Difference between revisions of "Paper - Early differentiation of the foregut in the dog"

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=Early Differentiation of the Foregut in the Dog=
 
=Early Differentiation of the Foregut in the Dog=
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by
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Andrew George Hendrickx
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B. A., Concordia College Moorhead, Minnesota, 1959
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A Master's Thesis
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submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE
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Department of Zoology
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Kansas State University Manhattan, Kansas
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1961
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LP
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2UUf ..
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TV
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7 £ TABLE OF CONTENTS
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INTRODUCTION AND LITERATURE REVIEW 1
 +
 +
Thyroid t 4
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Liver mt 7
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 +
Respiratory System 8
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 +
METHODS AND MATERIALS 10
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OBSERVATIONS 12
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Free Blastocyst Stage 12
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Primitive Streak Stage 12
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Preforegut Stage: 4-5 Somites 14
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Early Foregut Stage: 7 Somites 15
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Foregut Stage: 7-9 Somites 16
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Pharyngeal Pouch I and II Stage: 8-13 Somites 17
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Second Closing Membrane Stage: 15 Somites 19
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Liver Differentiation Stage: 17 - 19 Somites 21
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Rupture of the Oral Plate Stage: 21 - 23 Somites 23
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Lung Differentiation Stage: 26 - 27 Somites 25
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Thyroid Differentiation Stage: 29 - 30 Somites 27
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INTERPRETATIONS AND DISCUSSION 28
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Pharyngeal Pouches 30
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Thyroid , 32
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Lung Anlage , 33
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Liver Development 34
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SUMMARY 36
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ACKNOWLEDGMENTS 38
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LITERATURE CITED 39
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APPENDIX t 42
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INTRODUCTION AND LITERATURE REVIEW
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Knowledge of the embryological development of the mammalian pharynx is far from being complete. The pharyngeal regions of few mammals have been thoroughly investigated. This structure in the dog has only been studied to a limited extent (Godwin, 9). Critical literature dealing with pharyngeal derivatives in mammals consist of reports by Heuser and Streeter (13) on the human and the pig, Keibel (16), Kingsbury (17), Mall (20), Norris (23), and Weller (30) on the human and a report by Godwin (9) dealing strictly with the dog.
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Heuser and Streeter (13) reported that the appearance of endoderm cells which budded off from the inner cell mass of a pig's trophoblast characterized the initial appearance of endoderm formation. Soon after the endoderm cells budded off they became arranged in a plate. Hill and Hartman (according to Heuser and Streeter, 13) first observed the origin of endoderm from the inner cell mass in marsupials as cells arising from a formative area and spreading out from there.
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Descriptions of the early differentiation of the visceral regions of the human were lacking except for some modeled specimens at the four somite stage (Heuser and Corner, 12) which show a shallow foregut. The sides of the head, however, appeared smooth, without swellings, which would indicate the absence of pouches. Weller (30) described a two somite human embryo which according to his description possessed the first pharyngeal pouch. The beginning of the foregut was described by Lewis (19) as a slight elongation of the yolk sac. Grosser (10) stated that pharyngeal pouches grew both laterally and dorsally from the anterior part of the digestive tract with a "ventral pharyngeal groove" extending along the mid ventral wall of the pharynx, with lateral
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grooves connecting into it. At the seven somite stage of human embryos (Dandy, 5) a single pair of pharyngeal pouches in the form of dorso-lateral outgrowths was described. The endoderm of the first pharyngeal pouch was separated from the head ectoderm by only a narrow interval which was unoccupied by mesodermal cells. A buccopharyngeal membrane (oral plate) in the sense that ectoderm and endoderm were in contact was not found. The second pharyngeal pouch first showed in the seven and eight somite embryos and was clearly visible externally in the nine somite embryos (Bartelmez and Evans, 2) and at nine somites the first pharyngeal groove was seen as a distinct depression.
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Corner (4) described the foregut of a 10-somite human embryo, as being compressed dorso-ventrally with the anterior end immediately under the forebrain. The hypopharyngeal groove was represented as a low ridge continuous from an area posterior to the thyroid pouch to the anterior intestinal portal where it became a broad prominence. The first pharyngeal pouches and closing membranes were formed. The second and third pharyngeal pouches were interpreted as slight bulges on the right side only. The dorsal wall of the foregut showed two ridges formed by the dorsal aortae, separated by a groove produced by the notochord as it "projected" from the endodermal wall.
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According to Grosser (10) the first pouch appeared shortly after the separation of the anterior part of the digestive tract from the yolk sac and reached the ectoderm in the ten somite stage. At this time the third pouch was formed and it had reached the ectoderm in an embryo with 23 somites. The anlage of the fourth pouch also showed in the 23 somite embryo. External grooves were not evident when the pharyngeal pouches contacted the ectoderm. The first development of external grooves occurred during the period between
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the formation of the tenth and fifteenth pairs of somites. At the height of the development of the pouches the embryonic pharynx was flattened dorsoventrally, was convex dorsally and had a somewhat triangular outline, the base being directed dorsally and apex ventrally. Heuser and Streeter (13) found no evidence of a second branchial groove in the 11-somite embryo. In human embryos from 13 to 20 somites (Streeter, 27), the pharyngeal pouches appeared with endoderm contacting the ectoderm and a third pharyngeal pouch not yet contacting ectoderm. In human embryos of 21 to 29 somites, the fourth pharyngeal pouch may or may not be present but made no contact with the ectoderm. Davis (6) described a 20-somite human embryo in which the fourth pouch was a slight ventro-lateral outpocketing. The oral plate was reported (Streeter, 27) to have formed by the 13 somite stage and by the 20 somite stage it had two perforations.
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Rogers (25) first observed the foregut in rats of 3 somites. The first pharyngeal pouch formed by the 5 somite stage and by 10 somites the first closing membrane had formed. The second pharyngeal pouch formed by the 13 somite stage and its closing membrane by the 15 somite stage. The third pharyngeal membrane formed by 22 somites and its closing membrane by the 25 somite stage.
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According to Selle (26), bat embryos of seven or eight somites showed distinct head folds, pharyngeal membranes (oral plates) and the first pharyngeal pouch. The first closing membrane did not form until the eighth somite was complete but at that stage there was no sign of any other pouches. In 4 mm embryos the pharynx had flattened dorsoventrally, the pharyngeal membrane (oral plate) had broken and four pharyngeal pouches had formed, three of them in contact with the ectoderm. (Clapper (18) observed four pharyngeal pouches in guinea pigs of 5 mm. The closing membranes of the first three
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4
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pouches were observed, but the fusion of the ectoderm and endoderm was not intimate.
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Thyroid
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Weller (30) noted the first evidence of the presence of the median thyroid component was a well defined fold in the ventral wall of the pharynx before the second pair of somites had formed and consisted of a proliferation rather than a differentiation of pharyngeal cells. The thyroid anlage was recognized by Grosser (10) before the first closing membrane had formed. "It appeared as a prominence in the ventral wall of the pharynx, belonging primarily to the medial region between the first two ventral pharyngeal grooves, that is to say, to the oral portion of what is to be the area mesobranchiales. M It was at first anterior to the second branchial arch. In a four somite embryo studied by Bartelmez and Evans (2) the median thyroid was only H a questionable fold" in the ventral wall of the pharynx. Dandy (5) recognized no thyroid primordium in a seven somite embryo but mentioned a "ventral pouch" posterior to the first pair of pharyngeal pouches.
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The thyroid gland of a 10 somite human embryo (Corner, 4), appeared as a "laterally compressed protrusion from the median line fitted in between the diverging aortae, whose cells weren't differentiated from those of the pharyngeal area."
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The thyroid in a 5-somite rat embryo (Rogers, 25) was a "slightly concaved circumscribed area of epithelium" in the median part of the floor of the pharynx, dorsal to the aortic sac and caudal to the first aortic arches. By 10 somites it *as more concave and by 26 somites it was a "cup shaped evagination." By 29 somites it was being drawn back caudally with the aortic sac. As a result it became a "flask-shaped" structure attached to the pharynx
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by a stalk. The thyroid anlage of the guinea pig (Klapper, 18) formed at the ventral extremity of pouch one in the groove through the midline. The median thyroid (Weller, 30) undergoes changes in its external form becoming spherical. In a 4 mm embryo it appeared as a round, elongate evagination extending ventrally and caudally from the caudal floor of the first pharyngeal pouch, lined with typical pharyngeal endoderm cells while elongate cells with much cytoplasm were arranged peripherally with their long axes along the radii of the sphere. Streeter (27) illustrated the thyroid in a sagittal view of a 16-somite human embryo as a ventral evagination of the floor of the pharynx just caudal to the aortic bulb.
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Grosser (10) described the thyroid of a 23 somite embryo as a small, hollow, rounded diverticulum connected to the floor of the pharynx. Norris (23) described the thyroid anlage of the human as having been a fairly definite and rather extensive shallow evagination from the "mesobranchial region of the pharyngeal floor, H between the ventral extremities of the first two pairs of gill pouches.
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Godwin (9) considered the earliest thyroid anlage in the dog embryo as H a thickened area of epithelium in the midpharyngeal floor extending slightly caudad to the furrow formed at the mid-ventral junction of the mandibular arches to the caudal limit of the aortic sac. M A similar thickened area was described as projecting into the angle formed by tha union of the first and second arches with the aortic sac. The thyroid plate was depressed in the median portion and folded toward the anterior and posterior edges of the plate. The surface cells were columnar and laterally compressed, but the surface was irregular and sagged due to squeezing. In older stages the plate was thickened and buds of cells projected ventrally. At a later stage these buds elongated
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downward and the thyxoid plate decreased rapidly.
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In the cat Ramsay (24) found no indication of thyroid diverticulum at the 11 somite stage but a M mid-ventral longitudinal groove" not limited to the thyroid area appeared through the "propharynx" at 14 somites. In the 16 - 18 somite groups, a slight depression in the pharyngeal floor appeared immediately over the aortic sac between the tips of the first pharyngeal pouches. By 22 - 23 somites, a thyroid plate was recognized with a "thyroid pit". Growth in the "thyroid pit" area caused thickening of the plate, and resulted in reduction of the pit. With the increase of the thyroid plate activity, mesenchyme underlying the floor of the pharynx increased and migrated between the plate and the aortic sac, countersinking the plate and constricting the attachment to the pharyngeal floor.
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Moody (22) noted in his description of the thyroid gland of a 5 mm pig, "the median thyroid gland was a syncytium forming a bilobed, elongated mass irregular in outline lying in the mesodermal syncytium on the ventral and lateral walls of the aorta level with the second gill arch and was still attached to the pharynx by a cord of cells." Because of its position and the fact that the lateral elements of the gland were paired suggested that the pig's thyroid was already a paired organ.
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According to Selle (26) the thyroid anlage of bat embryos first appeared at eight somites (3 mm) as a cup-shaped groove in the floor of the pharynx anterior to the second pair of pharyngeal pouches. The endoderm of the ventral pharyngeal wall, including the "thyroglossal groove" was four or five layers of cells thick, but the dorsal wall was a single layer and the lateral walls were a double layer. In a 3.8 mm embryo the thyroid anlage was a solid oval mass ventral to the pharynx and dorsal to the bulbus, at the fork of the
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aortic arches. Its anteroposterior extension was 150 u of which only the medial portion was in contact with the pharyngeal floor. In the 4 mm embryo the entire thyroid mass was anterior to the second pharyngeal pouches, and its contact with the floor of the pharynx was about 40 cells wide at its widest point.
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Liver
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Streeter (27) described the first indications of liver development occurring at the level of the first somite in a 13-somite human embryo. There was an active proliferation of cells from the "coelomic tract" which was to form the early mesoblastic framework of the liver. "The gut epithelium lies under the more dorsal part of the liver primordium and doubtless is playing a role in its development".
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Lewis (19) described the liver in an early human embryo as a median outgrowth of the endodermal tube appearing as a well-defined cul-de-sac which showed extensive proliferation in the anterior and ventral walls in a 4 mm embryo. These proliferating cells formed irregular masses and anastomosing cords which by 4.9 mm formed a large crescentic mass to "an outpocketing of the intestine."
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Mall (20) in a description of a two week old human embryo measuring 2.1 mm, stated that the liver bud was well marked, encircling and invading the left omphalomesenteric vein, carrying the endothelial lining ahead of the sprouts and forming a series of sinuses as subdivisions of the lumen of the vein.
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Johnson (14) in an early account of liver development in the ground squirrel measuring 1.5 mm, stated that the anlage was a thickened area in the
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8
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ventral wall of the foregut, anterior to the yolk stalk, continuing laterally a short distance onto the walls of the digestive tube with the lateral extent being greater than the antero-posterior extent. As the embryo increased in length to 2 ran, the hepatic thickening was a distinct outpouching of the gut; by 2.5 mm it was a smooth walled outpouching of the foregut, connected to the ventral wall by a short stalk; by 4.5 mm the common bile duct had formed.
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Respiratory System
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The anlage of the lungs in the pig were described by Flint (7) as being unpaired and asymmetrical. He considered pulmonary anlage to arise from the ventral part of the head gut behind the sinus venosus, as a ventral outgrowth, preceded by a lateral flattening of the foregut posterior to the gill pouches at 3.5 mm. In the 4 mm embryo, he described "longitudinal furrows" dividing the foregut into two parts: a ventral respiratory portion and a dorsal digestive segment. From the posterior extremity of the pulmonary anlage, he found two lateral asymmetrical outgrowths, which gave rise to the stem bronchi. At this point he described the respiratory and digestive portions as separating, the separation beginning at the posterior end of the anlage and extending anteriorly along the line formed by the two longitudinal furrows. Corner (4) described the lung primordia of a 10-somite human embryo as an enlarged terminal portion of the laryngotracheal groove. In bat embryos measuring 3.8 to 4 mm the laryngotracheal groove and unpaired lung primordium was identified by Selle (26).
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The beginning of the respiratory apparatus according to Grosser (10) appeared prior to the formation of the last two closing membranes caudal to the pharyngeal pouches as a median ventral groove, the anterior portion of
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the groove forming a "ridge on the outer surface of the epithelium" correspond^ ing to the laryngotracheal groove. The posterior end of the groove was described as being rounded and was interpreted to be the unpaired anlage of the lungs; as growth continued the lung anlage and then the tracheal groove became separated from the esophagus.
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Corner (4) and Heuser (11) interpreted the laryngotracheal groove as a "low ridge" extending along the ventral pharyngeal wall. It extended between the posterior extremity of the thyroid pouch and the anterior intestinal portal, at which point it spread out into a wider prominence where it passed into the yolk sac. This enlarged terminal portion was interpreted as the unpaired primordium of the lungs. Photographed sections (Corner, 4) showed a medio-ventral evagination in the ventral wall of the foregut that appeared to be continuous with the thyroid anlage.
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Wallin (28) recognized no lung buds in a 13-somite human embryo. A thickening of the endoderm caudal to the venous end of the heart in a 17 somite embryo marked the beginning of the lungs (Atwell, 1). In twin embryos of 17 - 19 somites no digestive or respiratory anlage were identified by Watt (29) possibly because of extreme dorsoventral compression of the embryos he studied.
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In a 20-somite human embryo, the laryngotracheal groove was described (Davis, 6) as a ventral angle beginning cranial to the fourth pharyngeal pouch and opposite the sinus venosus, terminating in a swelling which was the lung anlage. In a 24-somite human embryo, Johnson (15) described a median longitudinal groove separating two elongate swellings: the lower portion was interpreted to be the lung bud and the dorsal part the esophagus
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10
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METHODS AND MATERIALS
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The 35 embryos used in this study were obtained from wild coyotes and from dogs of various breeds maintained for that purpose. The coyote embryos were removed from freshly killed animals obtained during coyote hunts and placed in the series according to the somite number and size of the uterine swellings. The ages of dog embryos were established with the aid of vaginal smears by which ovulation time was determined (Gier, 8). Some of the embryos used for this phase of dog embryology had been previously sectioned for other studies. Additional critical stages were selected from previously collected material or by laporatomies performed specifically for these stages. Embryos were stained with aceto-carmine, drawn by camera lucida or microprojection and then sectioned at 10 u. The embryos were stained with a modified Harris' hematoxylin or iron hematoxylin and counter stained with an orange G-acid fuchsin combination. Photographs were made of whole embryos and sections as needed.
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Comparisons between crown-rump measurements and somite number showed little consistency, consequently somite counts were used exclusively for stage designations. The incomplete somite immediately posterior to the tenth cranial nerve was counted as the first paired somite, and the last somite was considered to be the one anterior to the last intersomite groove.
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1 Kansas State Agriculture Experiment Station Project 321» Dog Embryology, under the direction of Dr. H. T. Gier.
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11
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Table 1. Embryos studied.
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■ 1
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 +
Somite
 +
 +
 +
 +
Age
 +
 +
s Type of i
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 +
 +
Collection
 +
 +
 +
 +
 +
Stage :
 +
 +
 +
Number
 +
 +
 +
 +
(days)
 +
 +
section j
 +
 +
 +
Number
 +
 +
 +
Blastocyst
 +
 +
 +
 +
 +
 +
 +
Transverse
 +
 +
 +
1123
 +
 +
 +
 +
 +
Primitive Streak
 +
 +
 +
 +
 +
 +
 +
Longitudinal
 +
 +
 +
65L
 +
 +
 +
 +
 +
Preforegut
 +
 +
 +
4
 +
 +
 +
16
 +
 +
 +
Transverse
 +
 +
 +
260LC
 +
 +
 +
 +
 +
 +
 +
4
 +
 +
 +
16
 +
 +
 +
Transverse
 +
 +
 +
117L
 +
 +
 +
 +
 +
 +
 +
5
 +
 +
 +
17
 +
 +
Transverse
 +
 +
 +
260Lb
 +
 +
 +
 +
 +
 +
 +
5
 +
 +
 +
17
 +
 +
Transverse
 +
 +
 +
117L
 +
 +
 +
 +
 +
Early Foregut
 +
 +
 +
7
 +
 +
17
 +
 +
Longitudinal
 +
 +
 +
260Rb
 +
 +
 +
 +
 +
Foregut
 +
 +
 +
7
 +
 +
 +
17
 +
 +
Transverse
 +
 +
 +
761C
 +
 +
 +
 +
 +
 +
 +
8+
 +
 +
 +
17
 +
 +
Transverse
 +
 +
 +
45L-6
 +
 +
 +
 +
 +
 +
 +
9
 +
 +
 +
17
 +
 +
 +
Transverse
 +
 +
 +
120LC
 +
 +
 +
 +
 +
Pharyngeal Pouch
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
1 & 2
 +
 +
 +
9
 +
 +
 +
17
 +
 +
 +
Transverse
 +
 +
 +
77L
 +
 +
 +
 +
 +
 +
 +
10
 +
 +
 +
17
 +
 +
 +
Transverse
 +
 +
 +
115La
 +
 +
 +
 +
 +
 +
 +
11
 +
 +
 +
17
 +
 +
 +
Longitudinal
 +
 +
 +
120L
 +
 +
 +
 +
 +
 +
 +
11
 +
 +
 +
17
 +
 +
 +
Transverse
 +
 +
 +
184
 +
 +
 +
 +
 +
 +
 +
12
 +
 +
 +
17+
 +
 +
 +
Transverse
 +
 +
 +
120 Rd
 +
 +
 +
 +
 +
 +
 +
12+
 +
 +
 +
17+
 +
 +
 +
Transverse
 +
 +
 +
115Rb
 +
 +
 +
 +
 +
 +
 +
12+
 +
 +
 +
17+
 +
 +
 +
Transverse
 +
 +
 +
118
 +
 +
 +
 +
 +
 +
 +
13
 +
 +
 +
17+
 +
 +
 +
Transverse
 +
 +
 +
154Rc
 +
 +
 +
 +
 +
Second Closing
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
Membrane
 +
 +
 +
15
 +
 +
 +
18
 +
 +
Longitudinal
 +
 +
 +
113Lb
 +
 +
 +
 +
 +
 +
 +
15+
 +
 +
 +
18
 +
 +
Transverse
 +
 +
 +
77L
 +
 +
 +
 +
 +
Liver Differentiation
 +
 +
 +
17
 +
 +
 +
18
 +
 +
Transverse
 +
 +
 +
SILa
 +
 +
 +
 +
 +
 +
 +
17
 +
 +
 +
18
 +
 +
Transverse
 +
 +
 +
77L-1
 +
 +
 +
 +
 +
 +
 +
17
 +
 +
 +
18
 +
 +
Longitudinal
 +
 +
 +
H3La
 +
 +
 +
 +
 +
 +
 +
17+
 +
 +
 +
18
 +
 +
Transverse
 +
 +
 +
121L
 +
 +
 +
 +
 +
 +
 +
18
 +
 +
 +
18
 +
 +
 +
Longitudinal
 +
 +
 +
81Lb
 +
 +
 +
 +
 +
 +
 +
18
 +
 +
 +
18
 +
 +
 +
Longitudinal
 +
 +
 +
81Lc
 +
 +
 +
 +
 +
 +
 +
19
 +
 +
 +
18
 +
 +
 +
Transverse
 +
 +
 +
77Ra
 +
 +
 +
 +
 +
Rupture of Oral Plate
 +
 +
 +
20
 +
 +
 +
18+
 +
 +
 +
Transverse
 +
 +
 +
121L
 +
 +
 +
 +
 +
 +
 +
21
 +
 +
 +
19
 +
 +
Longitudinal
 +
 +
 +
116Rb
 +
 +
 +
 +
 +
 +
 +
21
 +
 +
 +
19
 +
 +
Transverse
 +
 +
 +
42L
 +
 +
 +
 +
 +
 +
 +
21
 +
 +
 +
19
 +
 +
Transverse
 +
 +
 +
123L
 +
 +
 +
 +
 +
Lung Differentiation
 +
 +
 +
23
 +
 +
 +
19
 +
 +
 +
Transverse
 +
 +
 +
65R
 +
 +
 +
 +
 +
 +
 +
26
 +
 +
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20
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Transverse
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unknown
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Thyroid Differentiation
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29
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21
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Transverse
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83Ra
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30
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21
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Transverse
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83Rb
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12
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OBSERVATIONS
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Free Blastocyst Stage
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The youngest embryo studied was pre-somite, 14 day gestation, sectioned in the uterus. The uterine swelling measured 3 mm inside diameter, the uterine epithelium was continuous and extended outward into the uterine glands. Uterine milk occupied the area between the uterine epithelium and the blastocyst. The blastocyst itself was crumpled, so that exact measurements could not be determined but it obviously had conformed closely to the cavity within the uterine swelling. The single layer of ectoderm cells was dispersed in an irregular layer around the endodermal layer. The nuclei of the ectodermal layer took a dark blue stain and the abundant cytoplasm stained a light pink. The endoderm, also composed of a single layer of cells, lay immediately inside the ectoderm. It differed from the ectoderm in that the nuclei were spaced more widely and were almost twice the diameter of those in the ectodermal layer. The nuclei of the endoderm cells stained light blue and appeared granular. The cytoplasm, which was present in smaller amounts than that of the ectoderm, formed a thin continuous sheet between the nuclei. At this stage mesoderm had not appeared. A very thin, light staining fluid filled the blastocoel.
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Primitive Streak Stage
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In a 15 day old embryo, located in a uterine swelling measuring 4 mm inside diameter, three definite germ layers were identified (Plate II, Fig. 2) with a distinct embryonic disc and a primitive streak oriented transverse to the longitudinal axis of the horn of the uterus. The ectoderm (trophoderm)
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13
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was almost in contact with the uterine epithelium but the embryonic disc was distinctly cushioned by a film of uterine milk. At its medial portion, the primitive streak measured 1.3 mm in length and was three or four cell layers thick. At the most anterior extension of the primitive streak the primitive pit was identified as a depression in the dorsal wall 60^ in width (Plate II, Fig. 1). The area immediately ventral to the primitive pit was undifferentiated except for the thin loosely arranged endoderm layer, but posteriorly the three germ layers were reasonably distinct. The germ layers became more sharply identified posterior to the primitive streak.
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The neural plate, whose cells were closely packed and several cell layers thick, extended anteriorly 1.3 mm from the anterior tip of the primitive pit. The endodermal layer ventral to the primitive streak was a light staining, continuous layer of cytoplasm with few nuclei. Extending both anteriorly and posteriorly for a limited distance nuclei of endoderm cells were widely scattered and stained lightly. As the germ layers became more distinct posteriorly, the nuclei were more numerous and stained somewhat darker. Beyond the mesodermal layer the nuclei of the endodermal layer again became widely spaced and stained lightly like those described in the previous stage.
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More anteriorly, ventral to the neural plate, the nuclei of the endoderm gradually increased until they became two to three layers thick under the anterior most extension of the neural plate (Plate II, Fig. 3). The cells at this point had a columnar appearance. This thickened layer was 15 to 20 cells (200 ^j) in length and approximately 570 /i wide. The single layer of endodermal cells extended anteriorly where the nuclei gradually decreased in number and in staining intensity at the level of the anterior most extension of the mesoderm. In the mesoderm free area, the ectoderm and endoderm were
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14
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each only one cell layer in thickness with the layers in close apposition. The endodexm and ectoderm layers were continuous around the entire vesicle. The endoderm in the mesoderm free area retained the more primitive appearance described in the previous stage.
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Preforegut Stage: 4-5 Somites
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In the youngest embryos of this stage the endodermal layer was continuous under the entire embryonic area. The endodermal cells were cuboidal with round, darkly staining nuclei surrounded by a light staining granular cytoplasm. Immediately anterior, and also ventral to the anterior edge of the neural plate, the nuclei of the endoderm were tightly packed and pseudostratified. Pseudostratification gradually decreased posteriorly so that the endoderm was a single cell layer posteriorly from the last point of fusion with the neural plate.
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The endoderm was unevenly fused with the neural plate from the anterior tip of the plate through the region of the heart anlage (Plate I, area 1 and 2). The area of union between endoderm and neural plate was thus a strip approximately 12 cells wide in the middle, diminishing in width and in closeness of contact both anteriorly and posteriorly. Throughout the primitive streak the endoderm had again thickened to a low columnar type.
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In the five somite embryo the medial endodermal cell mass extended from the anterior edge of the proamnion to beyond the head fold (anterior part Plate I, area 1) increasing to a thickness of five or six cell layers for a short distance.
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Posterior to the head fold (Plate I, area 1) fusion was evident between the neural plate and the endoderm of the yolk sac. The heart anlage had
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15
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differentiated as endocardial tubes considerably further anteriorly than in the younger stages. The heart anlage (epimyocardial plate and endocardial mass) in the older embryos had been pulled anteriorly over the forming head fold. From its first appearance the heart anlage (Plate X» ^ area 2) pushed ventrally and medially resulting in a fold in the endodermal layer (Plate II, Fig. 4). The fold in the endoderm was secured under the medial edge of the splanchnic mesoderm which bordered the medial edge of the embryonic coelom. The fold of endoderm was continuous throughout the length of the heart anlage, but disappeared posteriorly. Medially to the fold described above the endoderm cells were cuboidal, but through the fold and continuing laterally to the heart anlage it was a high columnar epithelium. Immediately ventral to the heart anlage it again became cuboidal.
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Posterior to the heart anlage, throughout the somite region, and into the primitive streak area (Plate I, area 2, 3, 4), the endoderm lay close to the neural plate, but fusion was not distinct. In the posterior end of the somite region (Plate I, area 3) the endoderm ventral to the flat neural plate was a low columnar epithelium and the width of fusion between the layers decreased. Within the central area of the primitive streak the cells were undifferentiated, although laterally three germ layers were recognized. As in the younger embryos the endoderm cells were cuboidal posterior to the heart anlage and low columnar in the medial region through the primitive streak areas, changing to a simple cuboidal epithelium laterally.
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Early Foregut Stage: 7 Somites
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At this stage the endoderm of the proamnion varied from a pseudostratified columnar epithelium in the anterior half to a mass of cells four nuclei
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16
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thick in the posterior half. The endoderm under the hesdfold and forebrain area was a double pseudostratified columnar epithelium, similar to the thickened area in the previous stage, but from this point posteriorly it was a simple cuboidal layer. Immediately posterior to the head fold and medioventral to the neural plate the endoderm formed a sharp but shallow fold (Plate II, Fig. 5) marking the anterior most tip of the foregut. The anterior edge of the thickened endodermal mass of the proamnion marked the area of the margin of the anterior intestinal portal (MAIP).
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The MAIP represents the posterior extension of the foregut and the point where it becomes continuous with the yolk sac. The endoderm was closely fused to the neural plate from the tip of the foregut through the region of the metencephalon, although posterior to this area it was in close association but not fused to the neural plate.
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Laterally the heart anlage caused the ventro-medial folding of the endoderm, similar to that described in the preceding stage.
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Foregut Stage: 7-9 Somites
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The foregut appeared as a dorso-ventrally flattened pocket, with the anterior tip slightly posterior to the head fold, and the lateral edges projecting slightly dorsally. The length of the foregut was 120 p, slightly less than the thickness of the fifth cranial nervet the anterior tip of the foregut was almost directly ventral to the anterior edge of the fifth cranial nerve and the MAIP was under the posterior part of the fifth cranial nerve. The dorsal wall of the foregut was characterized by the medially thickened mass ventral to the neural plate, described in the previous stage, and a single layer of cuboidal epithelium laterally from this line. Laterally on both sides of the medial thickened mass, the wall pushed ventrally with the dorsal
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17
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aortae located in the shallow concavities, forming the hyperpharyngeal groove between the aortae. The thickened mass of the dorsal wall was continuous through the MAIP but persistent fusion with the neural plate was observed only in the anterior half of the foregut.
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The oral plate appeared under the anterior tip of the foregut, consisting of single layers of ectoderm and endoderm cells which were closely fused, extended laterally a distance of 20 to 55 nuclei (70^) and 100 p anteroposterior^. In embryo 761c, because of external pressure, the oral plate was forced posteriorly so its actual length could not be determined. The MAIP was characterized by the change of the lateral walls of the foregut from a pseudostratified layer to a cuboidal layer. The fused endocardial tubes ventral to the foregut, became paired structures lateral to the endodermal fold with the flattening of the yolk sac.
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Pharyngeal Pouch I and II Stage: 8-13 Somites
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The shape of the foregut was unchanged from the previous stage but Sessel's pocket was identified as a blind pouch anterior to the oral plate. The oral plate extended posteriorly twice the distance that it did in the previous stage. Fusion between the neural plate and the foregut was continuous from a point dorsal to the oral plate to a point posterior to the second pharyngeal pouch in younger embryos (8 - 11 somites) and beyond the MAIP in the older embryos (12 - 13 somites). The fifth cranial nerve now occupied a position dorsal to the anterior edge of the oral plate with its mandibular branch ventral and anterior to the tip of the first pharyngeal pouch. Near the center of the oral plate there was a rounded medial thickening of endodermal cells that first appeared in the 8-somite stage and was persistent until the oral plate broke in 21 somite embryos. The first pharyngeal
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18
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pouches were distinguishable as lateral expansions of the pharynx posterior to the oral plate and fifth cranial nerve, pressing the head mesoderm aside and fusing with the head ectoderm, thus forming the first closing membrane (Plate II, Fig. 6). In the later stages (11, 12, 13 somites) the first closing membrane protruded beyond the lateral margin of the adjoining head ectoderm resulting in the formation of shallow external grooves at both margins, with complete fusion between the two points (Plate II, Fig. 6). The first closing membrane extended from an area posterior to the fifth cranial nerve postero-ventrally to a point ventral to the anterior edge of the auditory placode. The head fold varied somewhat but in all stages it was located ventral to the anterior edge of the first closing membrane.
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The hypopharyngeal groove was identified as a shallow, medially located evagination extending from the oral plate to the MAIP, although between the first and second pharyngeal pouches it was very shallow. It reached its deepest depression between a point posterior to the fifth cranial nerve and the anterior edge of the auditory placode, between the ventral ends of the first pharyngeal pouches. Ventro-lateral to the posterior edge of the auditory placode the pharynx again extended laterally forming the second pharyngeal pouch. In the eight to eleven somite stages, two to three layers of mesodermal cells separated the endodermal lining of the second pouch from head ectoderm. In the later stages (12-13 somites) the head mesoderm had been pushed aside and in one case (13 somite) the endoderm and ectoderm were adjacent to each other but fusion was not definite. The ninth cranial nerve was located dorsal to and in the immediate region of the posterior edge of the second pharyngeal pouch. At this same level the hypopharyngeal groove deepened, expanding laterally more posteriorly, forming the splanchnopleuric fold. The pseudostratified epithelium of the hypopharyngeal groove anterior
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19
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to the second pharyngeal pouch thinned posteriorly, undergoing a transition to the typical cuboidal epithelium of the yolk sac at the MAIP.
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Throughout the pharyngeal region, the dorsal wall was a single layer of nuclei compared to a double or triple layer of nuclei in the lateral and ventral walls. Fusion of the endoderm of the hyperpharyngeal groove with the neural tube occurred throughout the length of the pharyngeal region.
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In the older stages (12 - 13 somites) ventral to the aortic arch and posteriorly there was a proliferation of splanchnic mesoderm of the apparent yolk sac dorsally into the coelom. Posteriorly and laterally this proliferation was continuous with the paired epimyocardial plates. The "yolk sac" endoderm associated with this thickened splanchnic mesoderm was a low columnar epithelium fading laterally into the typical cuboidal epithelium of the yolk sac.
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In the latest stage (13 somites) in this series, the liver anlage was identified as a lateral extension of the hypopharyngeal groove on the left side only, immediately anterior to the MAIP. This anlage was a pseudostratified columnar evagination, about 70 p long ending posteriorly on the splanchnopleuric fold.
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Second Closing Membrane Stage: 15 Somites
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The general structure of the anterior pharynx closely matched that of the previous stage. The oral plate, however, had expanded posteriorly half way the length of the first pharyngeal pouch, and the previously described thickening of the oral plate was pronounced, extending posteriorly to the edge of the plate. The first closing membrane was broadened considerably, pushed further laterally and fusion of the two germ layers was more intense.
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20
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From a point just behind the oral plate to beyond the first pharyngeal pouch the ventral wall of the pharynx was pushed dorsally by the developing bulbus thus obliterating the hypopharyngeal groove.
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The second pharyngeal pouch was again ventral to the posterior edge of the auditory placode; just posterior to this point the second closing membrane had formed over a length of approximately 60 p. This closing membrane pushed laterally beyond the adjacent head ectoderm forming slight external grooves dorsal and ventral to the fused membrane.
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The thyroid anlage was first identifiable in this stage. It appeared as a small proliferation of cells extending ventrally from the hypopharyngeal groove into the expanded aortic sinus ventral to the anterior edge of the auditory placode. The thyroid anlage was approximately 50 u long, 40 p wide and 25 p deep.
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The hypopharyngeal groove was again continuous from the oral plate to the MAIP although it was almost obliterated between the oral plate and the second pouch. The MAIP was ventral to the second intersomitic groove at this stage.
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Dorsal to the posterior edge of the ventricle, where the common cardinal veins join the sinus venosus, the splanchnic mesoderm of the yolk sac was proliferated dorsally with filamentous strands extending between the yolk sac endoderm and splanchnic mesoderm demonstrating fusion of the two layers. Several concentrated masses of splanchnic mesoderm was located ventro-lateral to the hypopharyngeal groove just anterior to the MAIP. These mesodermal proliferations were interpreted to be the precursors of the connective tissue of the liver.
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21
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The yolk sac endoderm had not changed from the previous stage except at the MAIP it was seven or eight cell layers thick. The splanchnopleuric fold consisted almost entirely of endoderm except for a thin layer of endocardium adjoining.
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The liver anlage appeared as a lateral evagination from the left side of the hypopharyngeal groove, only slightly larger than that described in the previous stage. There was yet no modification of the left side and no differentiation beyond the splanchnopleuric fold although the evagination extended as a flat groove into the MAIP.
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Liver Differentiation Staget 17 - 19 Somites
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Cephalic flexion had markedly increased at this stage, but the relationship of the pharyngeal pouches to the cranial nerves and the auditory placode had not changed. At its posterior border the oral plate was only a single cell layer, in which both endoderm and ectoderm cells blended together, compared to the double germ layers anteriorly, and appeared to be near the point of rupture in the 19 somite stage. The ingrowth of the oral plate previously described had blended with the developing Rathke's pouch. The head fold had progressed to the posterior edge of the otocyst in all embryos in this group.
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The first pharyngeal pouch had increased in overall size, extended more dorsally, and the closing membrane was expanded more than in the previous stages. The first pharyngeal grooves were represented by a broad shallow depression, deeper dorsally and ventrally and only slightly depressed over most of the closing membrane. The second pharyngeal pouch, closing membrane and pharyngeal groove were similar to those of the first but not as extensive.
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22
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The third pharyngeal pouch had developed by the lateral expansion of the foregut a ntero- ventral to the first somite. The third closing membrane was not yet formed* several layers of mesoderm yet separating pharyngeal endoderm from head ectoderm. The expanding aortic sinus again pressed the floor of the pharynx dorsally, particularly in the 19 somite embryo where there was only a narrow slit of pharyngeal cavity. Posterior to the third pharyngeal pouch the hypopharyngeal groove reached its ventral-most extension, forming a deep elongated triangular groove which gradually widened near the MAIP. In one case (19 somites) the hypopharyngeal groove in this region was as wide as the foregut itself. The gut had elongated so that the MAIP was now ventral to the fourth intersomitic groove.
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The thyroid anlage was again ventral to the anterior edge of the otocyst, between the ventral ends of the first and second pharyngeal pouches, where the aortic sinus pushed farthest dorsally. The greatest area of thyroid proliferation was 80 ju wide, 80 ^ long and 40 )i deep although two embryos showed no such proliferation. The cells of the thyroid mass were more loosely arranged than those in the rest of the pharyngeal wall.
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Just posterior to the union of the common cardinal veins to the sinus venosus, the splanchnic mesoderm was a compact deeply stained multiple cell layer. The yolk sac consisted of a multiple cell layer at the MAIP diminishing gradually to a single cuboidal cell layer.
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In the early 17 somite stage, the groove of the liver anlage again appeared anterior to the MAIP on the left side only, continuing posteriorly through the splanchnopleuric fold and fading out ventral to the sinus anlage. The wall of the groove consisted of cuboidal epithelium thickening laterally to a low columnar epithelium medial and ventral to the sinus anlage (Plate III,
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23
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Fig. 1). No lateral evagination occurred on the right side but the endoderm was thickened comparable to that of the left side. The thickening of the endoderm decreased to the cuboidal yolk sac epithelium under the posterior limit of the sinus anlage.
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The late 17 and the 18 and 19 somite stages had lateral evaginations with thickened epithelium on both sides.
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In the 18+ and 19 somite embryos, the liver anlagen were considerably deeper than in the 17 somite stage. As the splanchnopleuric fold concressed, enclosing a greater length of the groove thus elongating the liver diverticulum anterior to the fold (Plate III, Fig. 2), posterior differentiation of the groove continued so there was as much liver anlage beyond the splanchnopleuric fold in the 19 somite embryo as in the 17 somite stage (Plate III, Fig. 3).
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The hyperpharyngeal groove was clearly identified, and fusion of pharyngeal endoderm, notochord, and neural tube was irregular but was evident posterior to the second somite in some embryos. Posterior to the second somite the three layers were separated by invasion of mesenchyme, and the hyperpharyngeal groove faded out until only a shallow notch remained beyond the fifth somite.
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Rupture of the Oral Plate: 21 - 23 Somites
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By this stage the oral plate had broken and only its lateral margin remained in the older stages. In the 21 somite embryo the anterior portion of the oral plate was intact and 4 - 5 cell layers thick but the posterior portion was gone, connecting the pharyngeal cavity with the oral cavity (Plate III, Fig. 5). Sessel's pocket was somewhat enlarged as compared to
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the previous stage and the anterior edge was at the level of the middle of the fifth cranial nerve. The hypopharyngeal groove was yet distinct.
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The first and second pharyngeal pouches essentially were unchanged. The third closing membrane was now formed ventral to the anterior part of somite one. The dorso-ventral expanse of the closing membrane was as great as that of the preceding pouches but a ntero -posterior fusion was limited.
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The thyroid anlage varied between embryos from a single to a double mass (Plate III, Fig. 5) with the double mass located variably either one behind the other or lateral to one another. The greatest proliferation measured 100 p in length, 100 jli in width and 45 p in depth differing only from the pharyngeal wall in its loose, irregular arrangement. The thyroid anlage forced the endothelium of the aortic sinus ventrally in one case.
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Posterior to the third pharyngeal pouch the gut narrowed gradually as the lateral wall pushed medially forming a flattened tube the ventral half of which is now designated as the laryngotracheal groove. Short lateral extensions persisted on the dorso-lateral surface presaging the formation of the fourth pharyngeal pouch, resulting in a modified appearance. The mesodermal lung masses were first identified at this stage as thickened mesodermal areas lateral to the laryngotracheal groove and ventral to a point midway through the third somite and anterior to the liver anlage. Just medial to the mesodermal lung masses the paired endodermal lung primordia were identified as slight thickenings in the ventro-lateral wall of the larynotracheal groove. The lung primordia were about 200 ju long, but only 25 p wide and 45 p deep.
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Between the fourth and fifth intersomitic grooves the wall of the laryngotracheal groove thickened to a four or five cell layer (about 40 p)
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25
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immediately anterior to the liver anlage. At a point ventral to the middle of the fifth somite, the liver anlagen had rotated medially and fused into a large medial mass from which several small groups of cells (liver cords) proliferated into the thickened splanchnic mesoderm just posterior to the sinus venosus. Within the central liver mass, a central cavity opens broadly into the gut immediately anterior to the MAIP. From this' central cavity, paired ducts evaginate antero-laterally (Plate III, Fig. 4) nearly paralleling the larger central duct. The central duct joins the intestinal tract just anterior to the level of the fifth intersomitic groove, posteriorly to the MAIP.
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The entire splanchnopleuric fold was composed of the multiple pseudostratified epithelium comprising liver anlage. The liver anlage blended with the cuboidal epithelium of the yolk sac ventro-latarally and also blended with the pseudostratified cells of the intestinal groove dorsally.
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Lung Differentiation Stages 26-27 Somites
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In the 26 somite embryo the anterior portion of the oral plate was still intact and the medial growth of endoderm described in all previous stages appeared only as a slight rounded mass. A few remnants of the oral plate persisted along the lateral wall of the expanded oral cavity. The first, second and third pharyngeal pouches, closing membranes and pharyngeal grooves of the second and third pouches were distinct only dorsal and posterior and somewhat ventral to the third pharyngeal pouch, the anlage of the fourth pouch was identified as a slight lateral extension of the pharyngeal wall.
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26
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The thyroid anlage continued as a single bud over the dorsal wall of the protruding aortic sinus. It measured 90 p in length, 70 p in width and 45 p in depth at the point of largest proliferation in the 26 somite embryo. In the 27 somite stage the aortic sinus protruded into the pharynx considerably less than it did in the 26 somite stage but the thyroid had doubled in size, forming a rounded mass 100 p long, 130 p wide and 100 p deep.
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Ventral to and slightly anterior to the third intersomitic groove, the pseudostratified epithelium of the laryngotracheal groove thickened from two nuclei to four or five nuclei. The thickened portion expanded laterally forming a hollow bulbular structure (Plate IV, Fig. 1 and Plate V, Fig. 3), representing the endodermal lung buds.
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Posterior to the lung anlage, the dorsal portion of the gut extended laterally as described in some of the earlier stages (15 - 19 somites). The hyperpharyngeal groove was still present although mesenchyne completely separated the notochord from both neural tube and endoderm.
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The liver anlage was essentially the same as that described in the previous stage except that 1) it had been displaced slightly posteriorly, 2) the lateral ducts had disappeared leaving the central duct opening into the gut lumen and, 3) the posterolateral limit of the liver anlage had been moved medially by rolling concresence of the vitelline veins.
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The endoderm and mesoderm of the MAIP lateral to the liver anlage were now fused, forming a true "splanchnopleuric fold" ventral to the sixth intersomitic groove.
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27
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Thyroid Differentiation Staget 29 - 30 Somites
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The pharyngeal grooves had become prominent. Small fragments of the oral plate were still visible anteriorly. The hyperpharyngeal groove persisted posteriorly from the infundibulum. The pseudostratified endodermal epithelium of the pharynx changed in the region of the old oral plate to the cuboidal epidermal epithelium of the oral cavity.
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The fourth pharyngeal pouch was further developed but mesenchyme several cells thick remained between the head ectoderm and the pharyngeal endoderm (Plate IV, Fig. 3).
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The thyroid diverticulum had expanded into a three lobed mass (Plate V, Fig. 1) 100 p long, 200 ju wide and 200 p deep, protruding into the aortic sinus (Plate IV, Fig. 4). The thyroid anlage, except for the ventral lobulation was a solid mass of randomly oriented cells, continuous on the edges with the general pharyngeal floor. There was a major proliferation of thyroid posteriorly from the main anlage, lying between the pharyngeal floor and aortic sinus and separated from them by layers of mesenchyme. The aortic sinus at this level was considerably reduced, no longer protruding into the pharynx.
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Posterior to the fourth pharyngeal pouch, the gut narrowed to an oblong cavity similar to the laryngotracheal groove of the previous stages. At the level of the third intersomitic groove the thickened epithelium of the laryngotracheal groove had expanded forming two distinct paired endodermal lung buds (Plate V, Fig. 3) with a triple layer of nuclei in the pseudostratified epithelium. In the 29 somite embryo only the right bud had expanded posteriorly far enough to contain a separate lumen in transverse section (Plate IV, Fig. 2); both were lumenated in the 30 somite embryo. Posterior and dorsal
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to the endodermal lung masses the internal pleural cavities had been formed between the mesodermal lung masses and the mediastinum.
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The liver showed only slight advances over that described for the 26-27 somite embryos. The MAIP had closed more posteriorly, now occurring ventral to the seventh intersomitic groove, thus becoming completely separated from the liver anlage which was connected to the floor of the gut ventral to the fifth intersomitic groove. There was no indication of subdivision of liver anlage into anterior and posterior liver diverticulae, rather there was a growth of liver cord cells from the single median liver diverticulum which lay between the vitelline veins.
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INTERPRETATIONS AND DISCUSSION
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Although the general outline of development observed in dog embryos follows roughly that described for other species, many points either conflict with descriptions by previous workers or were completely overlooked.
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The thickened endodermal mass, ventral to the anterior edge of the primitive streak stage, which represented the first appearance of the pharynx had not been described previously. By the five somite stage the thickened endodermal mass had fused with the anterior part of the neural plate. With the formation of the head fold and folds medial to the heart anlage (Plate II, Fig. 4), the endoderm was pulled forward forming a shallow foregut by the seven somite stage (Plate II, Fig. 5). The processes of foregut formation observed here compare favorably with that described for 2 somite rat embryos (Rogers, 25).
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In late seven somite dog embryos the shape of the foregut was generally similar to that of seven and ten somite human embryos (Dandy, 5; Corner, 4)
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29
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and early rat embryos (Rogers, 25), but the medially thickened area ventral to the neural plate was interpreted as the point of fusion between two germ layers, although Dandy (5) and Corner (4) considered it to be the notochord embedded in the endoderm. Rogers (25) failed to mention the notochord and considered the germ layers to be only in "close contact". His illustrations bear this out.
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Elongation of the foregut resulted from the continuous forward extension of the head region, and the posterior concresence of the endoderm medioventral to the heart anlage. This interpretation essentially agreed with Rogers (25) who considered the active factors to be expansion of the anterior part of the neural plate, mitotic divisions and a "tucking in" of the adjacent endoderm.
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The dorso-ventral flattening of the foregut was obvious from the first appearance of the notch in the endoderm foretelling the formation of a foregut, and was probably the result of the various forces involved in original formation rather than by lateral expansion as described in the rat (Rogers, 25). The foregut in both cases, nevertheless, was a definite dorso-ventrally flattened pocket before the appearance of the first pharyngeal pouches.
 +
 +
The oral plate was first recognizable as an area of distinct fusion between the head ectoderm and the floor of the foregut in seven-somite dog embryos. It expanded continuously up to the 21 somite stage at which time it ruptured (Plate III, Fig. 5) near the center and rapidly disappeared. Time of formation was thus comparable to the first appearance in the rat at six somites (Rogers, 25) and humans after seven somites (Dandy, 5$ Corner, 4). The size and thickness of the oral plate in the dog agreed with that found by Davis (6) and McNurrich (21) in the human. A description of the multi-cell
 +
 +
 +
 +
30
 +
 +
 +
 +
thickening in the center of the oral plate so consistently present in 10 to 20 somite dog embryos was not found. The life of the oral plate was found closely comparable to that reported by Davis (6) and Lewis (19) in the human, and was more persistent than in the rat (Rogers, 25) in which it is a formed structure only in embryos of 6 to 10 somites.
 +
 +
Pharyngeal Pouches
 +
 +
The first pharyngeal pouch (Plate II, Fig. 6) was not distinguished in dog embryos earlier than the nine somite stage although Weller (30) described it in a two somite human embryo, Rogers (25) in a 6 somite rat embryo and Selle (26) for the bat reported first pharyngeal pouches in a seven somite embryo.
 +
 +
The first closing membrane was formed in the dog at nine somites, comparing favorably in time of development with eight somite bat embryos (Selle, 26) and 10 somites in rat (Rogers, 25) and humans (Grosser, 10; Corner, 4).
 +
 +
The second pharyngeal pouch developed in close sequence with the first pouch, in comparison to its first appearance in 10 somite human embryos (Corner, 4) and 12 or 13 somite rat embryos (Rogers, 25). Even after fusion of endoderm and ectoderm forming the first and the second closing membranes, there was no indication of an indentation forming pharyngeal grooves. Both the first and second grooves were first indicated in the 17 somite stage, after which they became more pronounced by expansion of the arches. In contrast, Heuser (11) reported distinct first pharyngeal grooves in nine somite human embryos.
 +
 +
The third pharyngeal pouch formed at the 17 somite stage (Plate V, Fig. 2), the closing membrane and pharyngeal groove by the 21 somite stage,
 +
 +
 +
 +
31
 +
 +
 +
 +
while in the rat Rogers (25) stated the pouch formed by 21 somites and its closing membrane by 25 somites.
 +
 +
The fourth pharyngeal pouch (Plate IV, Fig. 3) was first recognizable in dog embryos at the 26 somite stages and were clearly identified in 29 to 30 somite embryos (Plate V, Fig. 3). In the human, Watt (29) described three pharyngeal pouches at the 17 to 19 somite stage, Davis (6) reported a possible fourth pouch at the 20 somite stage and Johnson (15) in a 24 somite embryo identified four pharyngeal pouches of which only the first two formed closing membranes. Rogers (25) described no fourth pharyngeal pouch in the rat.
 +
 +
The first and second pharyngeal pouches of the dog were of equal size while the third pouch was smaller in its antero-posterior extension and the fourth pouch definitely smaller than the three preceding it. In the guinea pig Klapper (18) found a successive decrease in size of the pharyngeal pouches, Johnson (15) reported the second pharyngeal pouch of humans to be larger than the first with the third and fourth successively smaller, but Davis (6) reported the second pouch of the human to be smaller, although resembling the first in shape.
 +
 +
The dorsal wall of the pharynx was a single cuboidal layer while the lateral and ventral walls were a pseudostratified columnar epithelium with a double layer of nuclei. This was comparable to the observations of Johnson (15). Davis (6) stated the ventral wall was a stratified columnar layer three nuclei thick increasing to six nuclei at various levels.
 +
 +
The hyperpharyngeal groove of dog embryos was comparable to the "dorsal recesses" of human embryos (Davis, 6), and the "notochordal groove" described by Watt (29). It resulted from the fusion of endoderm with the floor of the neural tube and was considerably deepened by expansion of a dorsal aorta on
 +
 +
 +
 +
32
 +
 +
 +
 +
either side. As the notochord developed, the groove became shallower and gradually disappeared.
 +
 +
The hypopharyngeal groove was continuous from the posterior edge of the oral plate to the MAIP in the earlier stages (up to 21 somites) and continuous with the laryngotracheal groove in the later stages (21 to 30 somites). It was nearly obliterated in the region of the first and third pharyngeal pouches in 15 to 26 somite embryos by a dorsal protrusion of the aortic sinus into the pharynx. The dorsally protruding aortic sinus resembled the swelling molded over the ventral aorta in humans (Davis, 6) and Watt (29), with the most posterior swelling dorsal to the atrium closely resembling the "heart swelling" in human embryos (Davis, 6). The hypopharyngeal groove seemed to remain as a distinct depression in the line of concresence of the splanchnopleuric fold, and later differentiated locally into thyroid anlage, laryngotracheal groove and liver diverticulae.
 +
 +
Thyroid
 +
 +
The thyroid anlage in the dog was first identifiable in the 15 somite embryos as a thickening in the ventral wall of the pharynx, extending ventrally into the aortic sinus between the ventral tips of the first and second pouches. It slowly increased in size up to the 27 somite stage but nearly doubled in volume between 27 and 30 somite development while the aortic sinus regressed around it. By the 29-30 somite stage the thyroid was composed of three main buds (Plate V, Fig. 1) pushing down into the aortic sinus (Plate IV, Fig. 4), a large medial lobe and two minor lateral buds. The thyroid anlage was a loosely arranged mass of cuboidal cells comprising a portion of the floor, continuous around its margins with the
 +
 +
 +
 +
33
 +
 +
 +
 +
pharyngeal epithelium. Only on its posterior surface had it proliferated free from the pharyngeal floor (Plate V, Fig. 3). Thyroid development to this stage was comparable to that described for the human (Weller, 30) and failed to show a thyroid pit as described for the cat (Ramsay, 24).
 +
 +
Caylor and Schlotthauar (3) described the pig thyroid anlage as a bilobed "syncytium" between the second pharyngeal arches. Rogers (25) in the rat and Selle (26) in the bat described the thyroid anlage as a cup-shaped evagination anterior to the second pharyngeal pouch in five and eight somite stages, respectively. Davis (6) described its location between the second and third pharyngeal pouches, but Watt (29) identified the thyroid anlage between the first and second pouches just posterior to the bulging aortic sinus. Norris (23) described individual variations of human thyroid anlage. No loss of cells at the pharyngeal surface as observed by Godwin (9) in the dog and Ramsay (24) in the cat was found in the species studied. Such diversity of descriptions for thyroid development obviously involve more than species variation. Some authors (Weller, 30; Godwin, 9) have almost certainly confused non-specific folds for thyroid anlagen, others (Davis, 6} Johnson, 15) have reached unjustifiable conclusions from the study of one or only a few specimens.
 +
 +
Lung Anlage
 +
 +
The lung anlage was first identified in 21 somite embryos as slight thickenings in the ventro-lateral wall of the laryngotracheal groove. By 27 somites the lung anlage was four or five nuclei thick forming hollow lateral buds with the lumen continuous with the digestive tract (Plate IV, Fig. 2). At the 29 to 30 somite stage the lung buds were hollow, paired, near-spherical masses with walls composed of a pseudostratified epithelium
 +
 +
 +
 +
34
 +
 +
 +
 +
with a triple layer of nuclei (Plate IV, Fig. 2).
 +
 +
The anlage of the lungs in the pig (Flint, 7) develops essentially the same as in the dog. The left lung bud (primitive bronchi) of the pig was described as being higher and less prominent than the right lung bud, while in the dog they appeared to be at the same level, although the lumen of the right lung bud was not identified at 29 somites.
 +
 +
Corner (4) in a 10 somite, Atwell (l) in 17 somites, and Watt (29) in 17 - 19 somite human embryos described the terminal portion of the laryngotracheal groove, posterior to the sinus venosus as the unpaired lung anlage, Davis (6) in a 20 somite embryo described a large right and a small left lobe separated by a sulcus. Johnson (15) failed to identify the lung anlage at 24 somites, but Streeter (27) at 25 somites described it as an unpaired anlage separated from the esophagous by a multiple layer of columnar cells which was continuous around the anlage. Grosser (10), agreeing with Streeter (27), stated further that the unpaired condition remained only a short time, forming a paired "pulmonary sac". This differed from the dog in that the paired lung buds formed before being completely separated from the digestive tract.
 +
 +
Liver Development
 +
 +
The liver anlage first appeared at the 13 somite stage as a ventrolateral extension of the foregut on the left side. The 15 somite stage was unchanged but by 17 somites the epithelium was thickened from a single pseudostratified columnar layer to a double pseudostratified columnar layer extending to the lateral edges of the vitelline veins (Plate III, Fig. 1). By 18 19 somites sharp grooves (Plate III, Fig. 2) were visible in the lateral wall
 +
 +
 +
 +
35
 +
 +
 +
 +
of the foregut dorsal to its ventral wall as a result of medial rolling of splanchnopleure and the posterior extension of the gut. In the 21 - 23 somite stages the liver had rotated further medially. Small groups of cells extended into the thickened splanchnic mesoderm from a large medially located mass in which three small ducts had formed. The two lateral ducts blended with the medial duct forming a single common duct anterior to the MAIP where it became continuous with the intestinal tract. The 26 - 27 and 29 - 30 somite stages were like that of the 21-23 somite stages although further growth occurred and only one duct was present. As the liver rolled medially with the splanchnopleure and vitelline veins, it became located within the embryo.
 +
 +
Streeter (27) described the mesoblastic framework of the liver in 13 somite human embryos as being derived from the "coelomic tract" and the liver epithelium at 25 - 23 somite stages spreading into the stroma (mesoblastic framework) as columnar extensions in contrast to the medial rolling concresence of liver endoderm found in the dog. The stroma capillaries which the columnar extensions enmesh, described in the human at this stage, were not identified in the dog.
 +
 +
The description of human liver development by Streeter (27) essentially agreed with previous reports by Lewis (19), Davis (6), Atwell (l), and Heuser (11). Johnson (15) stated the liver diverticulum was not in close relation to the vitelline veins in a 24 somite human embryo. This agrees with other work on the human as well as the present findings in the dog where the anlage was immediately ventral and medial to the vitelline veins. Mall (20) described the liver bud in both man and dog as encircling and invading the omphalomesenteric vein (sinus anlage).
 +
 +
 +
 +
36
 +
 +
 +
 +
Early stages of liver development, as well as thyroid and lung development have been so completely ignored in most of the descriptions of mammalian development that few actual comparisons can be made. More thorough studies of later development of these same structures can be better interpreted with the analysis of early development provided by this study.
 +
 +
SUMMARY
 +
 +
In a study of differentiation of the foregut in the dog, 35 embryos, free blastocyst stage to the 30 somite stage were used. The embryos were drawn, photographed and sectioned.
 +
 +
The first indication of endodermal differentiation appeared at the primitive streak stage as a thickened mass, which by the five somite stage had fused with the neural plate. In the seven somite stage the endoderm was pulled forward with the formation of the head fold forming a shallow foregut. Further elongation resulted from posterior concresence of the endoderm ventro-medial to the heart anlage.
 +
 +
The oral plate appeared in the seven somite stage persisting to the 21 somite stage. The first pharyngeal pouch and closing membrane formed at the nine somite stage; the second pharyngeal pouch developed in close sequence with the first but the second closing membrane was not formed in embryos of less than 15 somites. Both the first and second pharyngeal grooves were first indicated in the 17 somite stage. The third pharyngeal pouch also developed in the 17 somite stage with the third closing membrane and pharyngeal groove formed by the 21 somite stage. The fourth pharyngeal pouch was recognizable at the 26 somite stage and clearly identified in 30 somite embryos.
 +
 +
 +
 +
37
 +
 +
 +
 +
The thyroid anlage was first identifiable in the 15 somite stage, as a thickening in the ventral wall of the pharynx between the ventral tips of the first and second pouches. It slowly increased in size up to 27 somite stage, nearly doubling in volume between 27 and 30 somite development. The three main buds which pushed into the aortic sinus were continuous with the ventral wall of the pharynx. Proliferation free from the pharyngeal floor occurred only on its posterior surface.
 +
 +
The lung anlage was first identified at the 21 somite stage as slight thickenings in the ventro-lateral wall of the laryngotracheal groove. Hollow lateral buds continuous with the digestive tract formed by the 26 somite stage. By the 30 somite stage the lung buds were hollow, paired, nearspherical evaginations from the floor of the foregut.
 +
 +
The liver anlage was first recognized at the 13 somite stage as a ventro-lateral extension of the thickened floor of the foregut on the left side. By 17 somites extensions were present on both sides to the lateral edges of the vitelline veins. By the 19 somite stage sharp grooves were visible in the medio-lateral wall of the foregut as a result of the rolling concresence of sphanchnopleure. The liver anlage continued to roll medially, forming a common duct from which liver cord cells extended into the surrounding splanchnic mesoderm.
 +
 +
 +
 +
38
 +
 +
 +
 +
ACKNOWLEDGMENTS
 +
 +
Acknowledgments and sincere gratitude are given to Dr. H. T. Gier for his understanding, supervision and guidance during this study; also for permission to use his excellent collection of dog embryos.
 +
 +
The author also wishes to thank the Kansas State Department of Zoology for laboratory space and equipment.
 +
 +
 +
 +
 +
 +
 +
 +
39
 +
 +
 +
c
 +
 +
 +
 +
 +
LITERATURE CITED
 +
 +
 +
 +
 +
1.
 +
 +
 +
Atwell, Wayne J.
 +
 +
A human embryo with seventeen pairs of somites. Carnegie Institution of Washington Publication No. 407. Contrib. to Embryol. 21(118): 1-24. 1930.
 +
 +
 +
 +
 +
2.
 +
 +
 +
Bartelmez, G. W. and H. M. Evans.
 +
 +
Development of the human embryo during the period of somite formation, including embryos with 2-16 pairs of somites. Carnegie Institution of Washington Publication No. 362. Contrib. to Embryol. 17(85): 1-67. 1926.
 +
 +
 +
 +
 +
3.
 +
 +
 +
Caylor, Harold and Schlotthauer, C. F.
 +
 +
The thyroid gland of swine. Anat. Rec. 34:331-340. 1927.
 +
 +
 +
 +
 +
4.
 +
 +
 +
Corner, George W.
 +
 +
A well preserved human embryo of 10 somites. Carnegie Institution of Washington Publication No. 394. Contrib. to Embryol. 20(112): 81102. 1929.
 +
 +
 +
 +
 +
5.
 +
 +
 +
Dandy, Walter E.
 +
 +
A human embryo with 7 pairs of somites. Amer. J. Anat. 10:85-100. 1910.
 +
 +
 +
»
 +
 +
 +
6.
 +
 +
 +
Davis, Carl L.
 +
 +
Description of a human embryo having 20 pairs of somites. Carnegie Institution of Washington Publication No. 332. Contrib. to Embryol. 15(72): 1-51. 1923.
 +
 +
 +
 +
 +
7.
 +
 +
 +
Flint, Joseph M.
 +
 +
Development of the lungs. Amer. J. Anat. 6:1-139. 1906.
 +
 +
 +
 +
 +
8.
 +
 +
 +
Gier, H. T.
 +
 +
Estrous cycle in the bitch: vaginal fluids. Vet. Scope. 5(2): 2-9. 1960.
 +
 +
 +
 +
 +
9.
 +
 +
 +
Godwin, Melvin C.
 +
 +
The early development of the thyroid in the dog with special reference to the origin and position of accessory tissue within the thoracic cavity. Anat. Rec. 66(2): 233-252. 1936.
 +
 +
 +
 +
 +
10.
 +
 +
 +
Grosser, Otto.
 +
 +
The development of the pharynx and organs of respiration. Manual of Human Embryology. Keibel, Franz and Franklin P. Mall. Vol. 2, Chap. 17, 446-497. 1912.
 +
 +
 +
 +
 +
11.
 +
 +
 +
Heuser, Chester H.
 +
 +
A human embryo with 14 pair of somites. Carnegie Institution of Washington Publication No. 414. Contrib. to Embryol. 22:135-154. 1930.
 +
 +
 +
 +
40
 +
 +
 +
 +
12. Heuser, Chester and George W. Corner.
 +
 +
Development horizons in human embryos, Age group X. 4-12 somites. Carnegie Institution of Washington Publication No. 611. Contrib, to Embryol. 36(244): 29-39. 1957.
 +
 +
13. Heuser, Chester and G. L. Streeter.
 +
 +
Early stages in the development of pigs embryos, from the period of initial cleavage to the time of the appearance of the limb buds. Carnegie Institution of Washington Publication No. 394. Contrib. to Embryol. 20(109): 1-29. 1929.
 +
 +
 +
 +
14. Johnson, Charles E.
 +
 +
On the development of the liver in the genus Citellus. Rec. 13(13): 169-176. 1919.
 +
 +
 +
 +
Anat.
 +
 +
 +
 +
15. Johnson, F. P.
 +
 +
A human embryo of 24 pair of somites. Carnegie Institution of Washington Publication No. 226. Contrib. to Embryol. 6(19): 125168. 1917.
 +
 +
16. Keibel, Franz and Franklin P. Mall.
 +
 +
Manual of Human Embryology. Philadelphia and London: J. B. Lippincott Co., 1912.
 +
 +
17. Kingsbury, B. F.
 +
 +
The development of the human pharynx 1. The pharyngeal derivatives. Amer. J. Anat. 18(3): 329-397. 1914.
 +
 +
18. Klapper, Clarence E.
 +
 +
The development of the pharynx of the guinea pig with special emphasis on the morphogenesis of the thymus. Amer. J. Anat. 78(2): 139-180. 1946.
 +
 +
19. Lewis, Frederic T.
 +
 +
The development of the intestinal tract and respiratory organs. Manual of Human Embryology. Keibel, Franz and Franklin P. Mall. Vol. 2, Chap. 17:291-334. 1912.
 +
 +
20. Mall, Franklin P.
 +
 +
A study of the structural unit of the liver. Amer. J. Anat. 5:227-309. 1906.
 +
 +
 +
 +
21. McMurrich, J. P.
 +
 +
The mouth and its organs. Manual of Human Embryology. Keibel, Franz and Franklin P, Mall. Vol. 2, Chap. 17:335-445. 1912.
 +
 +
22. Moody, R. O.
 +
 +
Some features of the histogenesis of the thyroid gland in the pig. Anat. Rec. 4(12): 429-452. 1910.
 +
 +
 +
 +
41
 +
 +
 +
 +
23. Norris, E. H.
 +
 +
The early morphogenesis of the thyroid gland. Amer. J. Anat.
 +
 +
24:443-465. 1918.
 +
 +
24. Ramsay, A. J.
 +
 +
Comments on the origin and growth pattern of thyroid parenchyma.
 +
 +
Anat. Rec. 70(3 )i 287-310. 1938.
 +
 +
25. Rogers, W. M.
 +
 +
The development of the pharynx and derivatives in the white rat.
 +
 +
Amer. J. Anat. 44(2): 283-329. 1927.
 +
 +
26. Selle, R. M. _
 +
 +
The embryology of the thyroid, parathyroid, and thymus of the Pacific pallid bat ( Antiozous pacificus merriam ). Amer. J. Anat. 56(2): 161-191. 1935.
 +
 +
27. Streeter, George L.
 +
 +
Developmental horizons in human embryology. Age group XI to xiii. Carnegie Institution of Washington. Washington, D. C. 1951.
 +
 +
28. Wallin, I. E.
 +
 +
A human embryo of 13 somites. Amer. J. Anat. 15:319-331. 1913.
 +
 +
29. Watt, J. C
 +
 +
Description of two young twin embryos with 17-19 paired somites. Carnegie Institution of Washington Publication No. 222. Contrib. to Embryol. 2(2): 5-44. 1915.
 +
 +
30. Weller, G. L.
 +
 +
Development of the thyroid, parathyroid and thymus glands in man. Carnegie Institution of Washington Publication No. 443. Contrib. to Embryol. 24(141): 93-142. 1933.
 +
 +
 +
 +
42
 +
 +
 +
 +
APPENDIX
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
43
 +
 +
 +
»
 +
 +
 +
ABBREVIATIONS USED IN THE PLATES
 +
 +
 +
 +
 +
AS
 +
 +
 +
 +
 +
aortic sinus
 +
 +
 +
 +
 +
CM
 +
 +
 +
 +
 +
closing membrane
 +
 +
 +
 +
 +
END
 +
 +
 +
 +
 +
endoderm
 +
 +
 +
 +
 +
END'C
 +
 +
 +
«i
 +
 +
 +
endocardium
 +
 +
 +
 +
 +
EPIMYO'C
 +
 +
 +
 +
 +
epimyocardium
 +
 +
 +
 +
 +
FG
 +
 +
 +
 +
 +
foregut
 +
 +
 +
 +
 +
HA
 +
 +
 +
 +
 +
heart anlage
 +
 +
 +
 +
 +
HE
 +
 +
 +
 +
 +
head ectoderm
 +
 +
 +
 +
 +
HE G
 +
 +
 +
m
 +
 +
 +
hyperpharyngeal groove
 +
 +
 +
 +
 +
HF
 +
 +
 +
J0
 +
 +
 +
head fold
 +
 +
 +
 +
 +
HO G
 +
 +
 +
 +
 +
hypopharyngeal groove
 +
 +
 +
 +
 +
LA
 +
 +
 +
«•
 +
 +
 +
lung anlage
 +
 +
 +
 +
 +
LIV
 +
 +
 +
 +
 +
liver anlage
 +
 +
 +
 +
 +
MAIP
 +
 +
 +
 +
 +
margin of the anterior intestinal portal
 +
 +
 +
 +
 +
N'CH
 +
 +
 +
 +
 +
notochord
 +
 +
 +
 +
 +
NP
 +
 +
 +
 +
 +
 +
neural plate
 +
 +
 +
 +
 +
OP
 +
 +
 +
 +
 +
oral plate
 +
 +
 +
 +
 +
OPR
 +
 +
 +
 +
 +
oral plate remnants
 +
 +
 +
 +
 +
PIT
 +
 +
 +
 +
 +
 +
primitive pit
 +
 +
 +
 +
 +
PG
 +
 +
 +
 +
 +
pharyngeal groove
 +
 +
 +
 +
 +
PP
 +
 +
 +
an
 +
 +
 +
pharyngeal pouch
 +
 +
 +
V
 +
 +
 +
PS
 +
 +
 +
 +
 +
primitive streak
 +
 +
 +
 +
 +
SOM
 +
 +
 +
«K
 +
 +
 +
somite
 +
 +
 +
~
 +
 +
 +
TFAM
 +
 +
 +
 +
 +
tail fold of amnion
 +
 +
 +
 +
 +
THY
 +
 +
 +
 +
 +
thyroid anlage
 +
 +
 +
 +
EXPLANATION OF PLATE I
 +
 +
 +
 +
Dorsal view drawing of a four somite embryo. The outline was made by projection of the whole mount of embryo 16R and the details filled in from transverse sections of 260 LC and 117L. The original drawing was made at 15X.
 +
 +
 +
 +
EXPLANATION OF PLATE II
 +
 +
Fig. 1. Longitudinal section of embryo 65L showing the primitive streak, primitive pit and neural plate.
 +
 +
Fig. 2. Longitudinal section of embryo 65L magnified from area '2 of Fig. 1, showing the three germ layers.
 +
 +
Fig. 3. Longitudinal section of embryo 65L magnified from area 3 of Fig. 1, showing the thickened columnar epithelium ventral to the anterior edge of the oral plate.
 +
 +
Fig. 4. Transverse section of five-somite embryo 117L showing the fold in the endoderm medial to the heart anlage.
 +
 +
Fig. 5. Longitudinal section of seven somite embryo 260 Rb showing the early foregut and the close association of endoderm to the neural plate.
 +
 +
Fig. 6. Transverse section of thirteen somite embryo 154Rc through the first pharyngeal pouch and closing membrane.
 +
 +
 +
 +
 +
EXPLANATION OF PLATif III
 +
 +
 +
 +
Fig. 1. Transverse section of early 17 somite embryo 81Ra immediately posterior to the MAIP showing the single lateral evagination of liver anlage on the left side and thickening on the right side.
 +
 +
Fig. 2. Transverse section of 19 somite embryo 77Ra just anterior to the margin of the anterior intestinal portal showing paired lateral evaginations of the liver anlage.
 +
 +
Fig. 3. Transverse section of 19 somite embryo 77Ra just posterior to the margin of the anterior intestinal portal showing liver anlage posterior to the splanchnopleuric fold.
 +
 +
Fig. 4. Median sagittal section of 21 somite embryo 116Rb showing the remnants of the oral plate and early stages of thyroid.
 +
 +
Fig. 5. Transverse section of 21 somite embryo 42L immediately anterior to the margin of the anterior intestinal portal, showing the relationship of the three liver ducts.
 +
 +
 +
 +
 +
EXPLANATION OF PLATE IV
 +
 +
 +
 +
Fig. !• Transverse section through the lung buds of 27 somite embryo 65R.
 +
 +
Fig. 2. Oblique transverse section through one lung bud of 29 somite embryo 83Ra.
 +
 +
Fig. 3. Frontal section through the pharyngeal pouch
 +
 +
region of 30 somite embryo 83Rb showing all four pharyngeal pouches.
 +
 +
Fig. 4. Frontal section through the thyroid anlage of 30 somite embryo 83Rb.
 +
 +
 +
 +
EXPLANATION OF PLATE V
 +
 +
 +
 +
Fig. 1. Frontal section through the floor of the pharynx of embryo 33Rb demonstrating tripartite nature of the thyroid anlage.
 +
 +
Fig. 2. Wax model approximately 50X of endodermal structures of dog embryos 81La, 77L-1, 121L, to illustrate spatial relationships between pharyngeal pouches and lung anlage.
 +
 +
Fig. 3. Wax model approximately SOX of endodermal structures of dog embryos 83Rb and 83Ra to illustrate changing relationship between pharyngeal pouches, liver anlage and lung anlage.
 +
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53
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V
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.. -~ L 1
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Fig. I
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P P 3 ; P P 2
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P P I
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P P 4
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P P 2
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P P 3
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^
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A S
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L I V
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L A
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L I V
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P P
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THY
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THE EARLY DIFFERENTIATION OF THE FOREGUT IN THE DOG
 +
 +
 +
 +
by
 +
 +
 +
 +
ANDREW GEORGE HENDRICKX
 +
 +
B. A., Concordia College Moorhead, Minnesota, 1959
 +
 +
 +
 +
AN ABSTRACT OF A MASTER'S THESIS
 +
 +
 +
 +
submitted in partial fulfillment of the
 +
 +
 +
 +
requirements for the degree
 +
 +
 +
 +
MASTER OF SCIENCE
 +
 +
 +
 +
Department of Zoology
 +
 +
 +
 +
KANSAS STATE UNIVERSITY Manhattan, Kansas
 +
 +
 +
 +
1961
 +
 +
 +
 +
A review of the literature was made and the only report found on the differentiation of the foregut in the dog was an incomplete report by Godwin (9) on thyroid development.
 +
 +
In a study of differentiation of the foregut in the dog, 35 embryos, free blastocyst stage to the 30 somite stage were used. The embryos were drawn, photographed and sectioned.
 +
 +
The first indication of endodermal differentiation appeared at the primitive streak stage as a thickened mass, which by the five somite stage had fused with the neural plate. In the seven somite stage the endoderm was pulled forward with the formation of the head fold forming a shallow foregut. Further elongation resulted from posterior concresence of the endoderm ventromedial to the heart anlage.
 +
 +
The oral plate appeared in the seven somite stage persisting to the 21 somite stage. The first pharyngeal pouch and closing membrane formed at the nine somite stage; the second pharyngeal pouch developed in close sequence with the first but the second closing membrane was not formed in embryos of less than 15 somites. Both the first and second pharyngeal grooves were first indicated in the 17 somite stage. The third pharyngeal pouch also developed in the 17 somite stage with the third closing membrane and pharyngeal groove formed by the 21 somite stage. The fourth pharyngeal pouch was recognizable at the 26 somite stage and clearly identified in 30 somite embryos.
 +
 +
The thyroid anlage was first identifiable in the 15 somite stage, as a thickening in the ventral wall of the pharynx between the ventral tips of the first and second pouches. It slowly Increased in size up to 27 somite stage, nearly doubling in volume between 27 and 30 somite development. The three main buds which pushed into the aortic sinus were continuous with the ventral wall of the pharynx. Proliferation free from the pharyngeal floor occurred only on its posterior surface.
 +
 +
The lung anlage was first identified at the 21 somite stage as slight thickenings in the ventro-lateral wall of the laryngotracheal groove. Hollow lateral buds continuous with the digestive tract formed by the 26 somite stage. By the 30 somite stage the lung buds were hollow paired, near spherical evaginations from the floor of the foregut.
 +
 +
The liver anlage was first recognized at the 13 somite stage as a ventrolateral extension of the thickened floor of the foregut on the left side. By 17 somites extensions were present on both sides of the lateral edges of the vitelline veins. By the 19 somite stage sharp grooves were visible in the medio-lateral wall of the foregut as a result of the rolling concresence of sphanchnopleure. The liver anlage continued to roll medially, forming a common duct from which liver cord cells extended into the surrounding splanchnic mesoderm.

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Early Differentiation of the Foregut in the Dog

by


Andrew George Hendrickx

B. A., Concordia College Moorhead, Minnesota, 1959

A Master's Thesis

submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE

Department of Zoology

Kansas State University Manhattan, Kansas


1961


LP

2UUf ..

TV

7 £ TABLE OF CONTENTS

INTRODUCTION AND LITERATURE REVIEW 1

Thyroid t 4

Liver mt 7

Respiratory System 8

METHODS AND MATERIALS 10

OBSERVATIONS 12

Free Blastocyst Stage 12

Primitive Streak Stage 12

Preforegut Stage: 4-5 Somites 14

Early Foregut Stage: 7 Somites 15

Foregut Stage: 7-9 Somites 16

Pharyngeal Pouch I and II Stage: 8-13 Somites 17

Second Closing Membrane Stage: 15 Somites 19

Liver Differentiation Stage: 17 - 19 Somites 21

Rupture of the Oral Plate Stage: 21 - 23 Somites 23

Lung Differentiation Stage: 26 - 27 Somites 25

Thyroid Differentiation Stage: 29 - 30 Somites 27

INTERPRETATIONS AND DISCUSSION 28

Pharyngeal Pouches 30

Thyroid , 32

Lung Anlage , 33

Liver Development 34

SUMMARY 36

ACKNOWLEDGMENTS 38

LITERATURE CITED 39

APPENDIX t 42


INTRODUCTION AND LITERATURE REVIEW

Knowledge of the embryological development of the mammalian pharynx is far from being complete. The pharyngeal regions of few mammals have been thoroughly investigated. This structure in the dog has only been studied to a limited extent (Godwin, 9). Critical literature dealing with pharyngeal derivatives in mammals consist of reports by Heuser and Streeter (13) on the human and the pig, Keibel (16), Kingsbury (17), Mall (20), Norris (23), and Weller (30) on the human and a report by Godwin (9) dealing strictly with the dog.

Heuser and Streeter (13) reported that the appearance of endoderm cells which budded off from the inner cell mass of a pig's trophoblast characterized the initial appearance of endoderm formation. Soon after the endoderm cells budded off they became arranged in a plate. Hill and Hartman (according to Heuser and Streeter, 13) first observed the origin of endoderm from the inner cell mass in marsupials as cells arising from a formative area and spreading out from there.

Descriptions of the early differentiation of the visceral regions of the human were lacking except for some modeled specimens at the four somite stage (Heuser and Corner, 12) which show a shallow foregut. The sides of the head, however, appeared smooth, without swellings, which would indicate the absence of pouches. Weller (30) described a two somite human embryo which according to his description possessed the first pharyngeal pouch. The beginning of the foregut was described by Lewis (19) as a slight elongation of the yolk sac. Grosser (10) stated that pharyngeal pouches grew both laterally and dorsally from the anterior part of the digestive tract with a "ventral pharyngeal groove" extending along the mid ventral wall of the pharynx, with lateral


grooves connecting into it. At the seven somite stage of human embryos (Dandy, 5) a single pair of pharyngeal pouches in the form of dorso-lateral outgrowths was described. The endoderm of the first pharyngeal pouch was separated from the head ectoderm by only a narrow interval which was unoccupied by mesodermal cells. A buccopharyngeal membrane (oral plate) in the sense that ectoderm and endoderm were in contact was not found. The second pharyngeal pouch first showed in the seven and eight somite embryos and was clearly visible externally in the nine somite embryos (Bartelmez and Evans, 2) and at nine somites the first pharyngeal groove was seen as a distinct depression.

Corner (4) described the foregut of a 10-somite human embryo, as being compressed dorso-ventrally with the anterior end immediately under the forebrain. The hypopharyngeal groove was represented as a low ridge continuous from an area posterior to the thyroid pouch to the anterior intestinal portal where it became a broad prominence. The first pharyngeal pouches and closing membranes were formed. The second and third pharyngeal pouches were interpreted as slight bulges on the right side only. The dorsal wall of the foregut showed two ridges formed by the dorsal aortae, separated by a groove produced by the notochord as it "projected" from the endodermal wall.

According to Grosser (10) the first pouch appeared shortly after the separation of the anterior part of the digestive tract from the yolk sac and reached the ectoderm in the ten somite stage. At this time the third pouch was formed and it had reached the ectoderm in an embryo with 23 somites. The anlage of the fourth pouch also showed in the 23 somite embryo. External grooves were not evident when the pharyngeal pouches contacted the ectoderm. The first development of external grooves occurred during the period between


the formation of the tenth and fifteenth pairs of somites. At the height of the development of the pouches the embryonic pharynx was flattened dorsoventrally, was convex dorsally and had a somewhat triangular outline, the base being directed dorsally and apex ventrally. Heuser and Streeter (13) found no evidence of a second branchial groove in the 11-somite embryo. In human embryos from 13 to 20 somites (Streeter, 27), the pharyngeal pouches appeared with endoderm contacting the ectoderm and a third pharyngeal pouch not yet contacting ectoderm. In human embryos of 21 to 29 somites, the fourth pharyngeal pouch may or may not be present but made no contact with the ectoderm. Davis (6) described a 20-somite human embryo in which the fourth pouch was a slight ventro-lateral outpocketing. The oral plate was reported (Streeter, 27) to have formed by the 13 somite stage and by the 20 somite stage it had two perforations.

Rogers (25) first observed the foregut in rats of 3 somites. The first pharyngeal pouch formed by the 5 somite stage and by 10 somites the first closing membrane had formed. The second pharyngeal pouch formed by the 13 somite stage and its closing membrane by the 15 somite stage. The third pharyngeal membrane formed by 22 somites and its closing membrane by the 25 somite stage.

According to Selle (26), bat embryos of seven or eight somites showed distinct head folds, pharyngeal membranes (oral plates) and the first pharyngeal pouch. The first closing membrane did not form until the eighth somite was complete but at that stage there was no sign of any other pouches. In 4 mm embryos the pharynx had flattened dorsoventrally, the pharyngeal membrane (oral plate) had broken and four pharyngeal pouches had formed, three of them in contact with the ectoderm. (Clapper (18) observed four pharyngeal pouches in guinea pigs of 5 mm. The closing membranes of the first three


4


pouches were observed, but the fusion of the ectoderm and endoderm was not intimate.

Thyroid

Weller (30) noted the first evidence of the presence of the median thyroid component was a well defined fold in the ventral wall of the pharynx before the second pair of somites had formed and consisted of a proliferation rather than a differentiation of pharyngeal cells. The thyroid anlage was recognized by Grosser (10) before the first closing membrane had formed. "It appeared as a prominence in the ventral wall of the pharynx, belonging primarily to the medial region between the first two ventral pharyngeal grooves, that is to say, to the oral portion of what is to be the area mesobranchiales. M It was at first anterior to the second branchial arch. In a four somite embryo studied by Bartelmez and Evans (2) the median thyroid was only H a questionable fold" in the ventral wall of the pharynx. Dandy (5) recognized no thyroid primordium in a seven somite embryo but mentioned a "ventral pouch" posterior to the first pair of pharyngeal pouches.

The thyroid gland of a 10 somite human embryo (Corner, 4), appeared as a "laterally compressed protrusion from the median line fitted in between the diverging aortae, whose cells weren't differentiated from those of the pharyngeal area."

The thyroid in a 5-somite rat embryo (Rogers, 25) was a "slightly concaved circumscribed area of epithelium" in the median part of the floor of the pharynx, dorsal to the aortic sac and caudal to the first aortic arches. By 10 somites it *as more concave and by 26 somites it was a "cup shaped evagination." By 29 somites it was being drawn back caudally with the aortic sac. As a result it became a "flask-shaped" structure attached to the pharynx


by a stalk. The thyroid anlage of the guinea pig (Klapper, 18) formed at the ventral extremity of pouch one in the groove through the midline. The median thyroid (Weller, 30) undergoes changes in its external form becoming spherical. In a 4 mm embryo it appeared as a round, elongate evagination extending ventrally and caudally from the caudal floor of the first pharyngeal pouch, lined with typical pharyngeal endoderm cells while elongate cells with much cytoplasm were arranged peripherally with their long axes along the radii of the sphere. Streeter (27) illustrated the thyroid in a sagittal view of a 16-somite human embryo as a ventral evagination of the floor of the pharynx just caudal to the aortic bulb.

Grosser (10) described the thyroid of a 23 somite embryo as a small, hollow, rounded diverticulum connected to the floor of the pharynx. Norris (23) described the thyroid anlage of the human as having been a fairly definite and rather extensive shallow evagination from the "mesobranchial region of the pharyngeal floor, H between the ventral extremities of the first two pairs of gill pouches.

Godwin (9) considered the earliest thyroid anlage in the dog embryo as H a thickened area of epithelium in the midpharyngeal floor extending slightly caudad to the furrow formed at the mid-ventral junction of the mandibular arches to the caudal limit of the aortic sac. M A similar thickened area was described as projecting into the angle formed by tha union of the first and second arches with the aortic sac. The thyroid plate was depressed in the median portion and folded toward the anterior and posterior edges of the plate. The surface cells were columnar and laterally compressed, but the surface was irregular and sagged due to squeezing. In older stages the plate was thickened and buds of cells projected ventrally. At a later stage these buds elongated


downward and the thyxoid plate decreased rapidly.

In the cat Ramsay (24) found no indication of thyroid diverticulum at the 11 somite stage but a M mid-ventral longitudinal groove" not limited to the thyroid area appeared through the "propharynx" at 14 somites. In the 16 - 18 somite groups, a slight depression in the pharyngeal floor appeared immediately over the aortic sac between the tips of the first pharyngeal pouches. By 22 - 23 somites, a thyroid plate was recognized with a "thyroid pit". Growth in the "thyroid pit" area caused thickening of the plate, and resulted in reduction of the pit. With the increase of the thyroid plate activity, mesenchyme underlying the floor of the pharynx increased and migrated between the plate and the aortic sac, countersinking the plate and constricting the attachment to the pharyngeal floor.

Moody (22) noted in his description of the thyroid gland of a 5 mm pig, "the median thyroid gland was a syncytium forming a bilobed, elongated mass irregular in outline lying in the mesodermal syncytium on the ventral and lateral walls of the aorta level with the second gill arch and was still attached to the pharynx by a cord of cells." Because of its position and the fact that the lateral elements of the gland were paired suggested that the pig's thyroid was already a paired organ.

According to Selle (26) the thyroid anlage of bat embryos first appeared at eight somites (3 mm) as a cup-shaped groove in the floor of the pharynx anterior to the second pair of pharyngeal pouches. The endoderm of the ventral pharyngeal wall, including the "thyroglossal groove" was four or five layers of cells thick, but the dorsal wall was a single layer and the lateral walls were a double layer. In a 3.8 mm embryo the thyroid anlage was a solid oval mass ventral to the pharynx and dorsal to the bulbus, at the fork of the


aortic arches. Its anteroposterior extension was 150 u of which only the medial portion was in contact with the pharyngeal floor. In the 4 mm embryo the entire thyroid mass was anterior to the second pharyngeal pouches, and its contact with the floor of the pharynx was about 40 cells wide at its widest point.

Liver

Streeter (27) described the first indications of liver development occurring at the level of the first somite in a 13-somite human embryo. There was an active proliferation of cells from the "coelomic tract" which was to form the early mesoblastic framework of the liver. "The gut epithelium lies under the more dorsal part of the liver primordium and doubtless is playing a role in its development".

Lewis (19) described the liver in an early human embryo as a median outgrowth of the endodermal tube appearing as a well-defined cul-de-sac which showed extensive proliferation in the anterior and ventral walls in a 4 mm embryo. These proliferating cells formed irregular masses and anastomosing cords which by 4.9 mm formed a large crescentic mass to "an outpocketing of the intestine."

Mall (20) in a description of a two week old human embryo measuring 2.1 mm, stated that the liver bud was well marked, encircling and invading the left omphalomesenteric vein, carrying the endothelial lining ahead of the sprouts and forming a series of sinuses as subdivisions of the lumen of the vein.

Johnson (14) in an early account of liver development in the ground squirrel measuring 1.5 mm, stated that the anlage was a thickened area in the


8


ventral wall of the foregut, anterior to the yolk stalk, continuing laterally a short distance onto the walls of the digestive tube with the lateral extent being greater than the antero-posterior extent. As the embryo increased in length to 2 ran, the hepatic thickening was a distinct outpouching of the gut; by 2.5 mm it was a smooth walled outpouching of the foregut, connected to the ventral wall by a short stalk; by 4.5 mm the common bile duct had formed.

Respiratory System

The anlage of the lungs in the pig were described by Flint (7) as being unpaired and asymmetrical. He considered pulmonary anlage to arise from the ventral part of the head gut behind the sinus venosus, as a ventral outgrowth, preceded by a lateral flattening of the foregut posterior to the gill pouches at 3.5 mm. In the 4 mm embryo, he described "longitudinal furrows" dividing the foregut into two parts: a ventral respiratory portion and a dorsal digestive segment. From the posterior extremity of the pulmonary anlage, he found two lateral asymmetrical outgrowths, which gave rise to the stem bronchi. At this point he described the respiratory and digestive portions as separating, the separation beginning at the posterior end of the anlage and extending anteriorly along the line formed by the two longitudinal furrows. Corner (4) described the lung primordia of a 10-somite human embryo as an enlarged terminal portion of the laryngotracheal groove. In bat embryos measuring 3.8 to 4 mm the laryngotracheal groove and unpaired lung primordium was identified by Selle (26).

The beginning of the respiratory apparatus according to Grosser (10) appeared prior to the formation of the last two closing membranes caudal to the pharyngeal pouches as a median ventral groove, the anterior portion of


the groove forming a "ridge on the outer surface of the epithelium" correspond^ ing to the laryngotracheal groove. The posterior end of the groove was described as being rounded and was interpreted to be the unpaired anlage of the lungs; as growth continued the lung anlage and then the tracheal groove became separated from the esophagus.

Corner (4) and Heuser (11) interpreted the laryngotracheal groove as a "low ridge" extending along the ventral pharyngeal wall. It extended between the posterior extremity of the thyroid pouch and the anterior intestinal portal, at which point it spread out into a wider prominence where it passed into the yolk sac. This enlarged terminal portion was interpreted as the unpaired primordium of the lungs. Photographed sections (Corner, 4) showed a medio-ventral evagination in the ventral wall of the foregut that appeared to be continuous with the thyroid anlage.

Wallin (28) recognized no lung buds in a 13-somite human embryo. A thickening of the endoderm caudal to the venous end of the heart in a 17 somite embryo marked the beginning of the lungs (Atwell, 1). In twin embryos of 17 - 19 somites no digestive or respiratory anlage were identified by Watt (29) possibly because of extreme dorsoventral compression of the embryos he studied.

In a 20-somite human embryo, the laryngotracheal groove was described (Davis, 6) as a ventral angle beginning cranial to the fourth pharyngeal pouch and opposite the sinus venosus, terminating in a swelling which was the lung anlage. In a 24-somite human embryo, Johnson (15) described a median longitudinal groove separating two elongate swellings: the lower portion was interpreted to be the lung bud and the dorsal part the esophagus


10


METHODS AND MATERIALS

The 35 embryos used in this study were obtained from wild coyotes and from dogs of various breeds maintained for that purpose. The coyote embryos were removed from freshly killed animals obtained during coyote hunts and placed in the series according to the somite number and size of the uterine swellings. The ages of dog embryos were established with the aid of vaginal smears by which ovulation time was determined (Gier, 8). Some of the embryos used for this phase of dog embryology had been previously sectioned for other studies. Additional critical stages were selected from previously collected material or by laporatomies performed specifically for these stages. Embryos were stained with aceto-carmine, drawn by camera lucida or microprojection and then sectioned at 10 u. The embryos were stained with a modified Harris' hematoxylin or iron hematoxylin and counter stained with an orange G-acid fuchsin combination. Photographs were made of whole embryos and sections as needed.

Comparisons between crown-rump measurements and somite number showed little consistency, consequently somite counts were used exclusively for stage designations. The incomplete somite immediately posterior to the tenth cranial nerve was counted as the first paired somite, and the last somite was considered to be the one anterior to the last intersomite groove.


1 Kansas State Agriculture Experiment Station Project 321» Dog Embryology, under the direction of Dr. H. T. Gier.







11



Table 1. Embryos studied.






■ 1


Somite


Age

s Type of i


Collection



Stage :


Number


(days)

section j


Number


Blastocyst




Transverse


1123



Primitive Streak




Longitudinal


65L



Preforegut


4


16


Transverse


260LC




4


16


Transverse


117L




5


17

Transverse


260Lb




5


17

Transverse


117L



Early Foregut


7

17

Longitudinal


260Rb



Foregut


7


17

Transverse


761C




8+


17

Transverse


45L-6




9


17


Transverse


120LC



Pharyngeal Pouch







1 & 2


9


17


Transverse


77L




10


17


Transverse


115La




11


17


Longitudinal


120L




11


17


Transverse


184




12


17+


Transverse


120 Rd




12+


17+


Transverse


115Rb




12+


17+


Transverse


118




13


17+


Transverse


154Rc



Second Closing







Membrane


15


18

Longitudinal


113Lb




15+


18

Transverse


77L



Liver Differentiation


17


18

Transverse


SILa




17


18

Transverse


77L-1




17


18

Longitudinal


H3La




17+


18

Transverse


121L




18


18


Longitudinal


81Lb




18


18


Longitudinal


81Lc




19


18


Transverse


77Ra



Rupture of Oral Plate


20


18+


Transverse


121L




21


19

Longitudinal


116Rb




21


19

Transverse


42L




21


19

Transverse


123L



Lung Differentiation


23


19


Transverse


65R




26


20


Transverse


unknown



Thyroid Differentiation


29


21


Transverse


83Ra




30


21


Transverse


83Rb







12


OBSERVATIONS

Free Blastocyst Stage

The youngest embryo studied was pre-somite, 14 day gestation, sectioned in the uterus. The uterine swelling measured 3 mm inside diameter, the uterine epithelium was continuous and extended outward into the uterine glands. Uterine milk occupied the area between the uterine epithelium and the blastocyst. The blastocyst itself was crumpled, so that exact measurements could not be determined but it obviously had conformed closely to the cavity within the uterine swelling. The single layer of ectoderm cells was dispersed in an irregular layer around the endodermal layer. The nuclei of the ectodermal layer took a dark blue stain and the abundant cytoplasm stained a light pink. The endoderm, also composed of a single layer of cells, lay immediately inside the ectoderm. It differed from the ectoderm in that the nuclei were spaced more widely and were almost twice the diameter of those in the ectodermal layer. The nuclei of the endoderm cells stained light blue and appeared granular. The cytoplasm, which was present in smaller amounts than that of the ectoderm, formed a thin continuous sheet between the nuclei. At this stage mesoderm had not appeared. A very thin, light staining fluid filled the blastocoel.

Primitive Streak Stage

In a 15 day old embryo, located in a uterine swelling measuring 4 mm inside diameter, three definite germ layers were identified (Plate II, Fig. 2) with a distinct embryonic disc and a primitive streak oriented transverse to the longitudinal axis of the horn of the uterus. The ectoderm (trophoderm)


13


was almost in contact with the uterine epithelium but the embryonic disc was distinctly cushioned by a film of uterine milk. At its medial portion, the primitive streak measured 1.3 mm in length and was three or four cell layers thick. At the most anterior extension of the primitive streak the primitive pit was identified as a depression in the dorsal wall 60^ in width (Plate II, Fig. 1). The area immediately ventral to the primitive pit was undifferentiated except for the thin loosely arranged endoderm layer, but posteriorly the three germ layers were reasonably distinct. The germ layers became more sharply identified posterior to the primitive streak.

The neural plate, whose cells were closely packed and several cell layers thick, extended anteriorly 1.3 mm from the anterior tip of the primitive pit. The endodermal layer ventral to the primitive streak was a light staining, continuous layer of cytoplasm with few nuclei. Extending both anteriorly and posteriorly for a limited distance nuclei of endoderm cells were widely scattered and stained lightly. As the germ layers became more distinct posteriorly, the nuclei were more numerous and stained somewhat darker. Beyond the mesodermal layer the nuclei of the endodermal layer again became widely spaced and stained lightly like those described in the previous stage.

More anteriorly, ventral to the neural plate, the nuclei of the endoderm gradually increased until they became two to three layers thick under the anterior most extension of the neural plate (Plate II, Fig. 3). The cells at this point had a columnar appearance. This thickened layer was 15 to 20 cells (200 ^j) in length and approximately 570 /i wide. The single layer of endodermal cells extended anteriorly where the nuclei gradually decreased in number and in staining intensity at the level of the anterior most extension of the mesoderm. In the mesoderm free area, the ectoderm and endoderm were


14


each only one cell layer in thickness with the layers in close apposition. The endodexm and ectoderm layers were continuous around the entire vesicle. The endoderm in the mesoderm free area retained the more primitive appearance described in the previous stage.

Preforegut Stage: 4-5 Somites

In the youngest embryos of this stage the endodermal layer was continuous under the entire embryonic area. The endodermal cells were cuboidal with round, darkly staining nuclei surrounded by a light staining granular cytoplasm. Immediately anterior, and also ventral to the anterior edge of the neural plate, the nuclei of the endoderm were tightly packed and pseudostratified. Pseudostratification gradually decreased posteriorly so that the endoderm was a single cell layer posteriorly from the last point of fusion with the neural plate.

The endoderm was unevenly fused with the neural plate from the anterior tip of the plate through the region of the heart anlage (Plate I, area 1 and 2). The area of union between endoderm and neural plate was thus a strip approximately 12 cells wide in the middle, diminishing in width and in closeness of contact both anteriorly and posteriorly. Throughout the primitive streak the endoderm had again thickened to a low columnar type.

In the five somite embryo the medial endodermal cell mass extended from the anterior edge of the proamnion to beyond the head fold (anterior part Plate I, area 1) increasing to a thickness of five or six cell layers for a short distance.

Posterior to the head fold (Plate I, area 1) fusion was evident between the neural plate and the endoderm of the yolk sac. The heart anlage had


15


differentiated as endocardial tubes considerably further anteriorly than in the younger stages. The heart anlage (epimyocardial plate and endocardial mass) in the older embryos had been pulled anteriorly over the forming head fold. From its first appearance the heart anlage (Plate X» ^ area 2) pushed ventrally and medially resulting in a fold in the endodermal layer (Plate II, Fig. 4). The fold in the endoderm was secured under the medial edge of the splanchnic mesoderm which bordered the medial edge of the embryonic coelom. The fold of endoderm was continuous throughout the length of the heart anlage, but disappeared posteriorly. Medially to the fold described above the endoderm cells were cuboidal, but through the fold and continuing laterally to the heart anlage it was a high columnar epithelium. Immediately ventral to the heart anlage it again became cuboidal.

Posterior to the heart anlage, throughout the somite region, and into the primitive streak area (Plate I, area 2, 3, 4), the endoderm lay close to the neural plate, but fusion was not distinct. In the posterior end of the somite region (Plate I, area 3) the endoderm ventral to the flat neural plate was a low columnar epithelium and the width of fusion between the layers decreased. Within the central area of the primitive streak the cells were undifferentiated, although laterally three germ layers were recognized. As in the younger embryos the endoderm cells were cuboidal posterior to the heart anlage and low columnar in the medial region through the primitive streak areas, changing to a simple cuboidal epithelium laterally.

Early Foregut Stage: 7 Somites

At this stage the endoderm of the proamnion varied from a pseudostratified columnar epithelium in the anterior half to a mass of cells four nuclei


16


thick in the posterior half. The endoderm under the hesdfold and forebrain area was a double pseudostratified columnar epithelium, similar to the thickened area in the previous stage, but from this point posteriorly it was a simple cuboidal layer. Immediately posterior to the head fold and medioventral to the neural plate the endoderm formed a sharp but shallow fold (Plate II, Fig. 5) marking the anterior most tip of the foregut. The anterior edge of the thickened endodermal mass of the proamnion marked the area of the margin of the anterior intestinal portal (MAIP).

The MAIP represents the posterior extension of the foregut and the point where it becomes continuous with the yolk sac. The endoderm was closely fused to the neural plate from the tip of the foregut through the region of the metencephalon, although posterior to this area it was in close association but not fused to the neural plate.

Laterally the heart anlage caused the ventro-medial folding of the endoderm, similar to that described in the preceding stage.

Foregut Stage: 7-9 Somites

The foregut appeared as a dorso-ventrally flattened pocket, with the anterior tip slightly posterior to the head fold, and the lateral edges projecting slightly dorsally. The length of the foregut was 120 p, slightly less than the thickness of the fifth cranial nervet the anterior tip of the foregut was almost directly ventral to the anterior edge of the fifth cranial nerve and the MAIP was under the posterior part of the fifth cranial nerve. The dorsal wall of the foregut was characterized by the medially thickened mass ventral to the neural plate, described in the previous stage, and a single layer of cuboidal epithelium laterally from this line. Laterally on both sides of the medial thickened mass, the wall pushed ventrally with the dorsal


17


aortae located in the shallow concavities, forming the hyperpharyngeal groove between the aortae. The thickened mass of the dorsal wall was continuous through the MAIP but persistent fusion with the neural plate was observed only in the anterior half of the foregut.

The oral plate appeared under the anterior tip of the foregut, consisting of single layers of ectoderm and endoderm cells which were closely fused, extended laterally a distance of 20 to 55 nuclei (70^) and 100 p anteroposterior^. In embryo 761c, because of external pressure, the oral plate was forced posteriorly so its actual length could not be determined. The MAIP was characterized by the change of the lateral walls of the foregut from a pseudostratified layer to a cuboidal layer. The fused endocardial tubes ventral to the foregut, became paired structures lateral to the endodermal fold with the flattening of the yolk sac.

Pharyngeal Pouch I and II Stage: 8-13 Somites

The shape of the foregut was unchanged from the previous stage but Sessel's pocket was identified as a blind pouch anterior to the oral plate. The oral plate extended posteriorly twice the distance that it did in the previous stage. Fusion between the neural plate and the foregut was continuous from a point dorsal to the oral plate to a point posterior to the second pharyngeal pouch in younger embryos (8 - 11 somites) and beyond the MAIP in the older embryos (12 - 13 somites). The fifth cranial nerve now occupied a position dorsal to the anterior edge of the oral plate with its mandibular branch ventral and anterior to the tip of the first pharyngeal pouch. Near the center of the oral plate there was a rounded medial thickening of endodermal cells that first appeared in the 8-somite stage and was persistent until the oral plate broke in 21 somite embryos. The first pharyngeal


18


pouches were distinguishable as lateral expansions of the pharynx posterior to the oral plate and fifth cranial nerve, pressing the head mesoderm aside and fusing with the head ectoderm, thus forming the first closing membrane (Plate II, Fig. 6). In the later stages (11, 12, 13 somites) the first closing membrane protruded beyond the lateral margin of the adjoining head ectoderm resulting in the formation of shallow external grooves at both margins, with complete fusion between the two points (Plate II, Fig. 6). The first closing membrane extended from an area posterior to the fifth cranial nerve postero-ventrally to a point ventral to the anterior edge of the auditory placode. The head fold varied somewhat but in all stages it was located ventral to the anterior edge of the first closing membrane.

The hypopharyngeal groove was identified as a shallow, medially located evagination extending from the oral plate to the MAIP, although between the first and second pharyngeal pouches it was very shallow. It reached its deepest depression between a point posterior to the fifth cranial nerve and the anterior edge of the auditory placode, between the ventral ends of the first pharyngeal pouches. Ventro-lateral to the posterior edge of the auditory placode the pharynx again extended laterally forming the second pharyngeal pouch. In the eight to eleven somite stages, two to three layers of mesodermal cells separated the endodermal lining of the second pouch from head ectoderm. In the later stages (12-13 somites) the head mesoderm had been pushed aside and in one case (13 somite) the endoderm and ectoderm were adjacent to each other but fusion was not definite. The ninth cranial nerve was located dorsal to and in the immediate region of the posterior edge of the second pharyngeal pouch. At this same level the hypopharyngeal groove deepened, expanding laterally more posteriorly, forming the splanchnopleuric fold. The pseudostratified epithelium of the hypopharyngeal groove anterior


19


to the second pharyngeal pouch thinned posteriorly, undergoing a transition to the typical cuboidal epithelium of the yolk sac at the MAIP.

Throughout the pharyngeal region, the dorsal wall was a single layer of nuclei compared to a double or triple layer of nuclei in the lateral and ventral walls. Fusion of the endoderm of the hyperpharyngeal groove with the neural tube occurred throughout the length of the pharyngeal region.

In the older stages (12 - 13 somites) ventral to the aortic arch and posteriorly there was a proliferation of splanchnic mesoderm of the apparent yolk sac dorsally into the coelom. Posteriorly and laterally this proliferation was continuous with the paired epimyocardial plates. The "yolk sac" endoderm associated with this thickened splanchnic mesoderm was a low columnar epithelium fading laterally into the typical cuboidal epithelium of the yolk sac.

In the latest stage (13 somites) in this series, the liver anlage was identified as a lateral extension of the hypopharyngeal groove on the left side only, immediately anterior to the MAIP. This anlage was a pseudostratified columnar evagination, about 70 p long ending posteriorly on the splanchnopleuric fold.

Second Closing Membrane Stage: 15 Somites

The general structure of the anterior pharynx closely matched that of the previous stage. The oral plate, however, had expanded posteriorly half way the length of the first pharyngeal pouch, and the previously described thickening of the oral plate was pronounced, extending posteriorly to the edge of the plate. The first closing membrane was broadened considerably, pushed further laterally and fusion of the two germ layers was more intense.


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From a point just behind the oral plate to beyond the first pharyngeal pouch the ventral wall of the pharynx was pushed dorsally by the developing bulbus thus obliterating the hypopharyngeal groove.

The second pharyngeal pouch was again ventral to the posterior edge of the auditory placode; just posterior to this point the second closing membrane had formed over a length of approximately 60 p. This closing membrane pushed laterally beyond the adjacent head ectoderm forming slight external grooves dorsal and ventral to the fused membrane.

The thyroid anlage was first identifiable in this stage. It appeared as a small proliferation of cells extending ventrally from the hypopharyngeal groove into the expanded aortic sinus ventral to the anterior edge of the auditory placode. The thyroid anlage was approximately 50 u long, 40 p wide and 25 p deep.

The hypopharyngeal groove was again continuous from the oral plate to the MAIP although it was almost obliterated between the oral plate and the second pouch. The MAIP was ventral to the second intersomitic groove at this stage.

Dorsal to the posterior edge of the ventricle, where the common cardinal veins join the sinus venosus, the splanchnic mesoderm of the yolk sac was proliferated dorsally with filamentous strands extending between the yolk sac endoderm and splanchnic mesoderm demonstrating fusion of the two layers. Several concentrated masses of splanchnic mesoderm was located ventro-lateral to the hypopharyngeal groove just anterior to the MAIP. These mesodermal proliferations were interpreted to be the precursors of the connective tissue of the liver.


21


The yolk sac endoderm had not changed from the previous stage except at the MAIP it was seven or eight cell layers thick. The splanchnopleuric fold consisted almost entirely of endoderm except for a thin layer of endocardium adjoining.

The liver anlage appeared as a lateral evagination from the left side of the hypopharyngeal groove, only slightly larger than that described in the previous stage. There was yet no modification of the left side and no differentiation beyond the splanchnopleuric fold although the evagination extended as a flat groove into the MAIP.

Liver Differentiation Staget 17 - 19 Somites

Cephalic flexion had markedly increased at this stage, but the relationship of the pharyngeal pouches to the cranial nerves and the auditory placode had not changed. At its posterior border the oral plate was only a single cell layer, in which both endoderm and ectoderm cells blended together, compared to the double germ layers anteriorly, and appeared to be near the point of rupture in the 19 somite stage. The ingrowth of the oral plate previously described had blended with the developing Rathke's pouch. The head fold had progressed to the posterior edge of the otocyst in all embryos in this group.

The first pharyngeal pouch had increased in overall size, extended more dorsally, and the closing membrane was expanded more than in the previous stages. The first pharyngeal grooves were represented by a broad shallow depression, deeper dorsally and ventrally and only slightly depressed over most of the closing membrane. The second pharyngeal pouch, closing membrane and pharyngeal groove were similar to those of the first but not as extensive.


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The third pharyngeal pouch had developed by the lateral expansion of the foregut a ntero- ventral to the first somite. The third closing membrane was not yet formed* several layers of mesoderm yet separating pharyngeal endoderm from head ectoderm. The expanding aortic sinus again pressed the floor of the pharynx dorsally, particularly in the 19 somite embryo where there was only a narrow slit of pharyngeal cavity. Posterior to the third pharyngeal pouch the hypopharyngeal groove reached its ventral-most extension, forming a deep elongated triangular groove which gradually widened near the MAIP. In one case (19 somites) the hypopharyngeal groove in this region was as wide as the foregut itself. The gut had elongated so that the MAIP was now ventral to the fourth intersomitic groove.

The thyroid anlage was again ventral to the anterior edge of the otocyst, between the ventral ends of the first and second pharyngeal pouches, where the aortic sinus pushed farthest dorsally. The greatest area of thyroid proliferation was 80 ju wide, 80 ^ long and 40 )i deep although two embryos showed no such proliferation. The cells of the thyroid mass were more loosely arranged than those in the rest of the pharyngeal wall.

Just posterior to the union of the common cardinal veins to the sinus venosus, the splanchnic mesoderm was a compact deeply stained multiple cell layer. The yolk sac consisted of a multiple cell layer at the MAIP diminishing gradually to a single cuboidal cell layer.

In the early 17 somite stage, the groove of the liver anlage again appeared anterior to the MAIP on the left side only, continuing posteriorly through the splanchnopleuric fold and fading out ventral to the sinus anlage. The wall of the groove consisted of cuboidal epithelium thickening laterally to a low columnar epithelium medial and ventral to the sinus anlage (Plate III,


23


Fig. 1). No lateral evagination occurred on the right side but the endoderm was thickened comparable to that of the left side. The thickening of the endoderm decreased to the cuboidal yolk sac epithelium under the posterior limit of the sinus anlage.

The late 17 and the 18 and 19 somite stages had lateral evaginations with thickened epithelium on both sides.

In the 18+ and 19 somite embryos, the liver anlagen were considerably deeper than in the 17 somite stage. As the splanchnopleuric fold concressed, enclosing a greater length of the groove thus elongating the liver diverticulum anterior to the fold (Plate III, Fig. 2), posterior differentiation of the groove continued so there was as much liver anlage beyond the splanchnopleuric fold in the 19 somite embryo as in the 17 somite stage (Plate III, Fig. 3).

The hyperpharyngeal groove was clearly identified, and fusion of pharyngeal endoderm, notochord, and neural tube was irregular but was evident posterior to the second somite in some embryos. Posterior to the second somite the three layers were separated by invasion of mesenchyme, and the hyperpharyngeal groove faded out until only a shallow notch remained beyond the fifth somite.

Rupture of the Oral Plate: 21 - 23 Somites

By this stage the oral plate had broken and only its lateral margin remained in the older stages. In the 21 somite embryo the anterior portion of the oral plate was intact and 4 - 5 cell layers thick but the posterior portion was gone, connecting the pharyngeal cavity with the oral cavity (Plate III, Fig. 5). Sessel's pocket was somewhat enlarged as compared to


the previous stage and the anterior edge was at the level of the middle of the fifth cranial nerve. The hypopharyngeal groove was yet distinct.

The first and second pharyngeal pouches essentially were unchanged. The third closing membrane was now formed ventral to the anterior part of somite one. The dorso-ventral expanse of the closing membrane was as great as that of the preceding pouches but a ntero -posterior fusion was limited.

The thyroid anlage varied between embryos from a single to a double mass (Plate III, Fig. 5) with the double mass located variably either one behind the other or lateral to one another. The greatest proliferation measured 100 p in length, 100 jli in width and 45 p in depth differing only from the pharyngeal wall in its loose, irregular arrangement. The thyroid anlage forced the endothelium of the aortic sinus ventrally in one case.

Posterior to the third pharyngeal pouch the gut narrowed gradually as the lateral wall pushed medially forming a flattened tube the ventral half of which is now designated as the laryngotracheal groove. Short lateral extensions persisted on the dorso-lateral surface presaging the formation of the fourth pharyngeal pouch, resulting in a modified appearance. The mesodermal lung masses were first identified at this stage as thickened mesodermal areas lateral to the laryngotracheal groove and ventral to a point midway through the third somite and anterior to the liver anlage. Just medial to the mesodermal lung masses the paired endodermal lung primordia were identified as slight thickenings in the ventro-lateral wall of the larynotracheal groove. The lung primordia were about 200 ju long, but only 25 p wide and 45 p deep.

Between the fourth and fifth intersomitic grooves the wall of the laryngotracheal groove thickened to a four or five cell layer (about 40 p)




25


immediately anterior to the liver anlage. At a point ventral to the middle of the fifth somite, the liver anlagen had rotated medially and fused into a large medial mass from which several small groups of cells (liver cords) proliferated into the thickened splanchnic mesoderm just posterior to the sinus venosus. Within the central liver mass, a central cavity opens broadly into the gut immediately anterior to the MAIP. From this' central cavity, paired ducts evaginate antero-laterally (Plate III, Fig. 4) nearly paralleling the larger central duct. The central duct joins the intestinal tract just anterior to the level of the fifth intersomitic groove, posteriorly to the MAIP.

The entire splanchnopleuric fold was composed of the multiple pseudostratified epithelium comprising liver anlage. The liver anlage blended with the cuboidal epithelium of the yolk sac ventro-latarally and also blended with the pseudostratified cells of the intestinal groove dorsally.

Lung Differentiation Stages 26-27 Somites

In the 26 somite embryo the anterior portion of the oral plate was still intact and the medial growth of endoderm described in all previous stages appeared only as a slight rounded mass. A few remnants of the oral plate persisted along the lateral wall of the expanded oral cavity. The first, second and third pharyngeal pouches, closing membranes and pharyngeal grooves of the second and third pouches were distinct only dorsal and posterior and somewhat ventral to the third pharyngeal pouch, the anlage of the fourth pouch was identified as a slight lateral extension of the pharyngeal wall.


26


The thyroid anlage continued as a single bud over the dorsal wall of the protruding aortic sinus. It measured 90 p in length, 70 p in width and 45 p in depth at the point of largest proliferation in the 26 somite embryo. In the 27 somite stage the aortic sinus protruded into the pharynx considerably less than it did in the 26 somite stage but the thyroid had doubled in size, forming a rounded mass 100 p long, 130 p wide and 100 p deep.

Ventral to and slightly anterior to the third intersomitic groove, the pseudostratified epithelium of the laryngotracheal groove thickened from two nuclei to four or five nuclei. The thickened portion expanded laterally forming a hollow bulbular structure (Plate IV, Fig. 1 and Plate V, Fig. 3), representing the endodermal lung buds.

Posterior to the lung anlage, the dorsal portion of the gut extended laterally as described in some of the earlier stages (15 - 19 somites). The hyperpharyngeal groove was still present although mesenchyne completely separated the notochord from both neural tube and endoderm.

The liver anlage was essentially the same as that described in the previous stage except that 1) it had been displaced slightly posteriorly, 2) the lateral ducts had disappeared leaving the central duct opening into the gut lumen and, 3) the posterolateral limit of the liver anlage had been moved medially by rolling concresence of the vitelline veins.

The endoderm and mesoderm of the MAIP lateral to the liver anlage were now fused, forming a true "splanchnopleuric fold" ventral to the sixth intersomitic groove.


27


Thyroid Differentiation Staget 29 - 30 Somites

The pharyngeal grooves had become prominent. Small fragments of the oral plate were still visible anteriorly. The hyperpharyngeal groove persisted posteriorly from the infundibulum. The pseudostratified endodermal epithelium of the pharynx changed in the region of the old oral plate to the cuboidal epidermal epithelium of the oral cavity.

The fourth pharyngeal pouch was further developed but mesenchyme several cells thick remained between the head ectoderm and the pharyngeal endoderm (Plate IV, Fig. 3).

The thyroid diverticulum had expanded into a three lobed mass (Plate V, Fig. 1) 100 p long, 200 ju wide and 200 p deep, protruding into the aortic sinus (Plate IV, Fig. 4). The thyroid anlage, except for the ventral lobulation was a solid mass of randomly oriented cells, continuous on the edges with the general pharyngeal floor. There was a major proliferation of thyroid posteriorly from the main anlage, lying between the pharyngeal floor and aortic sinus and separated from them by layers of mesenchyme. The aortic sinus at this level was considerably reduced, no longer protruding into the pharynx.

Posterior to the fourth pharyngeal pouch, the gut narrowed to an oblong cavity similar to the laryngotracheal groove of the previous stages. At the level of the third intersomitic groove the thickened epithelium of the laryngotracheal groove had expanded forming two distinct paired endodermal lung buds (Plate V, Fig. 3) with a triple layer of nuclei in the pseudostratified epithelium. In the 29 somite embryo only the right bud had expanded posteriorly far enough to contain a separate lumen in transverse section (Plate IV, Fig. 2); both were lumenated in the 30 somite embryo. Posterior and dorsal


to the endodermal lung masses the internal pleural cavities had been formed between the mesodermal lung masses and the mediastinum.

The liver showed only slight advances over that described for the 26-27 somite embryos. The MAIP had closed more posteriorly, now occurring ventral to the seventh intersomitic groove, thus becoming completely separated from the liver anlage which was connected to the floor of the gut ventral to the fifth intersomitic groove. There was no indication of subdivision of liver anlage into anterior and posterior liver diverticulae, rather there was a growth of liver cord cells from the single median liver diverticulum which lay between the vitelline veins.

INTERPRETATIONS AND DISCUSSION

Although the general outline of development observed in dog embryos follows roughly that described for other species, many points either conflict with descriptions by previous workers or were completely overlooked.

The thickened endodermal mass, ventral to the anterior edge of the primitive streak stage, which represented the first appearance of the pharynx had not been described previously. By the five somite stage the thickened endodermal mass had fused with the anterior part of the neural plate. With the formation of the head fold and folds medial to the heart anlage (Plate II, Fig. 4), the endoderm was pulled forward forming a shallow foregut by the seven somite stage (Plate II, Fig. 5). The processes of foregut formation observed here compare favorably with that described for 2 somite rat embryos (Rogers, 25).

In late seven somite dog embryos the shape of the foregut was generally similar to that of seven and ten somite human embryos (Dandy, 5; Corner, 4)


29


and early rat embryos (Rogers, 25), but the medially thickened area ventral to the neural plate was interpreted as the point of fusion between two germ layers, although Dandy (5) and Corner (4) considered it to be the notochord embedded in the endoderm. Rogers (25) failed to mention the notochord and considered the germ layers to be only in "close contact". His illustrations bear this out.

Elongation of the foregut resulted from the continuous forward extension of the head region, and the posterior concresence of the endoderm medioventral to the heart anlage. This interpretation essentially agreed with Rogers (25) who considered the active factors to be expansion of the anterior part of the neural plate, mitotic divisions and a "tucking in" of the adjacent endoderm.

The dorso-ventral flattening of the foregut was obvious from the first appearance of the notch in the endoderm foretelling the formation of a foregut, and was probably the result of the various forces involved in original formation rather than by lateral expansion as described in the rat (Rogers, 25). The foregut in both cases, nevertheless, was a definite dorso-ventrally flattened pocket before the appearance of the first pharyngeal pouches.

The oral plate was first recognizable as an area of distinct fusion between the head ectoderm and the floor of the foregut in seven-somite dog embryos. It expanded continuously up to the 21 somite stage at which time it ruptured (Plate III, Fig. 5) near the center and rapidly disappeared. Time of formation was thus comparable to the first appearance in the rat at six somites (Rogers, 25) and humans after seven somites (Dandy, 5$ Corner, 4). The size and thickness of the oral plate in the dog agreed with that found by Davis (6) and McNurrich (21) in the human. A description of the multi-cell


30


thickening in the center of the oral plate so consistently present in 10 to 20 somite dog embryos was not found. The life of the oral plate was found closely comparable to that reported by Davis (6) and Lewis (19) in the human, and was more persistent than in the rat (Rogers, 25) in which it is a formed structure only in embryos of 6 to 10 somites.

Pharyngeal Pouches

The first pharyngeal pouch (Plate II, Fig. 6) was not distinguished in dog embryos earlier than the nine somite stage although Weller (30) described it in a two somite human embryo, Rogers (25) in a 6 somite rat embryo and Selle (26) for the bat reported first pharyngeal pouches in a seven somite embryo.

The first closing membrane was formed in the dog at nine somites, comparing favorably in time of development with eight somite bat embryos (Selle, 26) and 10 somites in rat (Rogers, 25) and humans (Grosser, 10; Corner, 4).

The second pharyngeal pouch developed in close sequence with the first pouch, in comparison to its first appearance in 10 somite human embryos (Corner, 4) and 12 or 13 somite rat embryos (Rogers, 25). Even after fusion of endoderm and ectoderm forming the first and the second closing membranes, there was no indication of an indentation forming pharyngeal grooves. Both the first and second grooves were first indicated in the 17 somite stage, after which they became more pronounced by expansion of the arches. In contrast, Heuser (11) reported distinct first pharyngeal grooves in nine somite human embryos.

The third pharyngeal pouch formed at the 17 somite stage (Plate V, Fig. 2), the closing membrane and pharyngeal groove by the 21 somite stage,


31


while in the rat Rogers (25) stated the pouch formed by 21 somites and its closing membrane by 25 somites.

The fourth pharyngeal pouch (Plate IV, Fig. 3) was first recognizable in dog embryos at the 26 somite stages and were clearly identified in 29 to 30 somite embryos (Plate V, Fig. 3). In the human, Watt (29) described three pharyngeal pouches at the 17 to 19 somite stage, Davis (6) reported a possible fourth pouch at the 20 somite stage and Johnson (15) in a 24 somite embryo identified four pharyngeal pouches of which only the first two formed closing membranes. Rogers (25) described no fourth pharyngeal pouch in the rat.

The first and second pharyngeal pouches of the dog were of equal size while the third pouch was smaller in its antero-posterior extension and the fourth pouch definitely smaller than the three preceding it. In the guinea pig Klapper (18) found a successive decrease in size of the pharyngeal pouches, Johnson (15) reported the second pharyngeal pouch of humans to be larger than the first with the third and fourth successively smaller, but Davis (6) reported the second pouch of the human to be smaller, although resembling the first in shape.

The dorsal wall of the pharynx was a single cuboidal layer while the lateral and ventral walls were a pseudostratified columnar epithelium with a double layer of nuclei. This was comparable to the observations of Johnson (15). Davis (6) stated the ventral wall was a stratified columnar layer three nuclei thick increasing to six nuclei at various levels.

The hyperpharyngeal groove of dog embryos was comparable to the "dorsal recesses" of human embryos (Davis, 6), and the "notochordal groove" described by Watt (29). It resulted from the fusion of endoderm with the floor of the neural tube and was considerably deepened by expansion of a dorsal aorta on


32


either side. As the notochord developed, the groove became shallower and gradually disappeared.

The hypopharyngeal groove was continuous from the posterior edge of the oral plate to the MAIP in the earlier stages (up to 21 somites) and continuous with the laryngotracheal groove in the later stages (21 to 30 somites). It was nearly obliterated in the region of the first and third pharyngeal pouches in 15 to 26 somite embryos by a dorsal protrusion of the aortic sinus into the pharynx. The dorsally protruding aortic sinus resembled the swelling molded over the ventral aorta in humans (Davis, 6) and Watt (29), with the most posterior swelling dorsal to the atrium closely resembling the "heart swelling" in human embryos (Davis, 6). The hypopharyngeal groove seemed to remain as a distinct depression in the line of concresence of the splanchnopleuric fold, and later differentiated locally into thyroid anlage, laryngotracheal groove and liver diverticulae.

Thyroid

The thyroid anlage in the dog was first identifiable in the 15 somite embryos as a thickening in the ventral wall of the pharynx, extending ventrally into the aortic sinus between the ventral tips of the first and second pouches. It slowly increased in size up to the 27 somite stage but nearly doubled in volume between 27 and 30 somite development while the aortic sinus regressed around it. By the 29-30 somite stage the thyroid was composed of three main buds (Plate V, Fig. 1) pushing down into the aortic sinus (Plate IV, Fig. 4), a large medial lobe and two minor lateral buds. The thyroid anlage was a loosely arranged mass of cuboidal cells comprising a portion of the floor, continuous around its margins with the


33


pharyngeal epithelium. Only on its posterior surface had it proliferated free from the pharyngeal floor (Plate V, Fig. 3). Thyroid development to this stage was comparable to that described for the human (Weller, 30) and failed to show a thyroid pit as described for the cat (Ramsay, 24).

Caylor and Schlotthauar (3) described the pig thyroid anlage as a bilobed "syncytium" between the second pharyngeal arches. Rogers (25) in the rat and Selle (26) in the bat described the thyroid anlage as a cup-shaped evagination anterior to the second pharyngeal pouch in five and eight somite stages, respectively. Davis (6) described its location between the second and third pharyngeal pouches, but Watt (29) identified the thyroid anlage between the first and second pouches just posterior to the bulging aortic sinus. Norris (23) described individual variations of human thyroid anlage. No loss of cells at the pharyngeal surface as observed by Godwin (9) in the dog and Ramsay (24) in the cat was found in the species studied. Such diversity of descriptions for thyroid development obviously involve more than species variation. Some authors (Weller, 30; Godwin, 9) have almost certainly confused non-specific folds for thyroid anlagen, others (Davis, 6} Johnson, 15) have reached unjustifiable conclusions from the study of one or only a few specimens.

Lung Anlage

The lung anlage was first identified in 21 somite embryos as slight thickenings in the ventro-lateral wall of the laryngotracheal groove. By 27 somites the lung anlage was four or five nuclei thick forming hollow lateral buds with the lumen continuous with the digestive tract (Plate IV, Fig. 2). At the 29 to 30 somite stage the lung buds were hollow, paired, near-spherical masses with walls composed of a pseudostratified epithelium


34


with a triple layer of nuclei (Plate IV, Fig. 2).

The anlage of the lungs in the pig (Flint, 7) develops essentially the same as in the dog. The left lung bud (primitive bronchi) of the pig was described as being higher and less prominent than the right lung bud, while in the dog they appeared to be at the same level, although the lumen of the right lung bud was not identified at 29 somites.

Corner (4) in a 10 somite, Atwell (l) in 17 somites, and Watt (29) in 17 - 19 somite human embryos described the terminal portion of the laryngotracheal groove, posterior to the sinus venosus as the unpaired lung anlage, Davis (6) in a 20 somite embryo described a large right and a small left lobe separated by a sulcus. Johnson (15) failed to identify the lung anlage at 24 somites, but Streeter (27) at 25 somites described it as an unpaired anlage separated from the esophagous by a multiple layer of columnar cells which was continuous around the anlage. Grosser (10), agreeing with Streeter (27), stated further that the unpaired condition remained only a short time, forming a paired "pulmonary sac". This differed from the dog in that the paired lung buds formed before being completely separated from the digestive tract.

Liver Development

The liver anlage first appeared at the 13 somite stage as a ventrolateral extension of the foregut on the left side. The 15 somite stage was unchanged but by 17 somites the epithelium was thickened from a single pseudostratified columnar layer to a double pseudostratified columnar layer extending to the lateral edges of the vitelline veins (Plate III, Fig. 1). By 18 19 somites sharp grooves (Plate III, Fig. 2) were visible in the lateral wall


35


of the foregut dorsal to its ventral wall as a result of medial rolling of splanchnopleure and the posterior extension of the gut. In the 21 - 23 somite stages the liver had rotated further medially. Small groups of cells extended into the thickened splanchnic mesoderm from a large medially located mass in which three small ducts had formed. The two lateral ducts blended with the medial duct forming a single common duct anterior to the MAIP where it became continuous with the intestinal tract. The 26 - 27 and 29 - 30 somite stages were like that of the 21-23 somite stages although further growth occurred and only one duct was present. As the liver rolled medially with the splanchnopleure and vitelline veins, it became located within the embryo.

Streeter (27) described the mesoblastic framework of the liver in 13 somite human embryos as being derived from the "coelomic tract" and the liver epithelium at 25 - 23 somite stages spreading into the stroma (mesoblastic framework) as columnar extensions in contrast to the medial rolling concresence of liver endoderm found in the dog. The stroma capillaries which the columnar extensions enmesh, described in the human at this stage, were not identified in the dog.

The description of human liver development by Streeter (27) essentially agreed with previous reports by Lewis (19), Davis (6), Atwell (l), and Heuser (11). Johnson (15) stated the liver diverticulum was not in close relation to the vitelline veins in a 24 somite human embryo. This agrees with other work on the human as well as the present findings in the dog where the anlage was immediately ventral and medial to the vitelline veins. Mall (20) described the liver bud in both man and dog as encircling and invading the omphalomesenteric vein (sinus anlage).


36


Early stages of liver development, as well as thyroid and lung development have been so completely ignored in most of the descriptions of mammalian development that few actual comparisons can be made. More thorough studies of later development of these same structures can be better interpreted with the analysis of early development provided by this study.

SUMMARY

In a study of differentiation of the foregut in the dog, 35 embryos, free blastocyst stage to the 30 somite stage were used. The embryos were drawn, photographed and sectioned.

The first indication of endodermal differentiation appeared at the primitive streak stage as a thickened mass, which by the five somite stage had fused with the neural plate. In the seven somite stage the endoderm was pulled forward with the formation of the head fold forming a shallow foregut. Further elongation resulted from posterior concresence of the endoderm ventro-medial to the heart anlage.

The oral plate appeared in the seven somite stage persisting to the 21 somite stage. The first pharyngeal pouch and closing membrane formed at the nine somite stage; the second pharyngeal pouch developed in close sequence with the first but the second closing membrane was not formed in embryos of less than 15 somites. Both the first and second pharyngeal grooves were first indicated in the 17 somite stage. The third pharyngeal pouch also developed in the 17 somite stage with the third closing membrane and pharyngeal groove formed by the 21 somite stage. The fourth pharyngeal pouch was recognizable at the 26 somite stage and clearly identified in 30 somite embryos.


37


The thyroid anlage was first identifiable in the 15 somite stage, as a thickening in the ventral wall of the pharynx between the ventral tips of the first and second pouches. It slowly increased in size up to 27 somite stage, nearly doubling in volume between 27 and 30 somite development. The three main buds which pushed into the aortic sinus were continuous with the ventral wall of the pharynx. Proliferation free from the pharyngeal floor occurred only on its posterior surface.

The lung anlage was first identified at the 21 somite stage as slight thickenings in the ventro-lateral wall of the laryngotracheal groove. Hollow lateral buds continuous with the digestive tract formed by the 26 somite stage. By the 30 somite stage the lung buds were hollow, paired, nearspherical evaginations from the floor of the foregut.

The liver anlage was first recognized at the 13 somite stage as a ventro-lateral extension of the thickened floor of the foregut on the left side. By 17 somites extensions were present on both sides to the lateral edges of the vitelline veins. By the 19 somite stage sharp grooves were visible in the medio-lateral wall of the foregut as a result of the rolling concresence of sphanchnopleure. The liver anlage continued to roll medially, forming a common duct from which liver cord cells extended into the surrounding splanchnic mesoderm.


38


ACKNOWLEDGMENTS

Acknowledgments and sincere gratitude are given to Dr. H. T. Gier for his understanding, supervision and guidance during this study; also for permission to use his excellent collection of dog embryos.

The author also wishes to thank the Kansas State Department of Zoology for laboratory space and equipment.




39


c



LITERATURE CITED



1.


Atwell, Wayne J.

A human embryo with seventeen pairs of somites. Carnegie Institution of Washington Publication No. 407. Contrib. to Embryol. 21(118): 1-24. 1930.



2.


Bartelmez, G. W. and H. M. Evans.

Development of the human embryo during the period of somite formation, including embryos with 2-16 pairs of somites. Carnegie Institution of Washington Publication No. 362. Contrib. to Embryol. 17(85): 1-67. 1926.



3.


Caylor, Harold and Schlotthauer, C. F.

The thyroid gland of swine. Anat. Rec. 34:331-340. 1927.



4.


Corner, George W.

A well preserved human embryo of 10 somites. Carnegie Institution of Washington Publication No. 394. Contrib. to Embryol. 20(112): 81102. 1929.



5.


Dandy, Walter E.

A human embryo with 7 pairs of somites. Amer. J. Anat. 10:85-100. 1910.


»


6.


Davis, Carl L.

Description of a human embryo having 20 pairs of somites. Carnegie Institution of Washington Publication No. 332. Contrib. to Embryol. 15(72): 1-51. 1923.



7.


Flint, Joseph M.

Development of the lungs. Amer. J. Anat. 6:1-139. 1906.



8.


Gier, H. T.

Estrous cycle in the bitch: vaginal fluids. Vet. Scope. 5(2): 2-9. 1960.



9.


Godwin, Melvin C.

The early development of the thyroid in the dog with special reference to the origin and position of accessory tissue within the thoracic cavity. Anat. Rec. 66(2): 233-252. 1936.



10.


Grosser, Otto.

The development of the pharynx and organs of respiration. Manual of Human Embryology. Keibel, Franz and Franklin P. Mall. Vol. 2, Chap. 17, 446-497. 1912.



11.


Heuser, Chester H.

A human embryo with 14 pair of somites. Carnegie Institution of Washington Publication No. 414. Contrib. to Embryol. 22:135-154. 1930.


40


12. Heuser, Chester and George W. Corner.

Development horizons in human embryos, Age group X. 4-12 somites. Carnegie Institution of Washington Publication No. 611. Contrib, to Embryol. 36(244): 29-39. 1957.

13. Heuser, Chester and G. L. Streeter.

Early stages in the development of pigs embryos, from the period of initial cleavage to the time of the appearance of the limb buds. Carnegie Institution of Washington Publication No. 394. Contrib. to Embryol. 20(109): 1-29. 1929.


14. Johnson, Charles E.

On the development of the liver in the genus Citellus. Rec. 13(13): 169-176. 1919.


Anat.


15. Johnson, F. P.

A human embryo of 24 pair of somites. Carnegie Institution of Washington Publication No. 226. Contrib. to Embryol. 6(19): 125168. 1917.

16. Keibel, Franz and Franklin P. Mall.

Manual of Human Embryology. Philadelphia and London: J. B. Lippincott Co., 1912.

17. Kingsbury, B. F.

The development of the human pharynx 1. The pharyngeal derivatives. Amer. J. Anat. 18(3): 329-397. 1914.

18. Klapper, Clarence E.

The development of the pharynx of the guinea pig with special emphasis on the morphogenesis of the thymus. Amer. J. Anat. 78(2): 139-180. 1946.

19. Lewis, Frederic T.

The development of the intestinal tract and respiratory organs. Manual of Human Embryology. Keibel, Franz and Franklin P. Mall. Vol. 2, Chap. 17:291-334. 1912.

20. Mall, Franklin P.

A study of the structural unit of the liver. Amer. J. Anat. 5:227-309. 1906.


21. McMurrich, J. P.

The mouth and its organs. Manual of Human Embryology. Keibel, Franz and Franklin P, Mall. Vol. 2, Chap. 17:335-445. 1912.

22. Moody, R. O.

Some features of the histogenesis of the thyroid gland in the pig. Anat. Rec. 4(12): 429-452. 1910.


41


23. Norris, E. H.

The early morphogenesis of the thyroid gland. Amer. J. Anat.

24:443-465. 1918.

24. Ramsay, A. J.

Comments on the origin and growth pattern of thyroid parenchyma.

Anat. Rec. 70(3 )i 287-310. 1938.

25. Rogers, W. M.

The development of the pharynx and derivatives in the white rat.

Amer. J. Anat. 44(2): 283-329. 1927.

26. Selle, R. M. _

The embryology of the thyroid, parathyroid, and thymus of the Pacific pallid bat ( Antiozous pacificus merriam ). Amer. J. Anat. 56(2): 161-191. 1935.

27. Streeter, George L.

Developmental horizons in human embryology. Age group XI to xiii. Carnegie Institution of Washington. Washington, D. C. 1951.

28. Wallin, I. E.

A human embryo of 13 somites. Amer. J. Anat. 15:319-331. 1913.

29. Watt, J. C

Description of two young twin embryos with 17-19 paired somites. Carnegie Institution of Washington Publication No. 222. Contrib. to Embryol. 2(2): 5-44. 1915.

30. Weller, G. L.

Development of the thyroid, parathyroid and thymus glands in man. Carnegie Institution of Washington Publication No. 443. Contrib. to Embryol. 24(141): 93-142. 1933.


42


APPENDIX





43


»


ABBREVIATIONS USED IN THE PLATES



AS



aortic sinus



CM



closing membrane



END



endoderm



END'C


«i


endocardium



EPIMYO'C



epimyocardium



FG



foregut



HA



heart anlage



HE



head ectoderm



HE G


m


hyperpharyngeal groove



HF


J0


head fold



HO G



hypopharyngeal groove



LA


«•


lung anlage



LIV



liver anlage



MAIP



margin of the anterior intestinal portal



N'CH



notochord



NP


neural plate



OP



oral plate



OPR



oral plate remnants



PIT



primitive pit



PG



pharyngeal groove



PP


an


pharyngeal pouch


V


PS



primitive streak



SOM


«K


somite


~


TFAM



tail fold of amnion



THY



thyroid anlage


EXPLANATION OF PLATE I


Dorsal view drawing of a four somite embryo. The outline was made by projection of the whole mount of embryo 16R and the details filled in from transverse sections of 260 LC and 117L. The original drawing was made at 15X.


EXPLANATION OF PLATE II

Fig. 1. Longitudinal section of embryo 65L showing the primitive streak, primitive pit and neural plate.

Fig. 2. Longitudinal section of embryo 65L magnified from area '2 of Fig. 1, showing the three germ layers.

Fig. 3. Longitudinal section of embryo 65L magnified from area 3 of Fig. 1, showing the thickened columnar epithelium ventral to the anterior edge of the oral plate.

Fig. 4. Transverse section of five-somite embryo 117L showing the fold in the endoderm medial to the heart anlage.

Fig. 5. Longitudinal section of seven somite embryo 260 Rb showing the early foregut and the close association of endoderm to the neural plate.

Fig. 6. Transverse section of thirteen somite embryo 154Rc through the first pharyngeal pouch and closing membrane.



EXPLANATION OF PLATif III


Fig. 1. Transverse section of early 17 somite embryo 81Ra immediately posterior to the MAIP showing the single lateral evagination of liver anlage on the left side and thickening on the right side.

Fig. 2. Transverse section of 19 somite embryo 77Ra just anterior to the margin of the anterior intestinal portal showing paired lateral evaginations of the liver anlage.

Fig. 3. Transverse section of 19 somite embryo 77Ra just posterior to the margin of the anterior intestinal portal showing liver anlage posterior to the splanchnopleuric fold.

Fig. 4. Median sagittal section of 21 somite embryo 116Rb showing the remnants of the oral plate and early stages of thyroid.

Fig. 5. Transverse section of 21 somite embryo 42L immediately anterior to the margin of the anterior intestinal portal, showing the relationship of the three liver ducts.



EXPLANATION OF PLATE IV


Fig. !• Transverse section through the lung buds of 27 somite embryo 65R.

Fig. 2. Oblique transverse section through one lung bud of 29 somite embryo 83Ra.

Fig. 3. Frontal section through the pharyngeal pouch

region of 30 somite embryo 83Rb showing all four pharyngeal pouches.

Fig. 4. Frontal section through the thyroid anlage of 30 somite embryo 83Rb.


EXPLANATION OF PLATE V


Fig. 1. Frontal section through the floor of the pharynx of embryo 33Rb demonstrating tripartite nature of the thyroid anlage.

Fig. 2. Wax model approximately 50X of endodermal structures of dog embryos 81La, 77L-1, 121L, to illustrate spatial relationships between pharyngeal pouches and lung anlage.

Fig. 3. Wax model approximately SOX of endodermal structures of dog embryos 83Rb and 83Ra to illustrate changing relationship between pharyngeal pouches, liver anlage and lung anlage.


53






V






.. -~ L 1


Fig. I


P P 3 ; P P 2


P P I


P P 4


P P 2


P P 3


^




A S



L I V


L A


L I V




P P


THY





THE EARLY DIFFERENTIATION OF THE FOREGUT IN THE DOG


by


ANDREW GEORGE HENDRICKX

B. A., Concordia College Moorhead, Minnesota, 1959


AN ABSTRACT OF A MASTER'S THESIS


submitted in partial fulfillment of the


requirements for the degree


MASTER OF SCIENCE


Department of Zoology


KANSAS STATE UNIVERSITY Manhattan, Kansas


1961


A review of the literature was made and the only report found on the differentiation of the foregut in the dog was an incomplete report by Godwin (9) on thyroid development.

In a study of differentiation of the foregut in the dog, 35 embryos, free blastocyst stage to the 30 somite stage were used. The embryos were drawn, photographed and sectioned.

The first indication of endodermal differentiation appeared at the primitive streak stage as a thickened mass, which by the five somite stage had fused with the neural plate. In the seven somite stage the endoderm was pulled forward with the formation of the head fold forming a shallow foregut. Further elongation resulted from posterior concresence of the endoderm ventromedial to the heart anlage.

The oral plate appeared in the seven somite stage persisting to the 21 somite stage. The first pharyngeal pouch and closing membrane formed at the nine somite stage; the second pharyngeal pouch developed in close sequence with the first but the second closing membrane was not formed in embryos of less than 15 somites. Both the first and second pharyngeal grooves were first indicated in the 17 somite stage. The third pharyngeal pouch also developed in the 17 somite stage with the third closing membrane and pharyngeal groove formed by the 21 somite stage. The fourth pharyngeal pouch was recognizable at the 26 somite stage and clearly identified in 30 somite embryos.

The thyroid anlage was first identifiable in the 15 somite stage, as a thickening in the ventral wall of the pharynx between the ventral tips of the first and second pouches. It slowly Increased in size up to 27 somite stage, nearly doubling in volume between 27 and 30 somite development. The three main buds which pushed into the aortic sinus were continuous with the ventral wall of the pharynx. Proliferation free from the pharyngeal floor occurred only on its posterior surface.

The lung anlage was first identified at the 21 somite stage as slight thickenings in the ventro-lateral wall of the laryngotracheal groove. Hollow lateral buds continuous with the digestive tract formed by the 26 somite stage. By the 30 somite stage the lung buds were hollow paired, near spherical evaginations from the floor of the foregut.

The liver anlage was first recognized at the 13 somite stage as a ventrolateral extension of the thickened floor of the foregut on the left side. By 17 somites extensions were present on both sides of the lateral edges of the vitelline veins. By the 19 somite stage sharp grooves were visible in the medio-lateral wall of the foregut as a result of the rolling concresence of sphanchnopleure. The liver anlage continued to roll medially, forming a common duct from which liver cord cells extended into the surrounding splanchnic mesoderm.