIST OF TEXT-FIGURES

Text-figure

1 Comparison of the growth rate of the fetus
and the alimentary canal. . . . . . . . . . . . l7

Table

The Mean Weight
Age From Eleven

The Mean Weight
Fetuses Ranging

LIST OF TABLES

of Chick Fetuses Ranging in

to Twenty Days of Incubation .

of the Alimentary Canal of
in Age From Eleven to Twenty

Days of Incubation .

Geometric Rate of Growth

Measurements (in Microns) of Tissues in the

Esophagus of Developing Chick Fetuses.

The Influence of pH and Various Proteins Upon
Staining By Toluidine Blue in Developing

Chick Tissues.

O

l4

l5
16

26

37

 

II

III

IV

Fig. 9.

Fig.10.

LIST OF PLATES

Total fetuses (including yolk sac)

ranging in age from 20 to 15 days of

incubation; and

total fetuses (including yolk sac)

ranging in age from 14 to 11 days of

incubation.

Alimentary canals removed from fetuses

ranging in age from 20 to 12 days of

incubation.

Entire alimentary canal of a 20-day

fetus with the jejunum-ileum uncoiled.

Cross section through the esophagus of

an ll-day fetus;

cross section through the
an 11-day fetus;

cross section through the
a 16-day fetus;

cross section through the
an 18-day fetus;

cross section through the
a 20-day fetus; and

cross section through the
a 19-day fetus.

esophagus

esophagus

esophagus

esophagus

esophagus

of

of

of

of

of

63

65

67

69

 

VI

VII

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

13.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

LIST OF PLATES (Cont.)

. . . . . . . . . . . . . . . . . . . . 71

Cross section through the esophagus of
a 20-day fetus;

cross section through the proventriculus
of an ll-day fetus;

cross section through the proventriculus
of a 15-day fetus;

cross section through the proventriculus
of an 18-day fetus;

cross section through the proventriculus
of a 20-day fetus; and

cross section through the gizzard of an
ll-day fetus.

. . . . . . . . . . . . . . . . . . . . 73

Cross section through the gizzard of an
ll-day fetus (showing cilia);

cross section through the gizzard of a
15-day fetus;

cross section through the gizzard of an
18-day fetus;

cross section through the gizzard of a
20-day fetus;

cross section through the small intestine
of a 12-day fetus; and

cross section through the small intestine
of a 17-day fetus.

. . . . . . . . . . . . . . . . . . . . 75

Cross section through the small intestine
of a 20-day fetus;

 

Fig.

Fig.

Fig.

24.

25.

26.

LIST OF PLATES (Cont.)

cross section through the large intestine
of an ll-day fetus;

cross section through the large intestine
of a 14-day fetus; and

cross section through the large intestine
of a 20-day fetus.

INTRODUCTION

Calhoun (1954) extensively reviewed the literature and
described in detail variouscells and tissues in the avian
digestive tract (Galus domesticus) in specimens from 36 hours
subsequent to hatching to 2 years of age. Other workers have
made noteworthy contributions to the histology and gross
anatomy of the avian digestive tract. McLeod (1939), Bradley
and Grahame (1951) and Sturkie (1954) limited their descrip-
tion to the microscopic anatomy of adult birds, while on the
other hand, Kaupp (1918) limited his description to the gross
anatomy of the tract.

Other species have been used to limited extent for in-
vestigations of this type. Kendall (1947) described the
histological characteristics of the digestive tract in the
adult pigeon. Rosenberg (1941) identified chief cells,
mucous cells, and basal granular cells in the duodenal epi-
thelium of the adult turkey. An abundant number of basal
granular cells were also found in the tunica propia mucosa.

Embryology and organogenesis of the chicken digestive
tract has been studied in great detail. Patten (1952) sum-
marized and described the development of the tract in embryos
from fertilization of the ovum through 96 hours incubation.
Hamilton (1952) described both early development and organo-

genesis of the tract in various ages of fetuses.

Schumacher (1926) limited his study to the origin and
development of the epithelium and esophageal glands in
fetuses ranging in age from 5 to 21 days. Esophageal epi-
thelium was classified as columnar at 5 days, cuboidal at 9
days, and stratified at about 14 days. Glands arose as solid
outgrowths of epithelium which acquired a lumen by coalescence
and confluence of intercellular vacuoles. More recently,
Ivey and Edgar (l952)confirmed Schumacher's observations and
extended the study with a description of the development of
the lamina propia mucosa and the lamina muscularis mucosae.
Mayberry (1935) found that the distal end of the esophagus
evaginated to form a recognizable crop in 7-day fetuses and
that in 9-day fetuses its diameter was increased about 8
times. In 10-day fetuses the wall of the crop was thinner;
growth rate was retarded but it continued through 17 days of
incubation. Dilation of the crop on the right side was pro-
nounced on the 18th day of incubation. Epithelium lining the
crop consisted of three layers of cuboidal cells (7th day of
incubation); fully formed glands appeared on the 17th day of
incubation and stratified squamous epithelium was recogniz-
able on the 18th day of incubation.

Formation of the mucous and epithelial layers of the pro-
ventriculus and gizzard was described by Hibbard (1942).
Glands appeared in the proventriculus on the 7th day of incu-

bation, and the distal ends of the glands forked to form

simple branched tubular glands on the 10th day of incubation.
Compound and tubular glands with mucus secreting cells were
recognized on the 18th day of incubation and the glandular
layer was doubled by the time of hatching. On the 9th day of
incubation epithelial cells lining the gizzard were arranged
to form a stratified-like, columnar layer of tissue. On the
11th day of incubation a coagulum-like secretion appeared on
the distal surface of the epithelial cells. Secretory mate-
rial was first identified in the lumen of the tubular glands
on the 14th day of incubation. Keratinoid materials which
were produced by the glands rapidly increased to form as a
distinct layer on the epithelial surface in 18-day fetuses.

Innervation of the avian digestive tract was described
by Van Campenhout (1933). In the 6-day fetus the esophagus,
proventriculus, and gizzard were innervated by the pneumogas-
tric nerve and the sympathetic chain; the duodenum and remain-
der of the small intestine was innervated by the preaortic
plexus, and the large intestine by the nerve of Remak.

Minot (1900) found that in 4-day fetuses the mesoderm
of the large intestine was not differentiated into layers.
In 6-day fetuses the epithelium lining the large intestine
proliferated rapidly to occlude the lumen of the intestine.
On the 9th day of incubation the distal end of the rectum

was in contact with the epithelium of the proctodaeum and

differentiation of the muscular layers in the large intestine
was beginning.

Boyden (1922) found that the cloaca developed from aggre-
gates of disintegrating cells in the tail-bud region of 41-50
somite fetuses. Separation of the cloaca was initiated dur-
ing degeneration of these cells and completed on the 4th day
of incubation. The bursa appeared as a swollen region on the
caudal wall of the cloaca. It developed from coalesced vac-
uoles within the urodaeal membrane between the 5th and 6th
day of incubation. Boyden (1924) found that the cloaca con-
sisted of an anterior coprodaeum, a middle urodaeum and a
posterior proctodaeum. Development of the urodaeum was in-
duced when the Wolffian duct contacted the lateral wall of
the cloaca.

The cytology of the various portions of the digestive
tract of the chick has been studied by some investigators.
Chbéuiik' (1947) found that the golgi apparatus in epithelial
cells of the proventriculus was located between the nucleus
and lumen of the gland cells. However, in zymogenic cells
it was identified in the vicinity of the intercellular clefts.
Mitochondria appeared as delicate filaments in surface epi-
thelium and in the neck cells, and as thick rods and granules
in the zymogenic cells. Secretory granules arose in close
association with the golgi material. In the gizzard, the

golgi apparatus did not change subsequent to feeding.

Golgi bodies in intestinal epithelial cells, subsequent to
feeding, moved from the proximal to the distal surface of
the cells, and secretory granules increased in number and
were arranged in the meshes and on the surface of the golgi
apparatus. The mitochondria appeared as filaments, rods,
and granules and were in constant polar arrangement. In the
goblet cells the golgi material was distal to the nucleus
and the mitochondria collected on the border of the mucous
mass and in the cytoplasm of the narrow part of the cell.
Previously, Hibbard (1942) had described the golgi appara-
tus in the epithelial layer of the proventriculus and giz-
zard of the embryonic chick as an aqueous vacuole which is
an artefact. Argeseanu and May (1938) showed that chondri-
osomes were filamentus and uniformally distributed in the
cytoplasm of embryonic syncytial cells. In adult chickens
chondriosomes were concentrated and formed aggregates at the
distal end of the epithelial cells. The supra-nuclear chon-
driosomes remained in the vicinity of the nucleus throughout
the developmental period. Simard and Van Campenhout (1932)
observed that argentaffin cells appeared in the intestine on
the 11th day of incubation and they were first identified in
the epithelium near the umbilical pedical and ultimately
migrated into the submucosa. It was also demonstrated by
chorio-allantoic grafts that differentiation of argentaffin

cells occurs even in the absence of nervous elements, but

there was a hypertrophic development of connective tissue
and muscular tissue was reduced and this was attributed to
the absence of nervous elements. Dawson and Moyer (1948)
identified argentophile cells in the epithelium of the chick
proventriculus on the 8th day of incubation. The number of
argentophile cells reached a maximum on the 13th day of in-
cubation. They first appeared in the compound glands on the
12th day and in the gizzard on the 18th day.

Huxley (1932) and Weiss (1939) used Schmalhausen's data
and gave mathematical formulation of the relative growth
between some digestive organs and the total chick. They made
the interesting observation that some of the digestive organs
were growing at a faster rate than the total fetus.

Postnatal growth changes in single-comb white leghorn
chickens were studied by Latimer (1924, 1925). He observed
that in l-day chicks the digestive tract constituted 4 per-
cent of the net body weight; in 2-day chicks it was 12.1
percent and in 3-day chicks it was 15.8 percent. In the
adult it comprised only 2 percent of the total body weight.

“The chicken fetus has been used to only a limited extent
for histochemical investigation. Moog (1950) found that in
the duodenum of l8-day fetuses there was an accumulation of
alkaline phosphomonesterase in the striated borders and in
the crypts of Lieberkuhn. More recently Moog and Wenger

(1952) have found a "neutral" mucopolysaccharide which was

also found at sites of high alkaline phosphatase activity.

On the 17th day of incubation she noticed that a thin peri-
odic acid-Schiff-positive line appeared on the surface of the
developing villi in essentially the same position as alkaline
phosphatase was found. The Schiff material and the alkaline
phosphatase developed parallel until maximum levels were
reached. The Schiff-positive material was located at sites
of high phosphatase activity. It was difficult to hydrolyze
and it was resistant to diastase and hyaluronidase. Meta-
chromasia was absent and on the basis of this and other
observations, she concluded that the Schiff-positive material
was a "neutral" mucopolysaccharide. It functioned as a mesh-
anism for orientation of alkaline phosphatase and provided a
suitable environment for enzyme activity.

From the above review of the literature it is evident
that investigations concerned with the histogenesis and his-
tochemistry of the digestive tract in the embryonic chick
have been fragmentary.

It is the purpose of this study to: (I) ascertain
relative growth of the embryonic digestive tract, 1.6. the
esophagus, crop, proventriculus, gizzard, duodenum, Jejunum-
ileum, and rectum in comparison to the total embryo; (2) to
ascertain when tissue elements of the tract differentiate;

and (3) make a study of the histochemical nature of the

connective tissue of the tract for such things as basophilia
with establishment of "pH signatures", and the reaction of

these tissues to the periodic acid-Schiff reagents.

MATERIALS AND METHODS

Fetuses used in this study were obtained from single-
comb white leghorn hatching eggs. Incubation time ranged
from 11 through 20 days. In order to show the relation
between body weight and weight of the digestive tract, the
fetus was removed from the egg along with the yolk sac. The
latter was severed near its Junction with the small intestine
in all fetuses with the exception of 20-day chicks. Wet
weights of all fetuses were ascertained and recorded. The
alimentary canal in each fetus was severed Just posterior to
the pharynx and the entire tract including the anus was re-
moved. The liver was excised but the main portion of the
pancreas was left in the loop of the duodenum. The gut was
rinsed in cold physiological Ringer's solution and the excess
moisture was removed with a towel and then the entire tract
was weighed and the weight was recorded.

Tissues from the esophagus, crop, proventriculus, giz-
zard, duodenum, Jejunum-ileum, and large intestine were
Obtained from fetuses in which the incubation age varied
between 11 and 20 days. In all routine studies, tissues
were fixed from 8 to 12 hours in Smith's modification of
Bouin's picro-formol solution (Guyer, 1953). The tissues
were then dehydrated, cleared, and embedded in paraffin

(melting point 50-520 C.). Sections were cut at approximately

10

seven microns. They were affixed to clean glass slides and
were stained with hematoxylin and eosin. The exact location
of connective tissue, muscle, and epithelium in the digestive
tract was demonstrated with.various connective tissue stains,
namely: Mallory's connective tissue stain (Cason, 1950),
Masson‘s trichrome stain (Lillie, 1940), and with the Foot
(1933) modification of Masson's stain. In the latter, tis-
sues were left in Regaud's iron-hematoxylin for five minutes
prior to the trichrome staining procedure.

For more exact study of connective tissue components,
Weigert's resorcin fuchsin was used (McClung, 1939). The
orcein staining procedure of Dempsey 23 gl (1952) was also
used for the demonstration of elastic fibers. Collagenous
tissue was stained with Van Giesen's picro—acid fuchsin
(Guyer, 1953).

The periodic acid-Schiff method (Hotchkiss, 1948) was
used for study of polysaccharides. Some of the tissues used
for polysaccharide tests were fixed in Bouin's fixative and
others were fixed in acetic-alcohol-formalin for 24 hours at
0° (Lillie 1954). Tissues which were fixed in acetic acid-
alcohol-formalin were oxidized for 5 minutes in Hotchkiss'
periodic acid A and those fixed in Bouin's solution were
oxidized for 5 minutes in Hotchkiss' periodic acid B.

Basophilic components of the cytoplasm was studied with

0.1 percent toluidine blue. French and Benditt (1953) have

11

stated that mucopolysaccharides will react with basic dyes.
Tissues were left in various buffered solutions for 3 hours.
Acid phosphate buffer was used in solution maintained at

pH 2.5; acetate buffer in solutions maintained at pH 3.5 and
5.5, and phosphate buffer at pH 8.5. The slides were left
in toluidine blue from 10 minutes to 24 hours. The proteins
used for blocking staining reactions were protamine sulfate,
globulin (from serum), and albumin (bovine plasma). (These
were supplied by Bios Laboratories, Inc., New York 23, N. Y.)
The proteins were dissolved in a concentration of 1 mg. per
ml. of buffered solution. In order to determine if the
basophilia was due to ribonucleic acid, the slides were
treated with l:lO0,000 crystalline ribonuclease (supplied by
General Biochemicals, Inc. of Chagrin Falls, Ohio) which was

added to the buffered pre-treatment solutions.

RESULTS

1. GEOMETRIC GROWTH OF THE FETUS AND ALIMENTARY CANAL

Iatimer (1928) compared postnatal growth of the chick
with growth rate of the gizzard from date of hatching to 19
days of age. Further, this author completed his study by
the addition of Schmalhausen's data on growth of the fetus
(4th day of incubation to hatching) so that a comparison
could be made between prenatal and postnatal growth.

Table 2 shows the mean weights, standard deviation, and
the maximum and minimum weights of approximately 270 alimen-
tary canals from chick fetuses ranging in age from 11 to 20
days of incubation. It is evident in Table 1 that the mean
weight of the fetus increased from 3.35 grams on the 11th
day to reach a maximum of 41.96 grams on the 20th day.

Table 2 shows that the mean weight of the alimentary canal
increased from 0.12 grams on the 11th day of incubation to

a maximum of 3.44 grams on the 20th day. The geometric rate
of growth was calculated by dividing the mean weight of the
fetus at the end of any 24-hour period by the mean weight of
the fetus at the preceding 24-hour period.

Geometric growth rates of both the alimentary canal and
fetus are given in Table 3. It is evident in text-figure l

and Table 3 that the geometric growth rate of the alimentary

13

canal was greater than that of the fetus. For the period
from 11 to 12 days of incubation, geometric growth rate was
55 percent in the fetus and 58 percent in the alimentary
canal. In the succeeding 24-hour period (12th to 13th day
of incubation), the geometric growth rate of the alimentary
canal reached 68 percent while the geometric growth rate of
the fetus decreased to 32 percent. From 13 to 14 days of
incubation geometric growth rate in the fetus and alimentary
canal was 28 percent and 59 percent respectively. In the
subsequent 24-hour period (14th to 15th day of incubation)
geometric growth rate in both the fetus and alimentary canal
was 27 percent. This was succeeded by decreased geometric
growth rates in both the fetus and alimentary canal from the
15th to 19th day of incubation. In the former it decreased
from 36 percent to 2 percent while in the latter the decrease
was from 69 percent to 10 percent. 0n the 20th day of incu-
bation the geometric growth rate of the fetus was 67 percent
and in the alimentary canal it was 30 percent. The pro-
nounced increase in geometric growth rate of the fetus on
the 20th day was attributed to the withdrawal of the yolk
sac into the peritoneal cavity. For a further comparison

in size of the fetus and the alimentary canal see figures 1,

29 and 30

TABLE 1

THE MEAN WEIGHT OF CHICK FETUSES HANGING
IN AGE FROM 11 T0 20 DAYS INCUBATION

 

r L. L——*
—: r J—j

 

 

 

 

 

 

Days of Mean Weight Standard

Incubation of Fetus Deviation Maximum Minimum
11 3.35 [1.90 4.25 2.69
12 5.19 {0.40 5.61 4.52
13 6.85 50.66 7.65 4.72
14 8.78 50.18 10.40 8.14
15 11.18 {1.35 13.00 10.05
16 15.60 {1.56 17.86 12.23
17 17.64 51.87 20.93 12.08
18 24.57 52.76 31.00 18.70
19 25.12 {6.91 28.80 21.15
20 41.96 [4.56 53.69 32.29

 

TABLE 2

THE MEAN WEIGHT OF THE ALINENTARY CANAL OF FETUSES
HANGING IN AGE FROM 11 TO 20 DAYS INCUBATION

 

 

Days of Mean Weight of Standard

Incubation Alimentary Canal Deviation Maximum Minimum
11 .12 50.5 .07 0.15
12 .19 50.04 .25 0.13
13 .32 50.05 .42 0.18
14 .51 {0.08 .66 0.39
15 .65 {0.0002 .75 0.58
16 1.10 50.02 1.75 0.76
17 1.55 50.02 1.82 1.25
18 2.39 {0.19 3.48 1.37
19 2.63 {0.35 3.35 2.00
20 3.44 50.36 4.22 2.73

 

 

 

 

 

TABLE 3

GEOMETRIC RATE OF GROWTH

 

 

 

Period Fetus Alimentary Canal
11 12 55 % 58 %
12 13 32 % 68 %
13 14 28 % 59 %
14 15 27 % 27 z
15 16 36 % 69 %
16 17 16 % 41 %
17 18 39 % 54 %
18 19 2 % 10 5
19 20 67 % 30 %

 

 

 

17

TEXT-FIGURE 1

Comparison of the rate of growth between the fetus and
and the alimentary canal.

In the upper portion of the figure the logarithmic
weight in grams of the fetus is plotted against the age of
the fetus in days.

In the lower portion of the figure the logarithmic
weight in grams of the alimentary canal is plotted against
the age in days.

 

I '1'

20*-

     
  

IO WEIGHT OF FETUS

WEIGHT IN GRA MS

0

0.5

WEIGHT OF ALIMENTARY CANAL

0.2

 

411111111

 

 

I2 [4 [6 [3 20
TIME IN DA Y S

 

 

 

, A, 71— i i_ l i._ i
W _ —— _. _- _. _ _._ A- in l

19

2. GROSS MORPHOLOGY OF THE ALIMENTARY CANAL IN THE ELEVEN-
DAY FETUS

The various regions in the adult digestive tract are
established in the fetus on the 11th day of incubation.
The esophagus which is invested by loose connective tissue
was located on the right lateral surface of the neck. The
crop which develops on the right side in the distal third
of the esophagus was a thin walled sac-like structure lo-
cated Just anterior to the thoracic cavity. That portion of
the esophagus distal to the crop, Joined the proventiculus
and the latter was continuous with the gizzard. The gizzard
was a solid muscular organ about 0.5 cm. long and 0.5 cm.
wide which along with the liver, almost completely filled
the abdominal cavity of the fetus. The duodenal loop was
found inside the cavity but the JeJunum-ileum was coiled and
extended into the umbilical hernia. The distal end of the
small intestine Joined the large intestine at the Junction
of the caecum. The length of the small intestine was greater
but the width was essentially the same in both. The large
intestine was continuous with the cloaca. The latter con—
sisted of a single chamber. The bursa was seen on the dorsal
surface of the cloaca and was imbedded in the mesoderm of the

body‘wall.

2O

3. GROSS MORPHOLOGY OF THE ALIMENTARY CANAL OF THE TWENTY-
DAY'FETUS

The esophagus (fig. 4A) was cylindrical, approximately
5 cm. long, and extended from the pharynx to the proventric-
ulus. It was located in the right lateral region of the
thorax and neck. The crop which was about 1.5 cm. long and
1 cm. wide developed as a lateral outpocketing of the esoph-
agus in the right side of the thoracic cavity. The distal
end of the esophagus Joined the proventriculus (fig. 40)
which was 0.9 cm. long and 0.7 cm. wide and this in turn led
into the gizzard. The gizzard (fig. 4D) which was 2.5 cm.
long and 1.5 cm. wide was a solid muscular mass which with
the liver occupied the anterior portion of the abdominal
cavity. The duodenum (fig. 4E) springs from the anterior
lateral portion of the gizzard and formed a loop about 4 cm.
long. Within the loop was the maJor portion of the pancreas.
The JeJunum-ileum (fig. 4F) which occupies the distal por-
tion of the abdominal cavity, exhibited numerous coils and
was attached to the body cavity by the dorsal mesentery.
When uncoiled it measured about 20 cm. in length. The large
intestine (fig. 40) was somewhat wider than the small intes-
tine but was only 1.8 cm. in length. It was located distal
to the Junction of the small intestine and the caecum. The
distal end of the large intestine was continuous with the

cloaca (fig. 4H). The length of the cloaca was approximately

21

1.2 cm. and the width increased from 0.5 cm. at its anterior
end to approximately 0.7 cm. at the posterior end. It was
divided into three regions: an anterior coprodaeum, which
Joins the large intestine; a middle urodaeum which receives
the ureters and genital ducts; and a distal proctodaeum.

The bursa of Fabricius, a sac-like structure, is located on
the dorsal wall of the proctodaeum.

4. THE HISTOGENESIS OF THE DIGESTIVE TRACT OF THE VARIOUS
AGES OF THE FETUSES.

Stains. Ehrlich's hematoxylin and eosin, Mallory's
triple stain, and Masson's trichrome stain were used for
routine staining of tissue sections. Weigert's elastic tis-
sue stain and Dempsey's orcein procedure were utilized for
staining elastic tissue. Van Giesen's stain was used for
collagenous tissue.

Esophagus. The epithelium on the 11th day of incubation

 

(fig. 6) consisted of pseudostratified columnar epithelium
which was arranged to form numerous longitudinal folds of
tissue. 0n the 15th day of incubation it was essentially
the same as it was on the 11th day although there was an
inverse ratio between the number and size of the folds, i.e.,
the number decreased and the size increased. Between the
15th and 16th day (fig. 7) the pseudostratified columnar
epithelium differentiated into stratified epithelium. The

distal layer appeared to consist of a single layer of

22

cuboidal cells, while the proximal layer was columnar. 0n
the 17th day of incubation, the proximal layer of cells was
columnar and the intervening layers between this layer and
the most distal layer of cells appeared to be of the squamous
type. Between the 17th and 19th day inclusive, the differ-
entiation of the distal cells continued toward the squamous
type of epithelium. 0n the 19th day the epithelium was
established as stratified squamous and on the surface of the
most distal layer of cells, a horny layer of material was
recognizable.

An abundant number of cilia were evident on distal sur-
faces of the pseudostratified columnar epithelial cells of
ll-day fetuses. The number of cilia was greatly reduced in
the 16-day fetuses and they disappeared entirely in l7-day
fetuses.

The thickness of the various layers of tissue in the
esophagus of fetuses varying in age from 12 to 20 days of
incubation inclusive, was measured with an ocular micrometer.
All the measurements were made on sections of tissue seven
microns in thickness. Measurements of the thickness of
the various layers were made in five to six different sec-
tions of tissue.

It is evident in Table 4 that the thickness of the

stratified squamous epithelium of the esophagus increased

23

from about twelve microns on the 12th day to reach a maximum
thickness (60-100 microns) on the 20th day of incubation.

Mucous glands were first identified in l3-day fetuses
at which time the epithelium inpocketed to form small buds
of cells which differentiated to form simple tubular glands
with a distinct lumen in l6-day fetuses. By the 18th day of
incubation (fig. 8) the distal ends of the glands were
forked to form compound structures and the lumen in each
fork exhibited mucus. In 20-day fetuses (fig. 9) the glands
were differentiated into compound tubular glands which ex-
tended through the lamina propria mucosae to reach the sur-
face of the muscularis mucosae.

The lamina propria mucosae and muscularis mucosae con-
sisted of closely packed mesenchymal cells in 11 and l2-day
fetuses. There was a tendency for the mesenchymal cells to
loosen-up in 13-day fetuses, and in l6-day fetuses (fig. 7)
the mesenchymal tissue adJacent to the epithelium was loosely
arranged while the mesenchyme next to the external muscular
layer was more compact and exhibited small bundles of muscle
cells. Tissues in the former region (loosely arranged mes-
enchyme) differentiated into the lamina propria mucosae
while mesenchymal cells in the latter, differentiated to
form the muscularis mucosae and submucosa in l9-day fetuses.
The lamina propria mucosae became compact and vascularized

between the 16th and 19th day of incubation. In 20-day

24

fetuses (fig. 9) the mucosa was made up of stratified squa-
mous epithelium, compact lamina propria mucosae, a narrow
muscularis mucosae (consisting of longitudinal muscle fibers),
and a well defined submucosa. The lamina propria mucosae
increased in thickness from a minimum of forty-five microns
on the 15th day to a maximum of ninety microns on the 20th
day. The increase in thickness of the muscularis mucosae

was less pronounced, i.e., it increased from twelve microns
on the 16th day to a maximum of forty-five microns on the
20th day. The submucosa increased from a minimum with a
range of sixty to ninety microns, to a maximum with a range
of 150 to 200 microns, as the age of the fetus increased from
15 to 20 days.

The tunica muscularis externa was first identified in
l3-day fetuses. It consisted of an inner circular muscle
layer which was made up of short fibers. By the 19th day of
incubation the length of the fibers in this region had in-
creased and were well differentiated. Individual fibers
were invested by connective tissue fibers. Longitudinal
muscle fibers in the muscularis externa were first identified
in 15-day fetuses and differentiation was completed in 20-day
fetuses, i.e., bundles of muscle fibers were invested by
small amounts of connective tissues. The thickness of the
tunica muscularis externa on the 15th day of incubation was

165 microns and on the 17th day it decreased to sixty-five

25

microns. This decrease was attributed to an increase in
length of individual muscle fibers. A similar variation in
thickness of the muscular layer was observed between the
18th and 19th day of incubation.

The tunica adventitia, a loose connective tissue on the
outer surface of the adult esophagus, was essentially the
same in appearance as the underlying longitudinal layer of
muscularis externa in l3-day fetuses, i.e., both consisted
of mesenchyme. In 15-day fetuses the inner portion of the
mesenchyme exhibited bundles of longitudinal smooth muscle
fibers while the outer layer was less densely packed. The
latter layer of tissue continued to loosen and in 19-day
fetuses it was differentiated into a layer of loose connec-
tive tissue.

Auerbach's plexus was first identified between the
circular and longitudinal layers of the tunica muscularis
externa in 18-day fetuses.

It is evident from these observations that the sub-
mucosa was the most active growth center in the esophagus
in fetuses ranging in age from 15 to 20 days.

Esophageal connective tissue (lamina propria mucosae,
submucosa, and tunica adventitia) in all ages of fetuses
(11-20 days) did not show elastic tissue with either
Weigert's stain or Dempsey's g3 g1 (1952) modification of

orcein method.

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27

Crop. It appeared as a swollen region in the posterior

 

third of the esophagus in ll-day fetuses. At this stage in
development the histological structure was essentially the
same as the esophagus of a corresponding age, i.e., it con-
sisted of pseudostratified columnar epithelium and undiffer-
entiated mesenchyme. Mucous glands were restricted to the
region adJacent to the esophagus in 20—day fetuses. The
muscularis mucosae was narrow and was first identified in
20-day fetuses. Development of the external muscularis and
tunica adventitia, parallels the development of these regions
in the esophagus proper.

Proventriculus. Observation made in this study showed
that cross sections of the proventriculus exhibited a mucosa
with a small number of folds protruding into the lumen. The
number of folds rapidly increased until the 15th day of incu-
bation (fig. 13) and then they decreased but deepened through
the 16th day. On the 20th day (fig. 15) the folds were wider
and shallower.

The distal layer of cells in the stratified columnar
epithelium of the proventriculus (from the 11th through the
16th day of incubation) showed a well developed layer of
cilia on their distal surfaces. Between the 16th and 20th
day the cilia disappeared and the epithelium itself was
transformed from stratified columnar epithelium to simple

columnar epithelium on the 20th day of incubation.

28

The lamina propria mucosae consisted of loosely arranged
mesenchymal cells in ll-day fetuses (fig. 12). In 14-day
fetuses a portion of the mesenchymal cells were differen-
tiated into connective tissue and blood vessels. In 15
through l7-day fetuses the connective tissue underlying the
epithelium consisted of closely packed cells and fibers. In
the 18-day fetuses the lamina propria mucosae (fig. 14) con-
sisted of loosely arranged cells and fibers and between 18
and 20 days the number of longitudinally arranged muscle
fibers increased.

The muscularis mucosae was identified as a longitudinal
layer, lateral to the deep glands and almost in contact with
the circular layer of the tunica muscularis externa. This
layer appeared on the 12th day of incubation and differen-
tiation continued through the 20th day. Breaks were identi-
fied in the muscularis mucosae and at these sites the sub-
mucosa was confluent with the lamina propria mucosae.

The submucosa appeared on the 16th day of incubation in
a few isolated areas between the muscularis mucosae and the
tunica muscularis externa. In 20-day specimens the submucosa
was very thin and often absent.

The circular muscle layer was identified in the pro-
ventriculus on the 11th day of incubation (fig. 12). From
the 11th through the 16th day the circular muscle fibers

remained short and an abundance of connective tissue elements

29

were seen between the fibers. The longitudinal muscle layer
which differentiated on the 12th day was relatively thin and
even absent in some areas.

The serosa existed as compact mesenchymal tissue between
the llth and 15th day of incubation. Blood vessels appeared
within it on the 12th day. In 16-day fetuses it was thinner,
less compact, and collagenous tissue was identified within it
by means of Van Giesen's stain.

The proventricular glands were located in the lamina
propria mucosae. They were first seen as simple-branched
tubular glands in ll-day fetuses (fig. 12). The glands
brandhed again and again to form complex glandular struc-
tures in 14-day fetuses. Development of the glands was al-
most completed in the l6-day fetus and secretory droplets
were identified within the lumen of the glands of the 18-day
fetus (fig. 14).

Gizzard. Stratified ciliated columnar epithelium lined
the gizzard of the ll-day fetus (fig. 17). Between the 14th
and 15th day of incubation the ciliated border of the epi-
thelium was transformed into a striated border. The strati-
fied epithelium in lS-day fetuses (fig. 18) developed to form
crypts which marked the points where the gland ducts opened
to the surface. Between the 15th and 20th day the stratified
epithelium (fig. 20) was replaced by simple columnar epi-
thelium. An exudate of the glands was found in the crypts

and on the surface of the epithelium of l6-day fetuses.

30

The lamina propria mucosae was identified as loose con-
nective tissue in which the glands were located. On the llth
day of incubation (fig. 16) this layer appeared as a compact
mesenchymal tissue continuous with the submucosa. Many small
vacuoles appeared in cells of the lamina propria mucosae in
l2-day fetuses. These small vacuoles coalesed to form
larger ones on the 14th day. Between the 14th and 16th day
these vacuolated areas developed into glands which were sur-
rounded by loose connective tissue of the lamina propria
mucosae.

The muscularis mucosae was not identified in the gizzard
of 11 to 20-day fetuses (fig. 20).

The submucosa appeared as compact mesenchymal tissue
and was continuous with the lamina propria mucosae in ll-day
fetuses. By the 15th day of incubation (fig. 18) the sub-
mucosa appeared to be loosely arranged connective tissue
which stained positive for collagenous tissue with Van Giesen's
stain. The submucosa had developed into a compact layer of
connective tissue by the 20th day (fig. 20). On the ventral
and dorsal surfaces of the gizzard, the tunica muscularis
externa was absent and in these areas the submucosa came into
direct contact with the fibrous connective tissue (aponeurosis)
which covered the entire organ.

The tunica muscularis externa appeared as a large mass

of loose, circular fibers which extended from one aponeurosis

31

to the other in the ll-day fetus. By the 15th day of incu-
bation (fig. 18) the muscle bundles were invested with loose
connective tissue fibers. From the 15th to the 20th day the
muscle fibers lengthened and this accounted for the increase
in the size of the gizzard. There were no longitudinal
fibers in the gizzard of the 11 to 20—day fetuses.

The serosa was identified as a compact mesenchymal layer
on the llth day of incubation. This layer appeared very
thick on the dorsal and ventral surfaces where the tunica
muscularis externa was absent. This layer continued to
develop and from the 16th to 20th day was compact and stained
positive for collagenous fibers with Van Giesen's stain.

On the basis of the results obtained with Weigert's
stain and the orcein procedure of Dempsey gt a; (1952) elas-
tic tissue was not identified in the lamina propria mucosae,
the submucosa, or the tunica adventitia of the gizzard in 11
to 20-day fetuses, inclusive.

§mall_lntestine. The microscopic structure of the duo-
denum and JeJunum-ileum was essentially alike and from a
morphological point of view the regions were undistinguishable.

The stratified columnar epithelium exhibited numerous
folds in ll-day fetuses. These folds deepened and by the
13th day of incubation they were differentiated into villi.
The epithelium was transformed into simple columnar tissue

and it remained in this condition through the 20th day

32

(fig. 23). Goblet cells were first identified in epithelium
of l7-day fetuses (fig. 22) and glands of Lieberkuhn in 19-
day fetuses.

The lamina propria mucosae first appeared as compact
connective tissue in the villi of l7-day fetuses (fig. 22).
This layer stained positive for collagenous fibers with
Van Giesen's stain.

The muscularis mucosae of the small intestine consisted
of an inner longitudinal layer and an outer circular layer.
The former was identified as bundles of muscle fibers located
at the base of the villi on the 17th day of incubation. This
layer continued to develop through the 20th day (fig. 23) at
which time bundles of muscles from this layer were identi-
fied in the villi. It was difficult to identify the circular
layer of muscles from the circular layer of the tunica
muscularis externa. The circular layer was identified in
only a few areas of the small intestine of ZO-day fetuses.

The submucosa was seen in only a few areas of the duo-
denum of the 20-day fetus. By use of selective staining
(Mallory's, Masson's, and Van Giesen's), it was observed that
there was connective tissue between the circular muscle
layers of the muscularis mucosae and the lamina muscularis.

The tunica muscularis externa was made up of an inner
circular layer and an outer longitudinal layer. The circu-

lar layer was identified as bundles of short fibers and was

33

located in the mesenchymal walls of ll-day fetuses. The
outer longitudinal layer was first apparent on the 17th day
of incubation.

The serosa from the llth through the 16th day of incu-
bation appeared as undifferentiated mesenchymal tissue. By
the 17th day this layer was loosened and fibers were first
identified. Differentiation of fibers continued through the
20th day and a positive reaction for collagenous fibers was
obtained with Van Giesen's stain.

Large Intestine. The large intestine appeared morpho-
logically similar to the small intestine in most respects,
including the presence of villi and a muscularis mucosae
which consisted of a circular and longitudinal layer.

The epithelium on the llth day of incubation (fig. 24)
was identified as a stratified columnar layer of tissue. By
the 14th day folds appeared in the stratified columnar epi-
thelium which deepened on the 15th day and were differen-
tiated into villi by the 16th day. The epithelium at this
time had been transformed from stratified columnar into
simple columnar and remained in this condition through the
20th day (fig. 26). On the whole, the villi of the large
intestine appeared to be shorter than those found in the
small intestine on the 20th day.

The lamina propria mucosae was part of the undifferen-

tiated mesenchymal tissue which was located between the

34

epithelial layer and the circular muscle fibers of the tunica
muscularis externa of 11 through lS-day fetuses. By the 16th
day of incubation this mesenchymal layer was divided into a
compact area adjacent to the epithelium and a loose layer
adjacent to the circular muscle layer of the tunica muscularis.
The compact area adjacent to the epithelium sent fibers into
the villi and by the 20th day it had differentiated into the
lamina propria mucosae. The fibers of this compact layer

were found to stain positive for collagenous tissue with

Van Giesen's stain.

The muscularis mucosae consisted of an inner longitu-
dinal and an outer circular layer. In l6-day fetuses, bun-
dles of longitudinal muscle fibers first differentiated in
the loose mesenchymal tissue medial to the circular muscle
layers of the tunica muscularis externa. From the 17th to
20th day of incubation, differentiation of these bundles con-
tinued and a well developed layer of muscle fibers appeared
in 20-day fetuses (fig. 26). The circular layer first
appeared on the 18th day separate and distinct from the cir-
cular layer of the tunica muscularis externa.

The submucosa was identified as a very thin layer of
collagenous tissue between the circular muscle layer of the
muscularis mucosae and the circular layer of the tunica
muscularis externa, on the 18th day of incubation. In 20-

day fetuses the submucosa was very thin and in a few areas

35

it made contact with the lamina propria through spaces where
the muscularis mucosae was not a complete layer. This tissue
stained positive for collagenous fibers with Van Giesen's
stain.

The tunica muscularis externa was identified as a cir-
cular muscle layer in the compact mesenchymal tissue, forming
the wall of ll-day fetuses. On the 14th day of incubation
(fig. 25) bundles of longitudinal muscle fibers were seen and
by the 16th day these bundles had formed a complete layer
lateral to the circular fibers. The short circular fibers
continued to lengthen through the 20th day.

The very thin serosa which contained connective tissue
and blood vessels, developed from a thick mesenchymal layer
in the ll-day fetus. On the 16th day of incubation it began
to differentiate into a thin white fibrous connective tissue
and continued to differentiate through the 20th day.

Elastic tissue was not identified in the lamina propria
mucosae, submucosa, and the serosa, by Weigert's or Dempsey's
gt a; (1952) orcein modification stains.

5. STUDY OF METACHROMATIC STAINING ELEMENTS IN THE DIGESTIVE
TRACT OF CHICK FETUSES BY THE USE OF TOLUIDINE BLUE "pH SIGNA‘
TUBES", PROTEINS, AND RIBONUCLEASE.

Various regions in the digestive tract (esophagus, small

intestine, and large intestine) of chick fetuses varying in

age from 12 to 20 days, were used for study of metachromasia

36

in embryonic tissues. Tissues were stained with toluidine
blue according to the procedures of French and Benditt
(1953). To test the blocking of staining reactions with
proteins, tissues were left in either albumin, globulin, or
protamine solutions (1 mg./ml.) for two hours prior to stain-
ing with 0.1 percent toluidine blue. The hydrogen ion con-
centrations were controlled according to the procedures
described in the Materials and Methods section. In control
dye solutions, treatment with proteins was omitted but tis-
sues were placed in aqueous buffer solutions prior to
staining.

Estimation of basophilic materials was based on the
intensity of the staining reaction, i.e., deep purple stain-
ing reactions were given a value of 3; moderately purple, a
value of 2; and light blue reactions, a value of l. The
letter "G" was used for denoting blue-gray reactions and "P"
was used for weak metachromatic reactions. The results
obtained were presented in Table 5.

Staining intensity in sections of tissue from the

 

digestive tract. It is evident in Table 5 that in all
toluidine blue control solutions (pH 2.5 - 8.5 inclusive),
intensity of staining reactions was inversely related to
hydrogen ion concentration, i.e., as the hydrogen ion con-
centration decreased from pH 2.5 to 8.5, intensity of the

staining reaction increased from about 1 to 3.

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38

It is also evident that immersion of tissues in albumin
solution prior to staining, influenced the intensity of the
staining reaction. Staining reactions at a pH of 8.5, in
tissues from all regions of the digestive tract, were
essentially the same as the staining reactions in the corre-
sponding control solution. On the other hand, tissues which
were left in albumin prior to immersion in toluidine blue at
pH 5.5 stained more intensely than tissues that were trans-
ferred directly to toluidine blue solution at pH 8.5.

Albumin-treated tissues when stained with toluidine blue
at pH 3.5, stained less intensely than the tissues in tolui-
dine blue control solutions. Tissues in the esophagus of
20-day fetuses which were left in albumin and subsequently
stained in toluidine blue (pH 2.5) exhibited staining re-
actions essentially the same as those obtained in the tolui-
dine blue control solution (pH 2.5). Immersion of esophageal
tissues from 12 and l6~day fetuses and intestinal tissues
from 20-day fetuses in albumin, decreased the staining re-
action, i.e., in control solutions the tissues were stained
light blue while albumin-treated tissues stained gray.

Exposure of tissue sections to globulin prior to stain-
ing with toluidine blue influenced the intensity of the
staining reaction. At a hydrogen ion concentration of pH 8.5,
staining intensity of globulin-treated tissue was similar to

the untreated control tissues. However, globulin-treated

39

tissues at pH 5.5 stained more intensely than tissues from
control solutions (pH 5.5).

It is evident that at pH 3.5 a very marked difference
in staining intensity was_observed between tissues treated
with globulin and untreated control tissues. The globulin-
treated tissues stained a light gray, while on the other
hand, the tissues in control solution stained blue or light
purple.

Tissues stained with toluidine blue subsequent to treat-
ment with protamine sulfate also demonstrated that intensity
of staining was affected by protein. At pH 8.5 the staining
reactions in all tissues in the digestive tract were essen-
tially the same as corresponding regions which were stained
following control procedures. On the other hand, at pH 5.5
the staining was decreased in tissues treated with protamine
in comparison to those of control treated tissues. Tissues
which had been immersed in protamine prior to staining at
pH 3.5, were somewhat variable. The intensity of the stain-
ing (pH 3.5) reaction in the esophagus of l2-day fetuses and
the small intestine and large intestine of l6-day fetuses
was decreased, while staining in the other tissues was
similar to the controls. Tissues stained with toluidine
blue (pH 2.5) following protamine sulfate treatment were

similar to tissues stained following control treatment.

40

Intensity of staining reaction in_specific layers of
tissues of the digestive tract. The data presented in the
preceding paragraphs summarized the over-all basophilic
reactions obtained in tissues from various levels of the
digestive tract with experimental and control solutions.
Although the results presented in Table 5 are estimations of
staining reactions in a given section of tissue, individual
reactions in the various layers of tissue were subject to
considerable variation. For example, tissues in control
solution prior to staining at pH 8.5 stained the nuclei in
all layers of tissue (mucosa, submucosa, and the muscular
layers) and mucous glands deep purple. Muscle fibers stained
light blue and the lamina propria mucosae, submucosa and
tunica adventitia, stained metachromatically. Staining re-
actionsat pH 5.5 were essentially the same as those obtained
at pH 8.5.

At pH 3.5 the staining reaction in all layers of tissue
was less intense. Secretions within the lumen of the mucous
glands were stained light purple while the nuclei and cyto-
plasm of cells in the gland itself stained blue and light
blue respectively. The staining intensity of all tissues in
the esophagus, small intestine and large intestine were
fairly constant with the exception of goblet cells and glands
of Lieberkuhn in both the large and small intestine. The

former (goblet cells) stained metachromatically at pH 8.5

41

and 5.5, while at pH 3.5 and 2.5 these cells were colorless.
The latter (glands of Lieberkuhn) stained metachromatically
at pH 8.5. 5.5, and 3.5 and blue at pH 2.5.

Staining reaction in individual layers of tissues in
cross-sections of the digestive tract after immersion in
albumin and subsequent to staining in toluidine blue differed
from staining reactions obtained in the control solution. At
pH 5.5 and 8.5, nuclei in all layers of tissue in the diges-
tive tract stained deep purple. At the latter hydrogen ion
concentration, cytoplasm in connective tissue cells stained
metachromatically. At pH 5.5 the cytoplasm of connective
tissue cells was less metachromatic.

When the hydrogen ion concentration of the staining
solution was increased to pH 3.5, the intensity of staining
of nuclei in the esophagus and large intestine decreased from
about 3 (deep purple) to less than 1 (blue-gray). The cyto-
plasm of connective tissue cells was practically colorless
and the color of the cytoplasm of muscle cells was gray. In
20-day fetuses the nuclei continued to stain blue even at
pH 2.5 but in 12 and l6-day fetuses, the nuclei stained blue-
gray. The small intestine in all ages of fetuses stained
similarly to that of the controls.

Differences were observed in the staining intensity of
individual layers of tissues stained in toluidine blue, sub-

sequent to treatment with globulin, as compared with control

42

treated tissues. At pH 5.5 and 8.5, nuclei in all layers of
tissues observed (the mucous glands of the esophagus, goblet
cells and glands of Lieberkuhn of the large and small intes-
tine), stained deep purple. At these hydrogen ion concentra-
tions, the cytoplasm of the muscle fibers stained metachro-
matically as did the cytoplasm of connective tissue elements.

At pH 3.5 the staining reaction was markedly reduced in
all layers of tissue. The nuclei stained blue-gray; the
connective tissue cytoplasm and muscle fibers stained gray;
the mucous glands of the esophagus stained purple; and the
goblet cells and the glands of Lieberkfihn did not stain. At
pH 2.5 all tissue elements of the digestive tract in all ages
of fetuses stained similarly to that of the controls.

Sections of tissue which were treated with protamine
sulfate prior to staining with toluidine blue were subject
to considerable variation in staining reaction. At pH 8.5,
5.5, and 3.5, the staining which was observed appeared gran-
ular. In esophageal tissues of all fetuses, the nuclei
stained purple at pH 3.5, 5.5, and 8.5, and were blue at 2.5;
mucous glands were stained purple at pH 8.5 and 5.5, blue at
pH 3.5 and pink at pH 2.5; the cytoplasm of the connective
tissue (lamina propria mucosae, submucosa, the tunica adven-
titia in older fetuses, and the undifferentiated mesenchyme
in young fetuses) stained pink (metachromatic) at pH 8.5,

gray at pH 5.5 and 3.5 and colorless at pH 2.5. The nuclei

43

of the small intestine stained deep purple at pH 8.5, purple
pH 5.5 and 3.5, and blue at pH 2.5; the cytoplasm of the
connective tissues stained pink (metachromic) at pH 8.5,
gray at pH 5.5 and 3.5 and colorless at pH 2.5; while muscle
cytoplasm was blue at pH 8.5 and gray at pH 5.5, 3.5 and 2.5;
the goblet cells stained deep purple at pH 5.5 and 8.5 and
were unstained at pH 2.5 and 3.5; and the glands of
Lieberkuhn stained purple at pH 5.5 and 8.5 and were blue at
pH 2.5 and 3.5. The large intestine followed a similar pat-
tern as the small intestine except for the nuclei of all
layers of tissues which stained deep purple at pH 8.5, gray
at pH 5.5, purple at pH 3.5 and blue at pH 2.5.

The above observations indicate that staining of meta-
chromatic components was dependent upon the pH of the stain.
It is also evident from the above observations that staining
these components with a basic dye (toluidine blue) can be
modified by placing the tissues in the presence of protein at
a specified hydrogen ion concentration. It was observed that
albumin was most effective in blocking dye uptake at pH 2.5
and 3.5, globulin at pH 3.5 and protamine at pH 3.5 and 5.5.

The nucleic acids have a very strongIdTinity for basic
dyes (Singer, 1954). In order to ascertain whether or not
basophilia was due to ribonucleic acid, sections of the
esophagus, small intestine and large intestine of 20, 16,

and l2-day chick fetuses were treated for one and one-half

44

hours at 37° with ribonuclease at pH 5.5 prior to staining
with toluidine blue (pH 5.5). The nuclei in all tissue
layers (mucosa, submucosa, and muscular) stained deep blue.
The cytoplasm of epithelial cells stained pink; the connec-
tive tissue elements stained deep pink; and musCle cytoplasm
stained gray. The mucous glands of the esophagus, the goblet
cells and glands of Lieberkuhn of the small and large intes-
tine were stained pink. By comparing the above results with
the untreated control tissues which were stained at the same
pH, it was observed that the nuclei appeared lighter and the
mucous glands, goblet cells and the glands of Lieberkuhn
were stained pink instead of purple, and muscle cytoplasm

was gray instead of pink. On the other hand, the connective
tissues (lamina propria, submucosa and tunica adventitia) and
undifferentiated mesenchyme continued to stain pink (meta-
chromatic) in both experimentally treated tissues and in
control tissues.

6. THE PERIODIC ACID-SCHIFF TEST APPLIED TO TISSUES OF THE
DIGESTIVE TRACT OF CHICK FETUSES.

Sections of tissue were stained for polysaccharides by
the Hotchkiss (1948) method. In some experiments periodic
acid A (400 mg. periodic acid, 5 ml. M/5 acetate and 35 ml.
of ethyl alcohol) was used as an oxidizing agent for soluble
polysaccharidasand in other experiments periodic acid B

(400 mg. periodic acid, 5 ml. M/5 acetate and 45 ml. of

45

distilled water) was employed for oxidation of insoluble
polysaccharides. Some tissues were fixed in Bouin's fixative
for demonstration of insoluble polysaccharides. Soluble
polysaccharides were demonstrated after fixation of tissues
in acetic acid-alcohol-formalin.

Insoluble polysaccharides. The esophagus of 11 and 12-
day fetuses were negative for polysaccharides while esopha-
geal tissues in l3-day fetuses exhibited a strong acid-Schiff
reaction. The epithelium bordering the lumen and the cyto-
plasm of undifferentiated connective tissue cells were
stained uniformally light red. In lS-day fetuses the epi-
thelial border, basement membrane and connective tissue be-
tween the muscle fibers of the external muscular layers
stained light red. In fetuses from 18 to 20 days inclusive,
the intensity of the staining reaction was increased in
esophageal glands, i.e., they now stained deep red. The
underlying lamina propria mucosae and also the submucosa
stained less intensely, i.e., light pink.

The proventriculus of ll-day fetuses showed scattered
Schiff-positive granules in the cytoplasm of epithelial cells.
On the 12th day of incubation there was a thin layer of
Schiff-positive material on the distal border of the epithe-
lium in addition to the Schiff-positive cytoplasmic granules.
From the 14th to the 20th day Schiff-positive materials were

more extensive. The distal surface of the epithelium, the

46

lumen of the glands, the cytoplasm of cells and fibers in the
lamina propria, the submucosa, the connective tissue invest-
ing muscle in the external muscle layers, and the serosa,
stained light red.

On the llth and 12th days of incubation the epithelial
border and basement membrane of the gizzard exhibited Schiff-
positive reactions. In 14-day fetuses the epithelial border,
the lumen of the glands, submucosa, and serosa were Schiff-
positive. While in 17 - 20-day fetuses the Schiff—positive
reactions were more extensive, i.e., the lamina propria and
connective tissues investing the fasciculi of the external
muscular layers were also Schiff-positive.

The small intestine exhibited Schiff-positive granules
in the cytoplasm of epithelial cells from the llth through
the 16th day of incubation. On the 15th day Schiff-positive
materials were observed within the cytoplasm of connective
tissue cells and fibers which surrounded the muscle bundles.
By the 17th day the goblet cells and the connective tissues
were positive and this condition continued through the 20th
day.

Scattered granules of Schiff-positive material were seen
in the cytoplasm of epithelial cells of the large intestine
in chicks ranging from 11 to 18 days of incubation. The
basement membrane was positive from the llth through the 16th

day. The epithelial border showed a thin layer of

47

Schiff-positive material from the 12th to the 20th day. Both
the connective tissue fibers and the cytoplasm of connective
tissue cells exhibited positive Schiff reactions in 14-day
fetuses. In 19 and 20-day fetuses goblet cells, the epithe-
lial border, lamina propria, submucosa and connective tissue
elements around the muscle bundles were Schiff-positive.
Schiff—positive materials were first observed in the serosa
and goblet cells on the 18th day of incubation.

Soluble polysaccharides. The glands of the esophagus,
proventriculus, and gizzard, the epithelial borders of all
organs comprising the tract, and the goblet cells in the
intestine, stained red. All other tissues stained pink.

The connective tissue which surrounded the gland and serosa,
stained deep pink. All tissues were Schiff-positive in the
staining reaction.

Tissues stained f9; soluble pglysaccharides and treated
gith_ribonuclease. Red staining occurred in the epithelial
border; in the lumen of the glands of the esophagus, proven-
triculus, and gizzard; and also in the goblet cells of the
intestine. The other tissues stained pink. The serosa and
connective tissue around the glands stained deep pink. All
tissues were Schiff-positive in the staining reaction.

Tissues stained for soluble polysaccharides and treatei
Egth saliva. The connective tissue, epithelial border, lumen

of the glands of the esophagus, proventriculus, gizzard, and

48

the goblet cells of the intestine stained light red after
tissues were exposed to saliva for twenty minutes. The
epithelial and muscle layers showed a marked decrease in
staining intensity after exposure to saliva.

It can be seen in the above observations that only some
tissues exhibited a Schiff-positive reaction when Bouin's
solution was used as the fixative and periodic acid B was
used as the oxidizing agent. When acetic acid-alcohol-
formalin was used as a fixative and periodic acid A as the
oxidizing agent, all stained positive with the Schiff
reagent. The ribonuclease did not prevent the staining of
the Schiff reagent in any of the tissues observed. Saliva
did not abolish the staining although it did decrease the
intensity of it.

DISCUSSION

1. GROWTH AND HISTOGENESIS OF THE DIGESTIVE TRACT.

It is evident from results obtained in this study that
in fetuses of 11 to 20 days of incubation the geometric
growth rate of the alimentary canal exceeded the growth
rate of the fetus. These observations are in general agree—
ment with the calculations of Latimer (1928) who used the
data of Schmalhausen for estimation of prenatal growth rate.

Observations presented in preceding pages demonstrated
that the pattern of differentiation of the various layers
of tissue in the digestive tract was essentially the same in
the foregut, midgut, and hindgut regions. Epithelium lining
the tract differentiated first; circular muscle in the ex-
ternal muscular layer was next to appear; and lastly the
lamina propria and submucosa differentiated.

Differentiation of the various layers of tissue in the
esophagus was incomplete in ll-day fetuses but subsequent
rate of development in this region of the tract exceeded
differentiation in both the small and large intestine. Dif-
ferentiation of the gizzard and proventriculus closely par-
alleled the differentiation of the esophagus.

It was shown in the preceding pages that the morpho-

logical features of the esophageal epithelium were profoundly

50

modified between the llth and 20th day of incubation. In 11-
day fetuses it was classified as pseudostratified columnar
epithelium, in l6-day fetuses as stratified columnar and in
20-day fetuses it was differentiated into stratified squamous
epithelium. Other regions of the digestive tract (proven-
triculus, gizzard, small and large intestine) exhibited
stratified columnar epithelium lining the tract in ll-day
fetuses which was transformed into simple columnar epithelium
between the 16th and 20th day of incubation.

Cilia were identified on the free border of the epi-
thelial cells lining all regions of the tract in ll-day
fetuses. There was an inverse ratio in the number of cilia
to the age of the fetus, i.e., as the age of the fetus in-
creased the number of cilia decreased. By the 17th day of
incubation cilia were no longer identifiable on the distal
surfaces of the epithelial cells.

Glands were identified in the lamina propria of the
proventriculus of ll-day fetuses while in the esophagus and
gizzard, glands appeared on the 13th day of incubation. How-
ever, the glands of the esophagus and proventriculus devel-
oped into large compound glands, while those of the gizzard
appeared as a compact layer of parallel simple tubular glands
in the lamina propria mucosae.

The lamina propria mucosae, muscularis mucosae, and

the submucosa of the tract developed from a compact

51

mesenchymal layer located between the epithelial border and
the circular muscle bands of the tunica muscularis externa.
The only exception to the above was in the gizzard where no
muscularis mucosae was differentiated. This is in agreement
with Calhoun (1954) who found no muscularis mucosae in the
gizzard of adult chickens. The muscularis mucosae in both
the small and large intestine consisted of an inner longi-
tudinal layer and an outer circular layer, while that of the
esophagus and proventriculus consisted only of longitudinally
arranged fibers. Generally the lamina propria mucosae and
submucosa differentiated before the muscularis mucosae was
formed. The mesenchyme in the area where the lamina propria
formed, loosened up prior to the formation of the muscularis
mucosae and after the formation of the muscularis mucosae the
lamina propria mucosae developed into a compact layer of
collagenous tissue. On the other hand, the mesenchyme which
developed into the submucosa remained compact and after
formation of the muscularis mucosae the submucosa developed
into a very loose layer of connective tissue in the esopha-
gus; a rather compact layer in the gizzard; and a very thin
loose layer in the proventriculus and the small and large
intestine.

In all organs of the digestive tract with the exception
of the esophagus, the circular layer of the tunica muscularis

externa could be identified on the llth day of incubation.

52

However, it was clearly differentiating in the esophagus by
the 12th day. The tunica muscularis externa consisted of
an inner circular layer and an outer longitudinal layer of
muscle bundles in the esophagus, proventriculus, small
intestine, and large intestine in 20—day fetuses. However,
in the gizzard, only large amounts of circular muscle fibers
were identified but no longitudinal fibers were present in
11 to 20—day fetuses. Calhoun (1954) also found that there
were no lateral muscle layers in the tunica muscularis
externa of the gizzard in adult chickens. The outer longi-
tudinal layer of muscle developed from mesenchyme which lies
lateral to the circular muscle layer.

The tunica adventitia of the esophagus and the serosa
of the other organs developed from the mesenchyme which was
located lateral to the circular muscle layer of the tunica
muscularis externa. When fully differentiated it was found
to be composed of loose collagenous tissue with an abundance
of blood vessels.

2. HISTOCHEMICAL STUDIES OF THE CONNECTIVE TISSUES.

Wachstein (1949) and Leblond (1950) maintained with
Hotchkiss (1948) and Mc Manus (1948) that periodic acid trans-
forms polysaccharides into polyaldehydes. The latter were
found to stain various shades of red with fuchsin-sulfurous
acid reagent. Gibb and Stowell (1949) demonstrated that the

Schiff reagent will stain glycogen, mucin, mucoprotein,

53

hyaluronic acid and chitin. Gomori (1952) included with the
above mentioned Schiff-positive materials such substances as
starch, cellulose, glycolipids and mucopolysaccharides.

Dempsey gp.gl. (1947) found that mucopolysaccharides
could be identified on the basis of their acidic groups,
i.e., sulfate-containing mucopolysaccharides. Wislocki and
Sognnaes (1950) demonstrated that acid mucopolysaccharides
were not only metachromatic but also reacted intensely with
basic dyes at low pH.

Observations made in this study suggest that both acid
mucopolysaccharides and "neutral" polysaccharides exist in
the connective tissues of the embryonic digestive tract. At
pH 8.5 and 5.5 both the lamina propria and submucosa stained
metachromatically but when the pH of the dye solution was
decreased to 3.5 and 2.5 the staining reaction in these tis-
sues was weakly basophilic. The use of basic proteins such
as albumin, globulin and protamine, support this view. In-
tensity of the toluidine blue staining reaction was decreased
at pH 3.5 when tissues were treated with albumin and globulin
prior to staining while the staining reaction at pH 5.5 in
protamine treated tissues was less intense than it was in
the control solution. French and Benditt (1953) found that
basic proteins competed with the dye solution for the stain-

able groups in polysaccharides. Observations reported in the

54

preceding pages demonstrated that basophilia in the lamina
propria and submucosa could not be attributed to ribonucleo-
protein since immersion of tissues in ribonuclease did not
abolish the reaction. Moog and Wenger (1952) maintain that
polysaccharides containing sulfate groups (i.e., acid
mucopolysaccharides) generally take on a reddish coloration
when stained with toluidine blue. Observations reported in
this study showed that the connective tissue of the develop-
ing digestive tract stained pink with toluidine blue at

pH 5.5 and 8.5.

It was also observed that the periodic acid-Schiff
reaction was not completely abolished although the reaction
was decreased upon immersion of tissues in saliva. This
observation suggests that glycogen exists in combination
with other mucopolysaccharides. Wislocki gt._g;, (1947)
observed that a large amount of glycogen was present in fetal
tissues of mammals.

The intensity of the staining reaction with the periodic
acid-Schiff procedure for soluble polysaccharides and tolui-
dine blue remained fairly constant in the digestive tract of
fetuses between 11 and 20 days of incubation. On the other
hand, the periodic acid-Schiff procedure for insoluble poly-
saccharides did not produce a positive reaction in undiffer-
entiated mesenchyme but was positive in differentiated

tissues of the lamina propria, submucosa and serosa. The

55

intensity of staining with Schiff reagent for insoluble
polysaccharides was not diminished by exposing tissues to
saliva before staining. Jorpes gt. a}. (1948) maintained
that the periodic acid-Schiff procedure will not stain poly-
saccharides containing chondrdtin sulfuric acid. This would
suggest that from the results observed in this study the
material stained by the Schiff procedure was a "neutral"
polysaccharide.

Meyer (1950) found that both hyaluronic acid and chon-
dromfln sulfate were present in mesenchymal tissues. Bunting
(1950) maintained that acid mucopolysaccharides when stained
with toluidine blue stained metachromatically at low hydro-
gen ion concentrations. Further, he maintained that at low
pH values, tissues containing chondrdmdn sulfate exhibit
basophilia.

ENidence obtained in this study would indicate that the
fetal connective tissues were composed of acid mucopoly-
saccharides and "neutral" polysaccharides. The former were
present in undifferentiated mesenchyme while the latter were
not present until differentiation had taken place. The acid

mucopolysaccharides were demonstrated by staining with tolui-

dine blue at various hydrogen ion concentrations. The "neutral"

polysaccharides were demonstrated by use of the periodic acid-

Schiff reagent for insoluble polysaccharides (Hotchkiss, 1948).

These Schiff-positive materials were found to be resistant to

digestion by saliva.

SUMMARY

1. Fetuses were obtained from single-comb white leg-
horn hatching eggs. These were used for studies of the his-
togenesis of the digestive tract and the histochemistry of
the connective tissues found in the digestive tract.

2. Weights of fetuses of various ages were taken sub-
sequent to removal from the egg. The digestive tract of
various ages of chick fetuses was weighed and a comparison.
of the geometric growth rates of the fetus and the digestive
tract was made. In this study it was found that the geo-
metric rate of growth in the digestive tract was greater
than the geometric rate of growth in the fetus from the llth
to the 20th day of incubation.

3. Tissues stained with hematoxylin and eosin, Mallory's
triple stain, Masson's trichrome stain, Weigert's elastic
tissue stain, Dempsey's orcein procedure, and Van Giesen's
stain for collagenous tissue were used in the study of the
histogenesis of the digestive tract in chick fetuses.

4. It was observed in this study that the different
tissue layers which comprise the digestive tract differen-
tiated in an orderly sequence in all organs of the alimentary
canal. The layers differentiated in the following sequence:

first the epithelial layer which was followed by the circular

57

muscle fibers of the external muscle layer, then the lamina
propria and submucosa, and finally the muscularis mucosae.

5. When toluidine blue was used as a stain it was ob-
served that connective tissues stained either basophilic or
metachromatically depending upon the pH of the dye solution.
The staining intensity of the connective tissues could be
altered if the tissue sections were treated with basic pro-
teins prior to staining. Albumin, globulin and protamine
were the basic proteins used in this study.

6. It was possible with the use of toluidine blue and
the basic proteins, to demonstrate that the undifferentiated
mesenchymal connective tissues and the differentiated con-
nective tissues contain acid mucopolysaccharides.

7. The periodic acid-Schiff proceduresof Mo Manus
(1946) and Hotchkiss (1948) were used to study glycogen and
insoluble polysaccharides of the fetal tissues comprising
the digestive tract.

8. Observations made following the use of the Schiff
reagent demonstrated that glycogen was found in all tissues
of the digestive tract in chick fetuses of 11 to 20 days of
age. It was further observed that the differentiated connec-
tive tissues of the alimentary canal were Schiff-positive and
this was not soluble in saliva.

9. The conclusions reached in this study of the con-

nective tissue indicate that they contained an acid

58

mucopolysaccharide and a "neutral" polysaccharide. The
former were identified by staining with toluidine blue at
various hydrogen ion concentrations. These acid mucopoly-
saccharides were found in both undifferentiated mesenchyme
and in the differentiated connective tissues of the alimen-
tary canal. The "neutral" polysaccharides were observed
by staining with the Schiff reagent, were insoluble in
saliva, and were identified only in differentiated connec-

tive tissues and not in the undifferentiated mesenchyme.

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63

.. PLATE I
Fig. 1. Total fetuses (including yolk sac) ranging in age
from 15 to 20 days of incubation.

Fig. 2. Total fetuses (including yolk sac) ranging in age
from 11 to 14 days of incubation.

 

 

65

PLATE II

Fig. 3. Alimentary canals removed from fetuses ranging in
age from 12 to 20 days of incubation.

H

u a a, c, t ,2 ,. a: .223. E.,..if

_._____i__=__a_______aa__a______.:_r__,

t:
H .
ousés EHHnaHnii~a_.rs

 

67

Fig. 4.

PLATE III

Entire alimentary canal of a 20-day fetus with the
jejunum-ileum uncoiled.

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Esophagus

Crop
Proventriculus
Gizzard
Duodenum
Jejunum-ileum
Large intestine
Cloaca

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F16. 5.

Fig. 6.

Fig. 7.

Fig.,8.

Fig. 9.

Fig.10.

PLATE IV

Cross section through the esophagus of an ll-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Undifferentiated mesenchyme.

Cross section through the eSOphagus of an ll-day
fetus. Hematoxylin-eosin. Magnification X520.
A. Epithelium; B. Mesenchyme.

Cross section through the esophagus of a l6-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Lamina propria; C. Gland;
D. Muscularis mucosae; E. Submucosa;

F. Tunica muscularis externa; G. Tunica
adventitia.

Cross section through the esophagus of an l8—day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Lamina propria; D. Muscu-
laris mucosae; E. Submucosa; F. Tunica
muscularis externa.

Cross section through esophagus of a 20-day

fetus. Hematoxylin-eosin. Magnification X170.

A. Epithelium; B. Lamina propria; C. Gland;

D. Muscularis mucosae; E. Submucosa; F. Tunica
muscularis; G. Tunica adventitia.

Cross section.of esophagus of a 19-day fetus
using Masson's stain. Magnification X170.

Micrometer scale insert: 1 space= 0.01 mm.

 

71

Fig.

Fig.

Fig.
Fig.

Fig.

Fig.

ll.

12.

13.

14.

15.

16.

PLATE V

Cross section through the esophagus of a 20-day
fetus. Stained with toluidine blue at pH 8.5.
Magnification X170.

Cross section through proventriculus of an ll-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Gland; C. Undifferentiated
mesenchyme; E. Circular muscle layer;

F. Serosa.

Cross section of proventriculus of a 15-day fetus.
Hematoxylin-eosin. Magnification X170.

A. Epithelium; B. Gland; C. Lamina propria;
E. Tunica muscularis externa.

Cross section of proventriculus of an 18-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Gland; C. Lamina propria;
D.‘ Muscularis mucosae; E. Tunica muscularis.

Cross section through the proventriculus of a
20-day fetus. Hematoxylin-eosin. Magnification.
X170. (note opening of 1ar e gland into the
lumen of the proventriculus?

A. Epithelium; B. Gland; C. Lamina propria.

Cross section through the gizzard of an 11-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; C. Undifferentiated mesenchyme;
E. Tunica muscularis externa.

Micrometer scale insert: 1 space- 0.01 mm.

 

 

 

73

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

17.

18.

19.

20.

21.

22.

PLATE VI

Cross section through the gizzard of an ll-day
fetus (showing cilia). Hematoxylin-eosin.
Magnification X520.

A. Epithelium; B. Undifferentiated mesenchyme.

Cross section through the gizzard of a 15-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Glands; E. Tunica
muscularis.

Cross section through the gizzard of an l8-day
fetus. Hematoxylin-eosin. iagnification X170.
A. Epithelium; B. Glands; C. Submucosa;

E. Tunica muscularis; F. Serosa.

Cross section through the gizzard of a 20-day
fetus. Hematoxylin-eosin. Magnification X170.
A. Epithelium; B. Glands; C. Submucosa;

E. Tunica muscularis; F. Serosa.

,Cross section through the small intestine of a

12-day fetus. Hematoxylin-eosin. Magnification
X170.

A. Epithelium; C. Undifferentiated mesenchyme;
E. Circular muscles of tunica muscularis.

Cross section through the small intestine of a
l7-day fetus. Hematoxylin-eosin. Magnification
X170.

A. Epithelium; C. Lamina propria; E. Tunica
muscularis.

Micrometer scale insert: 1 space = 0.01 mm.

 

 

75

Fig.

Fig.

Fig.

Fig.

23.

24.

25.

26.

PLATE VII

Cross section through the small intestine of a
20-day fetus. Hematoxylin-eosin. Magnification
X170.

A. Epithelium; E. Tunica muscularis;

F. Serosa. .

Cross section through the large intestine of an
11-day fetus. Hematoxylin-eosin. Magnification
X170.

A. Epithelium; C. Undifferentiated mesenchyme;
E. Tunica muscularis.

Cross section through the large intestine of a
l4-day fetus. Hematoxylin-eosin. Magnification
X170.

A. Epithelium; C. Undifferentiated mesenchyme;
E. Tunica muscularis; F. Undifferentiated
mesenchyme.

Cross section through the large intestine of a
20-day fetus. Hematoxylin-eosin. Magnification
X170.

A. Epithelium; C. Lamina propria; E. Tunica
muscularis.

Micrometer scale insert: 1 Space - 0.01 mm.

 

 

 

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