A Morphological Study of the Development of the Human Liver I

Home , Foregut, Hepatic diverticulum, Septum transversum

A Morphological Study of the Development of the Human Liver I. DEVELOPMENT OF THE HEPATIC DIVERTICULUM ’

CHARLES B. SEVERN2 Department of Anatomy, University of Michigan, Ann Arbor, Michigan

ABSTRACT The development of the hepatic diverticulum was examined in 38 human embryos representing somite stages 1, 5, 8 and 10 through 29, inclu- sive. Interpretations were based on light microscopic study of serial sections of these embryos. The liver primordium was first identified in a five-somite embryo as a flat plate of endodermal cells continuous with, but lying ventral to, the endoderm of the foregut at the anterior intestinal portal. It is positioned caudal and ventral to the developing heart. This plate of endoderm subsequently undergoes a progres- sive folding due to differential growth of adjacent structures. During the folding process there is a close spatial relationship between the cells of the endodermal plate and the caudal and ventral endothelial lining of the atrium and the sinus venosus. The result of this folding is the establishment of a “T-shaped” diverticulum which projects ventrally and cephalically from the gut tract. The hepatic diverticulum is established by the 20 somite-stage embryo. This mode of development of the hepatic diverticulum is compared to the classical interpretation and to the development of other visceral organs.

The lack of an extensive sequential of the intrahepatic duct system, correla- series of human embryonic material has tion of the liver’s developmental pattern prevented past investigators from obtain- with its definitive architectural pattern, ing anything more than general and rather and comparison of the origin and develop- vague concepts as to how the human liver ment of the liver in various species of verte- develops. Due to this scarcity of material, brates. Whereas earlier works on liver de- most of the descriptions were based on velopment were somewhat imprecise, these observations of few embryos and repre- later investigations were quite specific in sented a limited series of developmental nature and the resulting interpretations stages. Extrapolations and interpolations yielded conflicting points of view. With the of the entire development of the liver were exception of Hammar, who used 88 human made from the stages available. The classical description of the develop- embryos ranging from 3.0 mm to 73.5 mm ment of the liver was written by Lewis in crown-rump length (CRL) the material (’12), who reviewed the observations made used covered a broad spectrum of develop- by previous authors. From this review it mental stages but the number of specimens appears that Thompson (’08), in his de- were few. Bloom described six embryos scription of a 2.5-mm embryo, coined the ranging from 10 mm to 40 mm CRL in term “outgrowth for hepatic diverticulum. size. Horstmann used nine embryos rang- From 1912 to the present several studies ing from 16 mm to 330 mm CRL. Elias’s have been made concerning the develop- study (’55) consisted of seven human em- ment of the human liver. They include bryos. those by Hammar (’26), Bloom (’26), 1This work was supported by the National Insti- Horstmann (’39), and Elias (’49, ’52, ’53, tutes of Health, Training grant GM312. ’55, ’57, ’64, ’67). These authors have been 2 Present address: University of Nebraska College of Medicine, Department of Anatomy, Omaha, Ne- primarily concerned with the development braska.

AM. J. ANAT., 131: 133-158. 133 134 CHARLES B. SEVERN

To my knowledge, no attempt has been ticulum of embryos of 17 and 23 somites made to trace the liver back to its primor- were made by an inverse wax-plate dial stage, nor has anyone traced the mor- method. phological development of the human liver Although the observations to be reported from the primordial stage to its adult con- begin with the youngest embryo in the figuration. series, the actual studies started with em- According to current accounts the liver bryos of Horizon XIII, in which the liver develops as a ventral “outgrowth” of the and its associated extrahepatic duct system gut endoderm in the region of the anterior were identifiable. Morphological develop- intestinal portal (Arey, ’65; Davies, ’63; ment was then traced backward, chrono- Hamilton et al., ’62; Patten, ’68; Du Bois, logically, to the stage where the liver ’63). The details of this process have not primordium could not be identified as a been described. The availability of an ex- specific morphological structure. Then the tensive series of human embryos made morphological changes were studied in de- possible this study of the morphological tail starting with this youngest embryo events involved in formation of the hepatic (one-somite, 1.5 mm, Horizon IX) and pro- diverticulum . gressed throughout the series of specimens. MATERIALS AND METHODS OBSERVATIONS This investigation was based on light Although the hepatic primordium could microscopic examination of serial sections not be specifically identified in the 1.5 mm of 38 human embryos in the Patten Em- embryo, the observations to be presented bryology Collection of the Department of begin with a description of this embryo as Anatomy, The IJniversity of Michigan, Ann a reference for further description of the Arbor, Michigan, and from the collection hepatic diverticulum in succeeding em- in the Department of Embryology, Carne- bryos. It was also necessary to include in gie Institution of Washington, Baltimore, the observations of the different staged Maryland. In table 1 are listed for all embryos, descriptions of the configuration embryos the source, collection number, of the foregut, heart, and septum trans- horizon stage, crown-rump length and versum as important interrelationships ap- number of somites, plane of section, sec- peared between the development of these tion thickness and stain used. Streeter’s structures and of the hepatic diverticulum. (’42) criteria based on both external form and internal structure for categorizing em- Horizon IX bryos into developmental age groups or One-somite embryos (1.5 mm CRL). “horizons” were used when possible to This embryo consisted of a flattened, elon- determine the developmental stages of the gated disc in which all three germ layers embryos. Embryos which fell within Hori- were present (figs. 1-4). The germ layers zons IX through XI1 were classified more were distinguishable only by their position specifically according to somite stages. and relationship to one another, since the When more than one embryo was available cells of these layers were not significantly at a developmental stage, the embryos were different from one another. cross-checked for morphological similari- The ectoderm of this embryo had the ties and/ or differences within that stage form of a thick middorsal plate of cells and also compared to the preceding and that was grooved longitudinally on its following stages. This allowed for a more dorsal surface. This dorsal plate was iden- accurate placement of embryos in a de- tified as the neural plate. The presence of velopmental seyuence. the dorsal groove was characteristic of this The morphological relations of the de- horizon. Ventral and contiguous to the veloping gut tract, heart, regional blood neural plate was a layer of endoderm, two vessels, septum transversum and liver to three cells thick, which extended lat- complex were observed. Photomicrographs erally as the single-cell layered yolk sac. were taken for clarification and substantia- Lateral to the midline zone of contiguity tion of some of the observations. Plaster between endoderm and ectoderm, these two models of the foregut and hepatic diver- germ layers were separated from each MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER 135

TABLE 1 Human embryos studied

Sections: plane 2/ Embryo no. 1 CRL (mm)/ thickness Horizon No. of somites (u) Stain 8 C-5080 IX 1.5/1 T/10 AlC C-2795 X 2.0/5 T/6 AlC-OG C- 391 X 2.0/8 T/10 A1C C-5074 X 3.3/10 T/10 HE C-3710 X 3.6/11 T/10 C-8970 X /12 T/5 HE C-6344 XI 2.5/13 T/6 AIC C-4529 XI 2.4/14 T/10 AIC-OG C-8962 XI 1.5/15 T/ C-7611 XI 2.4/16 T/8 HE (2-6784 XI 5.0/17 T/6 H and OG (2-8116 XI /17 S/8 A C- 164 XI 3.5/18 T/20 AlC C-6050 XI 3.0/19 F/10 AIC C-2053 XI 3.0/20 T/10 AIC C- 486 XI1 /21 T/10 AIC c-8943 XI1 3.9/22 T/8 HE C-4784 XI1 3.0/23 T/10

C-8505b XI1 3.7/23 Si8 ~ A C-8505a XI1 3.6/24 T/8 HE C-7852 XI1 3.6/25 T/10 HE C-8942 XI1 3.4/26 F/5 HE C-4736 XI1 3.0/26 T/10 AIC C-6144 XI1 3.3/27 T/10 AIC c-7999 XI1 3.3/28 T/10 HE C-8941 XI1 4.9/28 T-F/6 HE C-5923 XI1 4.0/28 T/10 AIC C-7724 XI1 3.5/29 S/8 HE C-1062 XI1 4.5/29 T/20 AIC P- 24 X 2.1/12 T/10 HE P- 71 X 2.5/6 T/10 HE P- 8 XI 3.0/ T/5 CB P- 195 XI 3.5/ Tilo HE P- 44 XI1 3.5/ T/10 HE P- 520 XI1 4.0/ T/15 M P- 36 XI1 4.5/28 T/10 HE P- 555 XI1 3.0 T/lO HE P- 483 XI1 4.5 T/10 HE

1 C, Carnegie collection; P, Patten collection. 2 T, transverse; F, frontal; S, sagittal. SAX, Alum cochineal; OG, orange G; HE, hematoxylin and eosin; A, azan; Co, carmine and blue; M, masson. other by a layer of mesodermal cells. The embryonic mesoderm of the amnion and layer of intervening mesoderm was two to yolk sac. five cells in thickness. In a caudal seg- The mesoderm which at the level of the ment of the embryo (region of the somite) somite was lateral to the midline juncture the mesoderm was present in its typical of endoderm and ectoderm, continued form and consisted of a medial mass, a across the midline and separated the endo- narrow intermediate portion and a lateral denn from the ectoderm at the cephalic mass which was divided into splanchnic end of the embryo (fig. 3). At this level and somatic layers (fig. 4). The space be- the endodermal layer was about eight cells tween splanchnic and somatic layers was thick. The cephalic end of the zone of con- presumptive coelom. In this region of the tact of endoderm and ectoderm (immedi- embryo the coelomic spaces were at the ately caudal to their separation by inter- lateral borders of the embryonic disc; the vening mesoderm) probably marked the mesoderm was continuous with the extra- site of the future stomodeum. For de- 136 CHARLES B. SEVERN scriptive purposes this site of the future in the form of a flat plate. The lateral ex- stomodeum was referred to as point a. tent of the plate corresponded to the lat- Rostral to this level the dorsal ectoderm eral extent of the pericardial cavity. consisted of a single layer of cells which The developing pericardium and heart, was identified as amnion. The endoderm which were rostral to the stomodeum in formed a midline plate of two to three cells the one-somite embryo, were present at the in thickness (fig. 2). The lateral extent of level of, and caudal to, the stomodeum in this endodermal plate paralled the medial the five-somite embryo. The lateral body borders of the coelomic spaces. The coe- wall had grown laterally and ventrally and lomic spaces appeared to approach each enclosed a major portion of the pericar- other in the midline at a still more rostral dium and heart,. The lateral edges of the level. Just caudal to the level at which pericardial mesoderm approximated each these coelomic spaces approximated each other in the midline ventrally and a space other and became a single cavity (peri- (pericardial coelom) was present. Parietal cardial coelom), the endoderm was a single pericardial and epimyocardial layers had cell layer and was identified as yolk sac. formed. An endothelial tube one cell in Again, for purposes of description, this thickness surrounded by a sheath of car- anterior point at which the thick two- to diac jelly was inside the epimyocardium. three-cell endodermal plate was continuous with the single cell layered yolk sac was Where the parietal pericardia were in con- designated as point b. The thickened plate tact in the midline there was a thickening of endoderm between points a and b was of mesodermal cells that separated the en- rostral to the body of the embryo, and dodermal plate from the epimyocardium represented the entire foregut primordium. and its endothelial lining. This mesoderm The primordial heart was also rostral to was continuous with the mesoderm of the the body of the embryo and dorsal to the parietal pericardium and the mesoderm endodenn between points a and b (fig. 1) . covering the amnion and yolk sac. Because In still more rostral sections the coelomic of its position and its relation to adjacent spaces were absent. All that remained was cellular layers, this mesodermal thickening a single cell layer of ectoderm (amnion) was interpreted as the developing septum and a single cell layer of endoderm (yolk- transversum. Caudal to this level, neither sac); between the two layers was inter- the epimyocardia nor the parietal pericar- posed a two- to three-cell thick layer of dia had fused; they were lateral to the sa- mesoderm. gittal plane. At this level the endothelium of the cardiac primordia was present as two Horizon X tubes whose medial walls were in juxta- Five-somite embryo (2 mm CRL). In position. the one-somite embryo the plate of endo- At a level caudal to that just described, derm between points a and b was entirely the two endothelial tubes were not in con- cephalic to point a; whereas, in the five- tact with each other, but projected laterally. somite embryo it lay entirely caudal to In this region the endothelial tubes ap- point a (fig. 5). In the region of the peared to straddle the cephalic surface of stomodeum of this older embryo (fig. 6), the endodermal plate. The cells of the en- points a and b appeared at the same trans- dodermal plate appeared to be in direct verse level and the intervening endoderm contact with the cells of the medial walls was dorsal, caudal and ventral to the de- of the endothelial tubes. Laterally, the en- veloping heart. pericardium and septum dothelial tubes were small in diameter and transversum. The endoderm dorsal to the appeared to be continuous with the de- developing heart formed the tubular fore- veloping capillary bed of the yolk sac. The gut, which extended from a point a, cau- endodermal plate was wider in this area of dally, to the caudal level of the developing contact. The area at which the endodermal heart. The caudal extent of the endodermal plate was in direct contact with the en- tube was identified as the anterior intes- dothelial lining of the developing heart tinal portal. The endoderm ventral and (fig. 7) was designated as point c. Just cranial to the anterior intestinal portal was dorsal to point c foregut and endodermal MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER 137 plate were continuous (anterior intestinal mesoderm covering the amnion and yolk portal) (fig. 8). sac. In the five-somite embryo cells of the At a more caudal level (level of the an- tubular foregut and those of the endoder- terior intestinal portal) the parietal peri- ma1 plate were cytologically similar. The cardia and epimyocardia of opposite sides plate was from two- to three-cells thick. were no longer in contact with each other These cells possessed little cytoplasm and in the midline (fig. 12). The thick epimyo- their tightly packed nuclei were large and cardial layer only vaguely outlined a spherical to ovoid in shape. The nucleo- division of the single tube into a bifid plasm was granular and contained darkly structure. The internal endothelial tube staining chromatin bodies. branched to the right and to the left and Eight-somite embryo (2 mm CRL). The disappeared laterally into the capillary bed foregut was more prominent than in the of the yolk sac. The fused medial endothe- five-somite embryo and was flattened dorso- lial wall, representing the fusion of paired ventrally. Its ventral and lateral walls were atrial primordia at this level in the five- thicker than its dorsal wall and were three- somite embryo, was not present in the to four-cells thick, the nuclei of these cells eight-somite embryo at any level. There was being compactly arranged and oval in no septum transversum in the midline and shape. The dorsal wall was a single layer the thick endodermal plate was in direct of cells possessing large, spherical nuclei. contact with the ventral and caudal sur- The endodermal plate, which was con- faces of the branching endothelial tube. At this point of contact (point c), the endo- tinuous with the foregut, projected ven- dermal plate was slightly wider than at its trally and cephalically from the anterior cephalic end. Although the thickness of the intestinal portal. It was morphologically endodermal plate was the same as that of and cytologically similar to the ventral and the ventral and lateral walls of the fore- lateral walls of the foregut. The most an- gut, the cell boundaries were more distinct terior extent of the endodermal plate in the plate than in the foregut. The nuclei (point b) appeared at the level at which were oval but the chromatin was not as the epimyocardium, parietal pericardium compact and the cells contained more cyto- and septum transversum were continuous plasm than those of the ventral wall of the with one another (fig. 11). In the eight- foregut. somite embryo point b was no longer at Ten-somite embryo (3.3 mm CRL). In the stomodeal level (fig. 9) as it was in the ten-somite embryo the neural folds had the five-somite embryo, but was at a more fused in the somite region and formed a caudal level where the ventricular portion neural tube. The neural tube was covered of the heart was continuous with the de- by dorsal ectoderm of the body wall. The veloping atrium. cephalic and caudal ends of the neural Just caudal to the level of the stomodeum tube were open (anterior and posterior (fig. lo), the developing heart was a dis- neuropores). The foregut had elongated tinct tubular structure situated within a and its cephalic end was still compressed well-defined pericardial cavity. It consisted dorsoventrally. The dorsal wall of the com- of two layers, an inner endothelial lining pressed foregut was one-cell thick. These surrounded by an epimyocardial layer. The cells were clearly definable. Their nuclei latter was continuous with the parietal were large and separated from each other pericardium by a middorsal attachment by distinct cytoplasm. The lateral and ven- (dorsal mesocardium). At this level there tral walls of the foregut were still two- to were also indications of the septum trans- three-cells thick, but the cells were no versum. This septum consisted of loosely longer cuboidal but columnar instead. They arranged mesodermal cells (3- to 4-cells contained more cytoplasm and the nuclei thick) which were located between the were more spherical than those of corre- parietal pericardium and the yolk sac en- sponding cells in the eight-somite embryo. doderm. These cells of the septum trans- At the cephalic end of the embryo the versum were continuous laterally and ven- endothelial tube of the developing truncus trally with the somatic and splanchnic region of the heart divided on either side 138 CHARLES B. SEVERN of the developing foregut (first aortic resulted in the formation of a head and arch). There was a ventral projection of tail fold. In the 11-somite embryo these the ventral wall of the foregut between the folds resulted in a C-shaped configuration divisions of the endothelid tube which was of the embryo, with its midventral portion identified as primordial thyroid. The endo- being constricted. dermal cells of this projection appeared to In the 11-somite embryo the septum be in contact with the endothelial cells of transversum had increased in volume. This the truncus. produced a greater separation between the In this embryo, the heart was still a parietal pericardium and the endodermal straight double-walled tube. The outer wall plate. The increase in mass of the septum consisted of the epimyocardium and the transversum was ventral and cephalic to inner wall the endothelial lining. In the the point at which the endoderm was in region of the anterior intestinal portal the direct contact with the endothelium of the outer epimyocardia and the parietal peri- developing heart (point c). Due to the in- cardia were not fused in the ventral mid- creased mass of the septum transversum line and the inner endothelial tube pro- ventral to point c and to the development jected to the right and left. At the point of the head and tail folds, the developing of this projection the cells of the endo- atrium appeared to be more dorsally posi- dermal plate were still in contact with the tioned. Despite this, the developing heart ventral and caudal surfaces of the endothe- still remained a straight tubular structure lium. in this embryo. The endodermal plate (between points The endodermal plate was widest in the b and c), which projected ventrally and region of caudal fusion of the heart. The more cephaIicaIIy in the five-somite and cells of this portion of the endodermal eight-somite embryos, was positioned more plate were adjacent to the lining cells of ventrally and 'was almost perpendicular to the caudal portion of the atrium and the the foregut at the anterior intestinal portal paired primordia of the sinus venosus. in the ten-somite embryo. The endodermal Summary of observations for Horizon X. plate was the same thickness as the ven- By the end of Horizon X, the following tral and lateral walls of the foregut. The morphological structures were established : greatest thickness and the widest extent of the foregut; the hepatic primordium (a this endodermal plate occurred along the thick endodermal plate which extended ventral and caudal surfaces of the endo- ventrally and cephalically from the anterior thelium at the point of the caudal fusion intestinal portal, and caudal and ventral of the paired primordia of the heart. to the developing heart) ; a straight tubular The two endothelium-lined vessels cau- heart consisting of truncus, ventricle and dal to the point of fusion were positioned atrium; the first aortic arch and the paired laterally at more caudal levels. These were primordia of the sinus venosus; septum the paired prirnordia of the sinus venosus. transversum; thyroid primordium; and a In both the five- and eight-somite embryos neural tube, open at cephalic and caudal these two channels were small and ap- ends. peared to break up close to the point of fusion and became continuous with the Horizon XI capillary beds of the yolk sac. In the ten- Thirteen-somite embryo (2.5 rnm CRL). somite embryo, however, these vessels, al- In the 13-somite embryo, in addition to though they were still continuous with the the greater length of the foregut, its ce- capillary bed of the yolk sac, were large phalic end had flattened out considerably, channels which extended caudally the being four to five times greater in width length of the embryo at the edges of the than in height (developing pharynx). lateral body wall. There also were connec- There was little change in the cytology of tions between these large vessels and small its cells, the only noticeable one being an blood vessels which appeared in situ in the increase in the quantity of cytoplasm in septum transversum. the ventral and lateral walls. This cyto- Eleven-sonzite embryo. Increased growth plasm was displaced to the luminal surface along the longitudinal axis of the cmbryo of the foregut lining. The middorsal wall MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER 139 of the foregut was flat, as was the ventral most perpendicular to the anterior intes- surface of the neural tube. These two sur- tinal portal. faces were separated by a rod-like structure Sixteen-somite embqo (2.4 mm CRL). of columnar cells (notochord) which ap- There was an increase in the overall size peared to be continuous with the endoder- of the heart, septum transversum, lateral mal cells of the dorsal wall of the foregut. body wall and foregut in the 16-somite As in the 10- and 11-somite embryos, the embryo. With the increased size of the midventral wall of the foregut (thyroid heart the cardiac loop was more pro- primordium) projected ventrally into the nounced. The ventricle bulged ventrally region in which the truncus appeared to and caudally to the atrium. The size of the divide around the developing pharynx. The atrium itself was increased. There was now endodermal cells appeared to be in con- a definite single sinus venosus which was tact with the endothelial cells of this bi- continuous caudally with paired omphalo- f urcation. umbilical trunks, These trunks represented The tubular heart had increased in the confluence of the primordia of the length and volume and fXed the pericardial omphalomesenteric and umbilical vessels. cavity. The cardiac loop was apparent in While in the 13-somite embryo the cells this embryo. The ventricle bulged ventrally of the thick endodermal plate were in direct contact with the endothelial cells of and caudally filling the right ventral por- the ventral and caudal walls of the atrium, tion of the pericardial sac, then curved to in the 16-somite embryo the cells of the the left and filled the left ventral portion endodermal plate were in direct contact of the pericardial sac. It then turned dor- with the endothelial cells of the ventral sally and cephalically and was continuous and caudal walls of the sinus venosus. No with the atrium which was positioned mid- midline septum was present in the sinus dorsally in the pericardial sac. The atrium venosus. was at the same level as the lowest point As previously stated, the size of the lat- of the bulging ventricle. At the level at eral body walls increased, thus enclosing which the ventricle was continuous with more of the lateral and ventral portions of the atrium the septum transversum was the embryo. The increased mass of the sep- thicker. The mass of the septum trans- tum transversum was lateral rather than in versum had increased both ventrally and the midline. The endodermal plate ventral laterally to point c. The increase appeared to point c was no longer flat, but rather to be in a dorsal direction, extending just dome-shaped (fig. 15). At point b it still ventral to the developing atrium, which in remained a flat plate (fig. 14). The overall this embryo, was more dorsally positioned. configuration of the thickened endodermal This dorsal extent coincided with the point plate was that of a gradual slope (fig. 13). at which the pericardial mesoderm divided Its ventral and cephalic tip (point b) was to the right and to the left. In the area in cephalic to point c, which was cephalic to which the pericardial mesoderm had not the anterior intestinal portal. The caudal fused in the ventral midline the endothe- position of the anterior intestinal portal lial lining of the atrium divided into ves- relative to point c was suggestive of an sels which branched laterally, right and increased growth in the caudal end of the left, representing the area of caudal fusion foregut and a correlative fixed position of of the heart. On the caudal and ventral the endoderm at point c. surfaces of this division the endodermal Seventeen-somite embryo (5 mm CRL). plate was in direct contact with the endo- The 17-somite embryo exhibited the first thelial lining and there was no intervening signs of the endodermal plate developing mesoderm of the septum transversum. The into the hepatic diverticulum. Whereas the lateral endodermal cells of the medial walls endodermal plate between points b and c of the vessels which branched right and was dome-shaped in the 16-somite embryo, left. Dorsal to this zone of contact the en- there were definite bilateral folds of the dodermal plate was continuous with the lateral edges of this endodermal plate in ventral and lateral walls of the foregut. In the 17-somite embryo( fig. 17). These folds this embryo the endodermal plate was al- occurred in the region where the omphalo- 140 CHARLES B. SEVERN umbilical trunks were confluent with the with the small blood channels of the yolk capillary bed of the yolk sac and at which sac and of the septum transversum. The there was an increased amount of meso- lateral extremes of the endodermal plate derm at the lateral edges of the septum were folded caudally. transversum. The ventral and caudal bulge In a three-dimensional view (figs. 25- of the ventricle had increased and there 32), the flat endodermal plate seen in had also been an increase in the cellular younger stages of embryos had begun to mass of the septum transversum laterally take the shape of a hollow “T-shaped’ and in the ventral midline. The midline structure. The walls of the right and left increase in inass further separated the extensions of the “T” were somewhat ventral endodermal plate from the layer thicker than the remaining walls. There of pericardial mesoderm. The endodermal appeared to be more nuclei in these areas; cells were still in contact with the endo- they were eccentrically displaced and thelial cells of the sinus venosus and of grouped peripherally with a relatively large the omphalo-umbilical trunks. The anterior amount of cytoplasm near the lumen. intestinal portal was caudal to the level of Nineteen-somite embryo (3.0 mm CRL). this zone of contact. The hollow “T-shaped diverticulum was Sagittal sections of another 17-somite positively identified in the 19-somite em- embryo verified the observations given bryo (fig. 19). The thickness of the walls above of a 17-somite embryo which was of this diverticulum was similar to that of sectioned transversely. Furthermore, they the walls of the caudal end of the foregut, brought out other facts for consideration. with which it was continuous. The lateral In the midline, the anterior intestinal por- walls of the cephalic end of the divertic- tal was caudal to the level of contact be- ulum were somewhat thicker and appeared tween endoderm and endothelium (point to project laterally into the septum trans- c). This caudal shift in the anterior in- versum. testinal portal created a fold in the endo- The only other morphological change to derm. The dorsal portion of this fold be noted was the development of the formed the ventral wall of the caudal por- omphalomeseriteric veins. The medially tion of the foregut. The ventral portion, positioned omphalomesenteric veins and between the anterior intestinal portal and the laterally positioned umbilical veins point c, formed the dorsal wall of the were confluent with the right and left hepatic diverticulum. The growth of the branches of the sinus venosus. These veins endoderm appeared to have been between were identified in previous embryos as the points a and c. Point c appeared to be omphalo-umbilical trunks. There was still relatively fixed as the site at which the a close relationship between the cephalic endoderm was in contact with the caudal end of the diverticulum and the sinus endothelial lining of the sinus venosus (fig. venosus. 18). In this 17-somite embryo there was Twenty-somite embryo (3.0 mm CRL). also a connection between the endodermal A cross sectional view of the ventral diver- plate and the mesodermal cells of the cau- ticulum showed an increased thickness of dal portion of the pericardium just ventral the septum transversum. The cells of the and dorsal to the sinus venosus. The endo- septum transversum were adjacent to the derm appeared continuous with the peri- lateral and ventral walls of the divertic- cardial mesoderm in these two areas. The ulum. The right and left growth of the rest of the endodermal plate, which pro- cephalic end of the diverticulum and the jected ventrally from the zone of contact, approximation of these sites of growth to was separated from the layer of parietal the developing veins were apparent. One pericardium by cells of the septum trans- could see the continuity between the fore- versum. There was a transverse groove in gut and the diverticulum (fig. 20). the endodermal plate located at its point Summary of observations for Horizon of contact with the endothelium of the XI. In Horizon XI (embryos having from sinus venosus and the omphalo-umbilical 13-20 somites) there was further overall vessels. Laterally the omphalo-umbilical growth and development of the embryos vessels were reduced in size and continuous as compared to those of the previous hori- MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER 141 zon. The heart had begun to rotate and at the stomodeal plate; b, the point at had increased in size: the ventricle bulged which the thick endoderm abruptly became ventrally and caudally, the atrium was the single-cell layer of the amnion; and c positioned more dorsally and cephalically the point at which there was contiguity to the ventricle and the single sinus between the endoderm and the endothe- venosus was established. The omphalo- lium of the developing caudal region of mesenteric veins and the umbilical veins the heart. In older embryos (8-somites) the were identified as separate vessels. There configuration of the endoderm showed a was an increased development of the sep- change in its morphological configuration. tum transversum and of the lateral body Instead of a “U-shaped” configuration the wall. The foregut had elongated and was endoderm had a “J-shaped’ configuration separable into pharyngeal and esophageal (fig. 26) which suggested an increase in regions. In the floor of the pharynx were the length of the thick segment of endo- signs of the developing thyroid. At the derm. This increase appeared to occur be- caudal end of the foregut the hepatic di- tween points a and c, with a developmental verticulum was established. lag in the growth of the endoderm between points b and c. The perpendicular position DISCUSSION of segment b-c to segment a-c appeared to In the present study it would appear that be due to an increased size and length of the hepatic diverticulum was formed con- the cephalic end of the embryo which re- currently with the establishment of the sulted in a ventral flexure of the head and gut tract and that it was established in an increase in the size and length of the part by differential growth of the endo- developing heart. In the 16-somite embryo derm, in part by differential growth of the endodermal plate was no longer flat surrounding structures and in part because but rather was related to an increased of a relatively fixed contact point between lateral mass of the septum transversum a segment of the endoderm and the en- and to increased foldings of the lateral dothelial lining of the heart. body wall (fig. 28). There was a continued It was noted in the observations that the folding of the endoderm primarily ventral endodermal primordium of the foregut and and lateral to point c in the 17-somite em- hepatic diverticulum was differentiated bryo, but the most striking change in the from the yolk sac by the thickness of its configuration of the endoderm was the cell layers. It was distinctly thicker than caudal shift of the anterior intestinal portal the single-cell layer of the yolk sac. In relative to point c. Whereas, the anterior tracing this thicker endodermal layer intestinal portal was at the same level as throughout the separate somite-staged em- point c in the younger embryo; it was bryos it was noted that there were no areas caudal to this point in the 17-somite and of “isolated cell proliferation and that this older embryos. Again this suggested an endoderm, although it increased in length, increase in length of the endoderm be- remained rather uniform in thickness until tween points a and c. This time, however, after the establishment of the hepatic di- due to the caudal position of the anterior verticulum. In following the morphological intestinal portal relative to point c, definite configuration of this thicker endodermal point of fixation was indicated in the area segment it was noted that in the one- of contiguity between the endoderm and somite embryo this endodermal segment endothelium of the sinus venosus. The con- consisted of a flat plate of cells which pro- sequence of this fixation and the increased jected rostral to the rostral end of the de- length of the endoderm between points a veloping embryo. In the five-somite embryo and c resulted in formation of the dorsal it had a “U-shaped” configuration with the wall of the hepatic diverticulum. As indi- dorsal limb of the “U” forming the ventral cated in the 19-, 20-, and 23-somite em- and lateral walls of the foregut (fig. 25). bryos, the endodermal segment between For purposes of description points a, b, and points c and b had folded caudally, ven- c were utilized as reference marks for the trally, and laterally to form the ventral hypothetical segmentation of the endo- and lateral walls of the hepatic divertic- derm. Point a represented the endoderm ulum (figs. 29-32). The folding of this 142 CHARLES B. SEVERN segment of endoderm appeared to be di- cephalic or caudal end, one would expect rectly related to an increase in mass of the that neither a thyroid nor a liver would cells in the septum transversum and to an develop. One would also suspect that the apparent area of fixation point c. The di- foregut would probably not develop. In two verticulum was “T-shaped and suggested cases of acardia amorphus recently ex- a bilobed structure. The bilobed configura- amined this was found to be the case. In tion of the hepatic diverticulum was at- neither instance was there a fused tubular tributed also to the area of contiguity of heart nor any evidence of a liver, dia- endoderm to endothelium and not only to phragm, thyroid, lung, or esophagus (figs. the sinus venosus, but also in part to the 23, 24). paired omphdo-umbilical trunks. A further observation concerning the The endothelial lining in contact with relationship of the development of the he- the endoderm at point c was the caudal patic diverticulum and the development of and ventral lining of the atrium in em- the septum transversum and the pericar- bryos up to 13-somite stage. In the 16- dium merits attention. In the five-somite somite embryo the endothelial lining in embryo the ventral segment of the thick contact with the endoderm was that of the endoderm (point b) was found at the same caudal and ventral surfaces of the sinus level as the point of fusion of the peri- venosus. No further fusion of the endo- cardium in the ventral midline. In em- thelial lining of the developing heart was bryos of 8, 10, 13, and 16 somites there observed in any of the embryos older than was a shift in the point of caudal closure five somites. This perhaps implies that the of the pericardial sac relative to point b. basic tubular structure of the heart, formed It appeared to move dorsally in relation to from a midventral fusion of paired pri- the ventral end of the endodermal plate, mordia, occurs prior to the eight-somite toward the zone of contact between endo- stage and that further growth of the tubular derm and the endothelial lining of the heart and the development of the sinus atrium (point c). If one considers the re- venosus from a bifid to a single structure lationships between the endodermal plate, occurs cephalad to the zone of contact be- the fusion of the pericardium and the ex- tween endothelium and endoderm. tent of the septum transversum at point b The area of endothelial-endodermal con- in the five-somite embryo and these same tiguity (point c) might have the following relationships in the 16-somite embryo, it significance. If this is a point of true fixa- would appear that, at least in part, the tion one might expect to find some type development of the septum transversum of malformation that would indicate this. was dependent on the development of the In subsequent studies two such abnormali- pericardium. In the same manner, growth ties were noted. The first of these was an of the endodermal plate between points b endodermal cyst located on the wall of the and c may be dependent to some extent on sinus venosus of a 15 mm pig embryo (fig. closure of the pericardium and/or the de- 22). One can assume that an area of con- velopment of the septum transversum. tiguity must have existed between the en- The observations and discussion pre- doderm and the endothelial lining of the sented in this study are contrary to the developing heart at some stage of develop- theory (which originated from Thompson’s ment and that, instead of the endoderm interpretation in 1908) that the hepatic detaching as jt normally does after estab- diverticulum is formed by an “outgrowth” lishment of the hepatic diverticulum, this of the gut tract. It has already been noted area of contact persisted and a segment that there were no notable localizations of of the hepatic diverticulum was broken off marked (cellular) proliferations until after and carried along with the dorsally and the establishment of the hepatic divertic- cephalically migrating sinus venosus. ulum. In the 25-somite embryo a localized If one speculates as to what might proliferation o€ cells occurred at the most happen if the paired cardiac primordia caudal end of the ventral wall of the di- failed to fuse in the ventral midline thus verticulum (point b). This localized pro- allowing no point of contiguity between the liferation created a bulge in the ventral endoderm and endothelium either at its wall of the diverticulum and was found MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER 143 in older embryos to be the developing - 1953 Observations on the general and regional anatomy of the human liver. Anat. cystic duct-gallbladder. In contrast to the Rec., 11 7: 377-394. mode of formation of the hepatic divertic- 1955 Origin and early development of ulum, the formation of the cystic duct- the liver in various vertebrates. Acta HeDato- gallbladder was an “outgrowth” typical of logica, 3: 1-56. 1957 Evolution and the liver. Chicago the primordia of the pancreas, parotid, sub- Med. School Quart., 19: 49-59. mandibular gland and lung. It was not 1964 Croissance appositionelle du foie until after the establishment of the bulge embryonaire. Rev. Int. Hepat., 14: 317-322. of the cystic duct-gallbladder that the 1967 Recruitment in human bile duct formation. Acta Hepatosplenologica, 14: 253- thickened endoderm continued caudal de- 260. velopment to the ventral walls of the Elias, H., and H. Bengelsdorf 1952 The struc- ductus choledochus and midgut. This ture of the liver of vertebrates. Acta Anat., 14: 297-337. would suggest that although points a and Hamilton, J. W., J. D. Boyd and H. W. Mossman b were hypothetical points of reference it 1962 Human Embryology. Williams and Wil- is quite possible that the endoderm which kins, Baltimore, pp. 240-244. formed the foregut and the hepatic diver- Hammar, J. A. 1926 Uber die erste Entstehung der nicht-kapillaren intrahepatischen Gallen- ticulum was established as early as the gange beim Menschen. 2. mikr.-anat. Forsch., one-somite embryo or perhaps even earlier. 5: 59-89. Horstmann, E. 1939 Entwicklung und Entwick- LITERATURE CITED lungsbedingunger des intrahepatischen Gallen- gangsystems. Roux’s Arch. Entwicklungsmech., Arey, L. B. 1965 Developmental Anatomy. W. 139: 364-392. B. Saunders, Philadelphia, pp. 255-259. Lewis, F. T. 1912 The development of the liver. Bloom, J. L. 1926 The embryogenesis of human In: Manual of Human Embryology. F. Keibel bile capillaries and ducts. Am. J. Anat., 36: and F. P. Mall, eds. J. B. Lippincott, Philadel- 451-465. phia, pp. 403428. Davies, J. 1963 Human Developmental Anat- Patten, B. M. 1968 Human Embryology. Mc- omy. Ronald Press, New York, 146-151. Graw-Hill, New York, pp. 390-394. Streeter, G. L. 1951 Developmental horizons in Du Bois, A. M. 1963 The embryonic liver. In: human embryos, Age groups XI to XXIII. Cont. The Liver. Vol. I. CH. Rouiller, ed. Academic Embry. Carnegie Inst., (Wash.) Embryology Press, New York, pp. 1-39. Reprint, vol. 11. Elias, H. 1949 A re-examination of the struc. Thompson, P. 1908 A note on the development ture of the mammalian liver. I. Parenchymal of the septum transversum and the liver. J. architecture. Am. J. Anat., 84: 311-333. Anat. Physiol., 42: 170-175. PLATE 1

EXPLANATION OF FIGURES

Schematic diagram and photomicrographs of a one-somite human embryo. Embryo 5080, Horizon IX. Photomicrographs are by courtesy of the Carnegie Institution of Washington. Diagram of a sagittal section showing the respective levels at which figures 2, 3 and 4 were taken. The endoderm is represented by the solid black area, neural ectoderm by the stippled area and mesoderm by the white area. Point a represents the area of the presumptive stomodeum. Point b represents the point at which the thickened endoderm is continuous with the single-cell layered yolk sac. (H) indicates presumptive heart. Photomicrograph of a transverse section immediately caudal to the level at which the thickened endoderm (E) is continuous with the yolk sac. x 75. Photomicrograph of a transverse section through the presumptive stomodeum. Note the mesoderm (M) extending across the midline and separating endoderm from ectoderm. x 75. Photomicrograph of a transverse section through the somite region. Note the contiguity of cells of the ventral neural plate with the dorsal archenteron (arrow), and also the position of the somite mesoderm. x 75.

144 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 1 Charles B. Severn

145 PLATE 2

EXPLANATION OF FIGURES

Schematic drawing and photomicrographs of a five somite human embryo. Embryo 2795, Horizon X. Photomicrographs are by courtesy of the Carnegie Institution of Washington. Diagram of a sagittal section showing the configuration of thickened endoderni (dark area) in relationship to the neural plate (stippled area) and developing heart (H). Observe the “U-shaped” configuration of the endoderm. Point a represents the presumptive stomodeum. Point b represents the site at which the thick endoderm is continuous with the single-cell layered yolk sac. Point c represents the area of contiguity of endoderm and the endothelial lining of the heart. AIP (anterior intestinal portal) is the point at which the flat plate of endoderm is continuous with the ventral and lateral walls of the foregut. Transverse dashed lines represent levels of figures 6, 7, and 8. Photomicrograpk of a transverse section through the stomodeum. Ob- serve the endothelial lining of the truncus arteriosus adjacent to the foregut (F), the fusion of the parietal pericardia in the ventral midline (arrow), and the mesodermal cells continuous with the somatic and splanchnic layers of mesoderm separating the thickened endoderm I:E) from the parietal pericardium. >: 75. Photomicrograph of a transverse section through the caudal level of the developing heart. Observe the fusion of the medial walls of the endothelial lining of the paired atria (A) and the lateral displacement of the epimyocardia and parietal pericardia (P). Also observe the con. tiguity of the thickened flat plate of endoderm with the caudal and ventral endothelial lining of the fused atria (arrow). x 75. Photomicrograph of a transverse section through the anterior intestinal portal. Observe the continuity of the thickened endodermal plate with :he ventral and lateral walls of the foregut (arrow). Also observe the paired endothelial primordia of the sinus venosus ( SV) immediately lateral and ventral to the anterior intestinal portal. The paired en- clothclial primordia of the sinus venosus are continuous with the blood island of the yolk sac. X 75.

146 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 2 Charles B. Severn

147 PLATE 3

EXPLANATION OF FIGURES

Schematic diagram and photomicrographs of an eight-somite human eni- byro. Enibryo 391, Horizon X. Photomicrographs are by courtesy of the Carnegie Institution of Washington. 9 Diagram of a sagittal section showing the “J-sh aped“ configuration of the thickened endoderm (dark area) and its relationship to the adjacent neural plate (stippled area) and the developing heart. Point a represents the presumptive stomodeum. Point b rep-esents the site at which the thick endoderm is continuous with the single-cell layered yolk sac. Point c represents the area of contiguity of the endoderm and endothelial lining of the heart. AIP (anterior intestinal portal) is the point at which the flat plate of endoderm is continuous with the ventral and lateral walls of the foregut. Transverse dashed lines represent levels of figures 10, 11 and 12. 10 Photoniicrograph of a transverse section through the level of the ventricle (V). Observe the well-defined pericardial sac (parietal pericardium, epimyocardium and dorsal mesocardium>s. Also observe the mesodermal cells of the septum transversum (S?’) which separate parietal pericardium from the yolk sac. There is no thickened endoderm ventral to the pericardium at this level. 11 Photomicrograph of a transverse section through the level at which the ventricle and atrium are continuous. Observe the continuity between epimyocardium, parietal pericardium and the mesodermal cells of the septum transversum (arrow). Observe the thickened endodermal plate at this level. x 75. 12 Photomicrograph of a transverse section through the caudal level of the undivided atrium (A). The epimyocardia and parietal pericardia are positioned laterally. Observe the contiguity between the endothelial lining of the atrium and the thickened endodermal plate (arrow). x 75.

148 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 3 Charles B. Severn

149 PLATE 4

EXPLANATION OF FIGURES

Schematic diagram and photomicrographs of a 16-soinite human em- byrcr. Embryo 7611, Horizon XI. Photomicrographs are by courtesy of the Carnegie Institution of Washington. 13 Diagram of a sagittal section showing the reversed “L-shaped” configuration of the thickened endoderm (dark area) in relationship to the adjacent neural plate (stippled area) and developing heart. Point b represents the site at which the thick endoderm is continuous with the single-cell layered yolk sac. Point c represents the area of contiguity of endoderm and endothelial lining of the heart. AIP (anterior intestinal portal) is the point at which the iiat plate of endoderm is continuous with the ventral and lateral walls of the foregut. Trans- verse dashed lines represent levels of figures 14, 15, and 16. 14 Photomicrograph of a transverse section through the level of the ventricle (V) and atrium (A). At this level the pericardium is well established and is separated from the thickened p.late of endoderm (E:) by mesodermal cells of the septum transversum (ST). The thickness of the endodermal plate ventral to the heart is approximately the same as that of the ventral and lateral walls of the pharynx. x 75. 15 Photomicrograph of a transverse section through the caudal level of the sinus venosus. Observe the contiguity between the endothelial lining of the sinus venosus and the endodermal plate (arrow). Also observe the mesoderm (M) between the lateral pericardia and the endodermal plate and the dome-shape of the thickened endoderm. x 75. 16 Photomicrograph of a transverse section through the level of the anterior intestinal portal. Observe the approximation of the paired omphalo-umbilical (OU ) trunks to the endoderm of the anterior intestinal portal. x 75.

150 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 4 Charles B. Severn

151 PLATE 5

EXPLANATION OF FIGURES

Phoiomicrographs are by courtesy of the Carnegie Institution of Wash- lll~ton 17 Photomicrograph of a transverse section of a 17-somite human embryo through the level of the sinus venosus (SV). Observe the ineso- derm lateral and dorsal to the thickened endoderm and the ventral folding (arrow) of the lateral edges of this endoderm. Embryo 6784. Horizon XI. x 75. 18 Photomicrograph of a sagittal section of a 17-somite human embryo. Observe the general configuration of the thickened endoderni. That segment of thickened endoderm (between the arrows) ventrai to the anterior intestinal portal is the presumptive hepatic diverticulum. Observe the area of contact between the endoderm and the endothelial lining of the sinus venosus (c, as appears in text). Observe the amount of mesoderm separating the thickened endoderm from the parietal pericardiuni ventral to c. Embryo 5116, H:orizon XI. x ’75. I9 Photomicrograph of a frontal section of a 19-sornite human embryo through the level of the heart and hepatic diverticulum. The diverticulum is a “T-shaped,” hollow structure. There is, an epithelial projection from its lateral walls. Separate omphaloniesenteric and umbilical veins can be identified. The diverticulum is in direct contact with the caudalmost extension of the endothelial lining of the sinus venosus (arrow). Embryo 6050, Horizon XI. x 75. 20 Photomicrograph of a transverse section of a 20..somite human eni- hryo imniediately caudal to the level of the anterior intestinal portal. The luinina of the hepatic diverticulum (D) and the foregut (F) are continuous. The diverticulum at this level exhibits a dilation to the right and to the left. Observe the contiguity between the lateral wails of the diverticulum and the medial endothelial linings of the omphalo- umbilical vessels (OU). Also observe the amount of mesoderm of the septum transversum (ST) between the ventral wall of the diverticuiuni and yolk sac endoderm. Embryo 2053, Horizon XI. r: 75.

152 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 5 Charles B. Severn

153 PLATE 6

EXPLANATION OF FIGURES

21 Photomicrograph of a sagittal section of a 23-somit19human embryo. The hepatic diverticulum (endoderm between arrows) is established. Observe the mass of the septum transversum (ST) between the ventral wall of the hepatic diverticulum and the parietal pericardium. Embryo 8505B, Horizon XII. Photomicrograph courtesy of the Car- negie Institution of Washington. ).: 75. 22 Photomicrograph of a sagittal section of a 15 min pig embryo. The heart (H), Liver (L), and foregut (F) are shown. Observe the endodermal cyst (C) in juxtaposition to the sinus venosus (SV). The in- sert is a higher magnification of the endodermal cyst. 23 Photomicrograph of the head, thorax and abdomen of a five-month acardia amorphus. Observe the paired vessels in the thorax (V) and also absence of heart, esophagus, lungs, diaphragm and liver. TE represents a rudimentary tracheoesophageal bud. 24 Photomicrograph of the head, thorax and abdomen of a full-term acxdia amorphus. Observe the paired vessels in the thorax (V) and absence of heart, esophagus, lungs, diaphragm antd liver. TE represents a rudimentary tracheoesophageal bud.

154 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 6 Charles B. Severn

155 PLATE 7

EXPLANATION OF FIGURES

Figures 25 through 28 are three-dimensional drawings of a ventrolateral view of different stages of the developing foregut and hepatic diverticulum made from reconstructions and models. Letter (a j indicates the stoniodeal area i~fthe tubular foregut. Letter (b) identifies the leading edge of thickened endoderin where it is continuous with the one-cell layered yolk sac. Letter (cj indicates the zone of contact between the endoderm and the caudal endothelial lining of the developing heart. 2:; Drawing shows the general configuration of the endoderm as it exists in a five-somite human embryo. 26 Drawing shows the general configuration of the ertdoderm as it exists in an eight-somite human embryo. 27 Drawing shows the general configuration of the endoderm as it exists in a 13-soinite human embryo. 28 Drawing shows the general configuration of the ertdoderm as it exists in a 16-somite human embryo.

156 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 7 Charles B. Severn

157 MORPHOLOGICAL DEVELOPMENT OF THE HUMAN LIVER PLATE 8 Charles B. Severn

29 Drawing shows the general configuration of the endoderm as it exists in a 19-somite human embryo. Letter (b) represents the leading edge of thickened endoderm where it is continuous with the one-cell layered yolk sac. Letter (c) represents the zone of contact between the endoderm and the caudal endothelial lining of the developing heart. 30 Drawing shows the general configuration of the eridoderm as it exists in a 23-somite human embryo. Letters (b) and (c) have the same representation as in figure 29. Arrows show the directions of the views for figures 31 and 32. 31 Drawing shows a section of figure 30 as viewed from the ventral side. Stippled lines and arrows show the direction of folding of the endoderm. Letter (c) represents the zone of contact between the endoderm and the caudal endothelial lining of the developing heart. 32 Drawing shows a section of figure 30 as viewed from the lateral side. Stippled lines and arrows represent the direction of folding of the endoderm. Letter (c) indicates the zone of contact between the endoderm and the caudal endothelial lining of the developing heart.

158