Download Development of the liver in the chicken embryo

THE ANATOMICAL RECORD 234555-567 (1992)
Development of the Liver in the Chicken Embryo.
I. HEPATIC CORDS AND SlNUSOlDS
GENE K. WONG AND MICHAEL J. CAVEY
Department of Biological Sciences, Division of Zoology, The University of Calgary,
Calgary, Alberta, Canada
ABSTRACT
Hemopoiesis in the liver of the chicken embryo begins on
day 7 of incubation (Hamburger and Hamilton Stage 30) and peaks on day
14 (Stage 40). During this time frame, the differentiation of hepatic cells was
examined by light microscopy, transmission and scanning electron microscopy, and morphometry. The avian liver is a closely packed mass of dendriform cords and discontinuous sinusoids. Hepatocytes are pyramidal in
shape, and they ring the bile canaliculi which run through the centers of
the cords. Semithin sections, made possible by infiltration and embedding
in glycol methacrylate, were stained with hematoxylin and eosin to assess
the general architecture of the organ and the lipid content of the hepatocytes and by the periodic acid-Schiff reaction and hematoxylin to visualize
the cytoplasmic stores of glycogen. The number of hepatocytes with demonstrable glycogen fluctuates erratically in early hemopoiesis, and the
proportion of glycogen-containing cells progressively increases as hemopoiesis climbs to a peak. Most differentiating hepatocytes are devoid of
lipid droplets until Stages 39 and 40. From Stage 30 to 35, hepatocyte volume falls to its lowest value. Subsequently (Stages 36 to 40), cell volume
increases and hepatocytes achieve a relatively uniform size. Ultrastructural changes in the differentiating hepatocytes, including alterations to
the mitochondria, endoplasmic reticulum, and Golgi apparatus, are documented. These morphological and morphometric findings on the prehepatocyte population and hepatic vasculature cover 2 of the 3 elements deemed
critical to hepatic hemopoiesis in many vertebrates. o 1992 Wiley-Liss, Inc.
Key words: Capillaries, Chick embryo, Vascular endothelium, Hematopoiesis, Kupffer cells, Liver, Microscopy, Morphogenesis
The first evidence of organogenesis of the liver appears in 20- to 22-somite chicken embryos (50-53
hours of incubation) when endodermal diverticula
arise from the floor of the foregut and extend forward
into the mesoderm of the septum transversum (Le
Douarin, 1975). Distal portions of the diverticula form
the liver, while proximal segments give rise to the biliary ducts and gall bladder (Romanoff, 1960). The septum transversum ostensibly provides the hepatic connective tissue, hemopoietic stem cells, and Kupffer
cells (fixed macrophages) for the hepatic sinusoids (Rifkind et al., 1969).
Structurally, the embryonic livers of birds and mammals appear very different. Hepatic sinusoids of a
mammal, for example, are smaller than those of a bird,
possibly due t o the presence of extravascular clusters of
hemopoietic cells in the mammal (Le Douarin, 1975).
In both birds and mammals, the discontinuous linings
of the sinusoids consist of endothelial cells and Kupffer
cells (Hodges, 1972; Bankston and Pino, 1980). The sinusoidal lumina and the perisinusoidal spaces may be
linked directly by fenestrae traversing the endothelial
cells or by intercellular gaps in the sinusoidal linings.
The mammalian liver is a network of anastomosing,
0
1992 WILEY-LISS, INC.
cribiform plates which are usually 1 or 2 cell layers
thick, and the bile canaliculi occur between the cells of
the plates (Elias, 1949a,b; Hodges, 1972). Hepatic
plates of mammals are arranged radially with respect
to central veins, organizing the tissue of the liver into
lobules (Elias, 1949a,b). The avian liver, on the other
hand, is a mass of branching, hollow cords, and the bile
canaliculi are represented by the lumina of the cords
(Romanoff, 1960). There is little or no evidence of lobulation in the avian liver (Romanoff, 1960; Hodges,
1972).
As a prelude to re-examination of hepatic hemopoiesis in the chicken embryo, we have studied the differentiation of hepatocytes and sinusoidal cells by light
Received August 20, 1991; accepted May 5, 1992.
Mr. G . K. Wong is now at Department of Anatomy and Cell Biology,
University of Alberta, Faculty of Medicine, Edmonton, Alberta, Canada T6G 2 H l .
Address reprint requests to Dr. M. J. Cavey, Division of Zoology,
Department of Biological Sciences, The University of Calgary, 2500
University Drive N.W., Calgary, Alberta, Canada T2N 1N4.
556
G.K. WONG AND M.J. CAVEY
microscopy, transmission and scanning electron microscopy, and morphometry. Our observations pertain
to the period between the onset and the peak of hepatic
hemopoiesis and illustrate a number of advantages afforded by semithin sections of glycol methacrylate-embedded tissues. Portions of this study have been reported in abstract form (Wong and Cavey, 1990).
ethylene glycol 400 (Yeung and Law, 1987) and allowed to harden for 24 h r a t ambient temperature.
Microtomy and Light Microscopy
Blocks of liver embedded in epoxy resin were sectioned with glass knives on a Sorvall MT-1 ultramicrotome a t a thickness of 1pm. Sections were stained with
a n alkaline combination of 0.5% azure II and 0.5%
MATERIALS AND METHODS
methylene blue (Richardson et al., 1960) and mounted
Specimens
in high-viscosity immersion oil.
Fertilized chicken (Gallus domesticus) eggs were obLivers embedded in glycol methacrylate were sectained locally (Agnew Chicks, Calgary, AB, Canada; tioned with glass knives on a n LKB Bromma 2218 HisCo-op Hatcheries, Calgary, AB, Canada), and embry- torange microtome (Stages 30 to 38) or a Reichert-Jung
onic development was arrested by refrigerating the 1140/Autocut microtome (Stages 39 to 42) a t a thickeggs at 2-4°C. At 12 h r intervals, batches of eggs were ness of 3 pm. Sets of spaced serial sections were made
transferred to a Marsh Turn-X tabletop incubator or from each liver, allowing different staining regimes to
Humidaire free-standing incubator a t 37°C and 70% be applied to comparable areas of the organ. Sections
relative humidity. Embryonic ages were estimated by were 1)stained with Harris’ hematoxylin and countercandling the eggs. Once freed of the egg envelopes, em- stained with eosin Y (Du Pont Company, 1981), 2) subbryos were staged according to the landmarks of Ham- jected to the periodic acid-Schiff reaction (Cavey and
burger and Hamilton (19511, using a stereomicroscope Cloney, 1972) and stained with Harris’ hematoxylin,
and fiber-optic illuminator.
and 3) stained with 0.5% azure I1 and 0.5% methylene
blue in 0.25% sodium borate (Cavey and Yeung, 1991).
Specimen Preparation
Cover glasses were attached with Fisher Permount.
The primary fixative applied to all specimens was 1%
Light-microscopic sections were viewed with a Nikon
paraformaldehyde and 2% glutaraldehyde (as recom- Optiphot compound microscope equipped with planamended by Bankston and Pino, 1980) in 0.05 M Mil- chromatic objective lenses and a Microflex HFX-I1 pholonig’s phosphate buffer a t pH 7.4. Primary fixation tomicrographic attachment. Micrographs were made
was conducted a t ambient temperature. The secondary on Kodak Technical Pan 2415 film (sometimes insertfixative used on some specimens was 2% osmium ing a green filter into the light path) and Kodak Kotetroxide in 1.25% sodium bicarbonate buffer at pH 7.4 dacolor Gold 100 film (always placing 80A and neutral(Wood and Luft, 1965). Secondary fixation was carried density filters in the light path).
out in a n ice bath.
Embedding in epoxy resin
Livers were excised from embryos which ranged from
Stage 29 to 42 and processed concurrently. The organs
were flooded with primary fixative, cut into slices, and
diced into small blocks. The blocks were then placed in
a container of fresh fixative, where they remained for 2
hr. Without washing, the blocks were exposed to the
secondary fixative for 45-60 min. They were subsequently rinsed with demineralized water for 10 sec,
dehydrated with graded ethanols, treated with propylene oxide, and infiltrated with LX-112 medium (Ladd
Research Industries, Burlington, VT). Polymerization
of the epoxy resin proceeded for 18 h r at 60°C.
Ultramicrotomy and Transmission Electron Microscopy
Blocks of liver embedded in epoxy resin were sectioned with a diamond knife on a Sorvall MT-6000 ultramicrotome at a thickness of 70 nm. The sections,
collected on naked copper grids, were stained serially
with aqueous solutions of uranyl acetate and lead citrate (Reynolds, 1963) and examined with a JEOL JEM100s transmission electron microscope a t 60 kV. Micrographs were made on Kodak Electron Microscope
4489 film.
Scanning Electron Microscopy
Specimens for scanning electron microscopy were selected from those being prepared for transmission elecEmbedding in glycol methacrylate
tron microscopy. Whole livers were cut into slices and
Whole livers were removed from chicken embryos immersed in the primary fixative for 3 hr. The slices
which ranged from Stage 30 to 42 and processed con- were slowly dehydrated with graded ethanols over a
currently. The organs were immersed in the primary period of 2 weeks, and final drying was accomplished
fixative for 8 h r with 1 change and dehydrated slowly with the fluorocarbon compound Peldri I1 (Pelco Interin graded ethanols: lo%, 30%, 50%, and 70% ethanols national, Redding, CA). Slices of liver were treated
for 12 h r each with two changes; 95% ethanol for 3 days with a solution of equal parts of fluorocarbon and abwith 2 changes; and absolute ethanol for 6 days with solute ethanol for 12-18 h r and then with pure fluorofour changes. Infiltration medium was prepared from a carbon (liquid phase) for 2-6 hr. The fluorocarbon was
Reichert-Jung Historesin kit (Cambridge Instruments, converted to its solid phase by refrigeration and alHeidelberg, Federal Republic of Germany). Over a 1 lowed to sublime overnight in a fume hood at ambient
week period, the ratio of infiltration medium-to-abso- temperature.
The dried slices of liver were attached to aluminum
lute ethanol was increased from 1:2 to 1:l. The specimens were next transferred to vials containing undi- stubs with double-sided cellophane tape and coated
luted medium, and the vials were placed on the with a gold-palladium film, approximately 28 nm
inclined table of a rotating mixer for 2 days. The em- thick, in a Polaron PS3 sputter coater. The specimens
bedding medium was supplemented with 0.6 ml poly- were observed with a JEOL JSM-35CF scanning elec-
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HEPATIC DEVELOPMENT I N T H E CHICKEN EMBRYO
TABLE 1. Stages of embryonic development with corresponding embryonic ages and
anatomical landmarks (after Hamburger and Hamilton, 1951)
Stage
29
30
31
32
33
34
Incubational age
(days)
6.0
6.5
7.0
7.5
7.5 to 8.0
8.0
35
36
37
38
8.0 to 9.0
10.0
11.0
12.0
39
40
13.0
14.0
41
15.0
16.0
42
Anatomical landmarks
Egg tooth appears on the beak
Scapular and flight feather germs form
Nictitating membrane extends from the outer ocular
rim to the scleral papillae
Claw primordia appear on the toes
Feather germs completely surround the external
auditory meati
Eyelids close
External features (except for lengths of the beak and
third toes) lose diagnostic value
tron microscope a t 10 kV. Micrographs were made on
Ilford FP4 film.
h
V
=
J~[f(x)]~dx
k!
Morphometry
Morphometric data on individual hepatocytes were
generated from transmission electron micrographs using Sigmascan software (Jandel Scientific, Corte Madera, CA) on a Zenith 2-157 personal computer linked to
a Hitachi HDG-1111C graphics tablet. For each stage
of embryonic development, 10 hepatocytes were analyzed. From vertical sections, cell perimeters and cell
areas were measured directly. Three sets of cell volumes were calculated in Quattro Pro software (Borland
International, Toronto, ON, Canada). Two of these sets
were generated with the formula for the volume (V) of
a sphere:
;
v = - PI-3.
In the first set of calculations, radius (r) was deter~ substituting the
mined from the equation r = C l 2 by
measured perimeter for circumference (C). In the second set, radius was determined from the equation r =
by inserting the measured area (A). A third set of
volumes was computed by axial rotation of linear equations. The vertical sections of cells were divided symmetrically. On one side of this long (apicobasal) axis
(x), the cell boundary was traced with straight lines to
produce a polygon. Lines perpendicular to the x-axis
were then raised to the corners of these polygons, creating smaller zones. The lengths of the perpendicular
lines and the intervening distances were recorded, and
zonal volumes were determined with linear equations
of the form f(x) = mx + b rotated about the x-axis. In
each linear equation, the positive slope (m) of a line
was calculated from the measured lengths and distances. The length of the shorter perpendicular line
served a s the i-intercept (b). The- integration steps
were as follows:
=
]P[mx
+ bl'dx
g
=
]r[m2xz
+ 2mbx + b2]dx
g
=
TI
=P
i:
-m2x3 + mbx'
h
+ b'x) 1
g
(:
-m2h3 + mbh2+ b'h)
The final step of the integration is a simplified formula
for zonal volume, where the upper limit (h) is the distance between the perpendicular lines and the lower
limit (g) is 0 (the value of x at the y-intercept). The sum
of the zonal volumes yields a cell volume.
Hepatocyte differentiation has often been correlated
with the presence or absence of glycogen deposits and
lipid droplets in the cytoplasm. Accordingly, 100 hepatocytes were sampled in a single embryo at each stage
of development, using black-and-white photomicrographs of sections stained with hematoxylin and eosin
and by the periodic acid-Schiff reaction and hematoxylin to ascertain the proportions of cells containing glycogen only, lipid only, both glycogen and lipid, or neither glycogen nor lipid.
RESULTS
Organogenesis of the Liver
Chicken embryos a t Stages 30 (n = 31, 32/33 (n = 41,
34 (n = 3), 35 (n = 3), 36 ( n = 61, 37 (n = 51, 38 ( n = 41, 39
Fig. 1. Dendriform hepatic cords (hc) of a Stage-35 embryo. Erythrocytes (ery) appear in the sinusoids
(hs) between the cords. X 1,550. Bar, 10 pm.
Fig. 2. Pyramidal hepatocytes ring the bile canaliculus (bc) within a cord from a Stage-35 embryo. hs,
hepatic sinusoid. x 2,900. Bar, 5 pm.
HEPATIC DEVELOPMENT IN T H E CHICKEN EMBRYO
(n=5), and 40 (n=6) were studied in detail. These
stages represent 24 hr intervals in embryonic development, beginning a t the onset of hepatic hemopoiesis
and extending to its peak (Medda et al., 1977). The
staging of Hamburger and Hamilton (1951) will be
used to indicate embryonic age. Relevant stages are
correlated with days of incubation and anatomical
landmarks in Table 1. Embryos at Stages 29 (n = 1),41
(n=3), and 42 (n=3) were also prepared for microscopic examination to validate the hemopoietic landmarks set by Medda et al. (1977). Hepatocyte ultrastructure a t Stage 29 (1 day before the onset of
hemopoiesis) was virtually the same as that in Stage
30. The hepatocytes a t Stages 41 and 42 (1 and 2 days
after the hemopoietic peak) could not be distinguished
ultrastructurally from Stage-40 cells.
Large semithin sections of glycol methacrylate-embedded tissues greatly facilitated the light-microscopic
survey of the developing liver. Sections stained with
hematoxylin and eosin revealed the general histology of
the organ as well as the lipid distribution among the
hepatocytes. (A lipid stain was not applied to the glycol
methacrylate sections, because the hepatic tissue was
not treated with osmium tetroxide before the lengthy
dehydration which preceded infiltration.) Cytoplasmic
regions which were periodic acid-Schiff-positive in
light-microscopicsections correlated well with cytoplasmic glycogen deposits in electron-microscopicsections.
Hepatic Cords
The liver of the chicken embryo consists of parenchymal cords interspersed with sinusoids (Fig. 1). The hepatic cords have a cylindrical configuration. The apical
(biliary) surfaces of the pyramidal hepatocytes adjoin
the bile canaliculus which runs through the center of a
cord. The basal (vascular) surfaces of the hepatocytes
flank the sinusoidal linings (Fig. 2). In the early cords
(Stages 17 and 18),6-13 hepatocytes ring each canaliculus (Fukuda, 1976). Between the onset and peak of
hemopoiesis, 6-8 hepatocytes encircle a canaliculus,
and the cell number tends to normalize at 6 in Stage 41
(Kingsbury et al., 1956).
A typical hepatocyte has a basal nucleus with one or
two large nucleoli (Fig. 3). Cisternae of the granular
endoplasmic reticulum (ER) are prevalent, flanking
the plasmalemma and partially encircling the mitochondria. Little agranular endoplasmic reticulum is
evident. Mitochondria tend to situate in the cell apex,
while ribosomes and polysomes are found throughout
the cytoplasm. Common cytoplasmic inclusions include
lipid droplets and glycogen granules. Some hepatocytes
border the sinusoids but fail to reach a canaliculus
(Fig. 4). The mitochondria of these cells, also flanked
by cisternae of granular ER, usually situate in the
basal cytoplasm. Such hepatocytes are relatively rare.
Two parenchymal cells can be identified in the developing livers of birds and mammals (Ganote and
Moses, 1968; Sandstrom and Westman, 1971; Fancsi,
1982). “Dark” cells have the ultrastructural characteristics listed above for typical hepatocytes (Fig. 5).
“Light” cells share many of these same characteristics,
but they present a paler cytoplasmic matrix which contains little or no granular ER. Vesicular cisternae of
agranular ER are prevalent in the “light” cells, but
they establish no intimate relationships with the plas-
559
malemmata or mitochondria. Extremely small vesicles
are interspersed with the glycogen granules of the hepatocytes. “Dark” and “light” cells are encountered
throughout hepatic development, and the former always outnumber the latter.
Organogenesis of the liver was examined closely in
embryos from Stage 30 to 40. Throughout this period,
the hepatocytes undergo subtle changes in shape, but
their nuclei, enclosing 1 or 2 large nucleoli, remain
spherical. The nucleoli are bipartite bodies, displaying
inner fibrous and outer granular zones (Hodges, 1972).
The outer membrane of the nuclear envelope binds ribosomes, but the ribosome distribution is uneven.
Changes in histology and ultrastructure are recorded
in the micrographs of Figures 6-14 and 15-23, respectively. The proportions of hepatocytes with or without
glycogen and lipid between the onset and peak of hepatic hemopoiesis are summarized in Figure 24. The
relative frequencies of glycogen-containing cells f luctuate during early hemopoiesis (Stages 30, 32/33, 34,
and 35). As hemopoietic activity climbs to a peak
(Stages 36,37,38,39, and 401, the proportion increases
until all cells carry glycogen. Substantial accumulation of lipid by the hepatocytes is not apparent until
Stages 39 and 40.
Hepatocytes at Stage 30 are tall cuboidal or low columnar cells (Figs. 6, 15). Their large, electron-lucent
mitochondria have few cristae and tend to localize in
the basal cytoplasm. Vesicular cisternae of granular
ER are poorly developed and show a low concentration
of bound ribosomes. Free ribosomes and polysomes are
dispersed throughout the cytoplasm but are not especially numerous. Glycogen deposits occur in relatively
few hepatocytes a t this time. Dense material in the
Golgi cisternae and vesicles is a morphological indication of activity.
At Stage 32/33, the hepatocytes are still tall cuboidal
or low columnar in shape, and cytoplasmic lipid droplets are observed in a few cells for the first time (Figs.
7, 16). In transmission electron micrographs, small
pockets of glycogen often adjoin the Golgi apparatus.
Although fewer cells contain glycogen in Stage 34
(Figs. 8,171, the cytoplasmic accumulations are larger.
Lipid droplets are also growing in size, and many of the
nascent droplets have electron-opaque mantles and
electron-lucent cores. These bipartite droplets accumulate until Stage 40, but it is still unknown if their images reflect compositional differences between the central and peripheral areas or result from differential
extraction and staining (Sandstrom and Westman,
1971). From Stage 41 onward, only the homogeneous
lipid droplets accumulate in the cytoplasm.
As hepatocytes differentiate, the electron opacity of
their mitochondria intensifies and the cristae increase
in number. The content of granular endoplasmic reticulum expands concurrently, establishing a network of
flattened cisternae with high concentrations of bound
ribosomes. These cisternae promptly affiliate with mitochondria and the undersurfaces of the plasmalemmata.
By Stage 35, the sinusoidal lumina have shrunk in
size, being reduced to the width of just 2 or 3 erythrocytes (Figs. 9, 18). Most hepatocytes have by now assumed pyramidal shapes, creating a network of dendriform cords (Fig. 1). Canaliculi develop in the cords
560
G.K. WONG AND M.J. CAVEY
Figs. 3-5.
HEPATIC DEVELOPMENT IN THE CHICKEN EMBRYO
while changes in hepatocyte shape are progressing
(Fukuda, 1976). Hepatocytes adjoining a bile canaliculus form apicolateral junctions, originally called “attachment zones” (Stephens and Bils, 1967) but subsequently recognized as occluding (tight) junctions
(Sandstrom and Westman, 1971). We have found no
evidence of intermediate junctions beneath the tight
junctions (Hodges, 1972). A few short microvilli from
the apices of the hepatocytes project into the canaliculi.
The first hepatocytes to contain both lipid and glycogen are encountered at Stage 36 (Figs. 10, 19). Microvilli are no longer confined to the apices of the cells.
They also extend into the extracellular spaces between
hepatocytes and into the perisinusoidal spaces separating hepatic cords from the sinusoidal linings. Except
for slight differences in the content of glycogen and
lipid, hepatocytes at Stages 37 (Figs. 11, 20) and 38
(Figs. 12, 21) are remarkably similar in histology and
ultrastructure. As the apices of hepatocytes retract and
the intercellular spaces unite, the bile canaliculi enlarge and extend farther along the hepatic cords. Material inside the canaliculi is observed for the first time
a t Stage 37, although bile secretion is known to begin
much earlier (Sandstrom and Westman, 1971).
Most hepatocytes begin to accumulate lipids at Stage
39 (Figs. 13, 22). Bipartite lipid droplets are observed
in some cells, but droplets with a uniform consistency
appear in others. By Stage 40, all hepatocytes possess
lipid and glycogen (Figs. 14, 23). The profusion of lipid
droplets led Romanoff (1960) to describe the intervening cytoplasm as trabecular. Increasing numbers of microvilli project into the expanding bile canaliculi.
Shortening of the microvilli during enlargement of the
canaliculi, equated with biliary stasis by Sandstrom
and Westman (19711, was not observed.
Changes in cell size also take place between the onset and peak of hepatic hemopoiesis (Fig. 25). The areas
of vertically sectioned hepatocytes decrease from Stage
30 to 35. From Stage 36 onward, the sectional areas
increase, reaching a plateau at Stage 38. All three
methods used to calculate volume confirm a decrease in
hepatocyte size from the onset of hemopoiesis to Stage
35, after which size increases to reach a maximum at
Stage 38 or 39. By using a radius derived from cell
area, the calculated volumes were, on average, 16%
higher than those generated by integration techniques.
With a radius obtained from cell perimeter, the vol-
Fig.3.Hepatocytes of a Stage-37 embryo. Granular cisternae of the
endoplasmic reticulum (er) appose the plasmalemmata and mitochondria (mt) of the hepatocytes (Id, lipid droplet; nl, nucleolus; nu, nucleus). The narrow apices of the hepatocytes adjoin a bile canaliculus
(bc), and the broad bases of the cells face the sinusoids (en, endothelial
cell). arrowhead, perisinusoidal cell; ery, erythrocyte; ps, perisinusoidal space. x 8,550. Bar, 1 pm.
Fig. 4.Many hepatocytes associate exclusively with the sinusoidal
linings (en, endothelial cell; nu, nucleus). Mitochondria (mt) tend to
localize in the basal cytoplasm of these cells. ps, perisinusoidal space.
x 15,400. Bar, 1 pm.
Fig.5.Parenchymal cells of the liver include “ d a r k cells and “light”
cells. Flattened cisternae of the granular endoplasmic reticulum (er)
are present in “dark” cells while vesicular cisternae of the agranular
endoplasmic reticulum are evident in “light” cells. gl, glycogen granules; mt, mitochondrion. x 6,300. Bar, 1 pm.
561
umes were about 41% higher than those determined by
either of the other two procedures.
Hepatic Sinusoids
Morphological changes of the hepatic sinusoids from
the onset to the peak of hemopoiesis are very subtle.
The sinusoidal linings consist of flattened endothelial
cells (Figs. 3 , 4 ) and cuboidal Kupffer cells. A very close
association exists between the sinusoidal cells and the
outlying perisinusoidal cells and hepatocytes. A basal
(external) lamina invests each sinusoid, and it, like the
vessel, is discontinuous.
In early stages of hepatic development, the sinusoidal linings are continuous epithelia, and there are no
obvious channels between the lumina and perisinusoidal spaces. In the Stage-32/33 liver, sectors of the
sinusoidal walls become quite thin, and epithelial discontinuities are apparent in electron-microscopic sections. These discontinuities, ranging from 0.5 to 17 pm
in width, are intercellular gaps rather than transcellular fenestrae (cf. Hodges, 1972).
Between Stages 29 and 42, endothelial cells show
some plasmalemmal activity, manifested as membrane
pits and cytoplasmic vesicles and disclosing a potential
mechanism (i.e., transcytosis) for transporting fluid
and macromolecules between the blood plasma and tissue fluid. The pits and vesicles become progressively
less prominent as intercellular gaps arise within the
sinusoidal lining. Gaps in the sinusoidal wall would
account for vascular permeability, and the larger ones
are likely routes by which extravascular hemopoietic
cells would enter the bloodstream. Hemopoietic roles of
the sinusoidal cells are considered in our companion
paper (Wong and Cavey, 1992).
DISCUSSION
Scanning electron microscopy confirms that the
avian liver is a mass of dendriform cords. It is understandable how longitudinal sections of the hepatic
cords were originally misinterpreted as cell plates and
how the central canaliculi of avian cords were mistakenly considered to be comparable to those of mammalian plates (Elias and Bengelsdorf, 1952; Hodges,
1972). As the concentration of cords increases during
organogenesis, it becomes increasingly difficult to discern their cylindrical nature in light-microscopic sections.
Two modes of canaliculus formation were proposed
by Fukuda (1976): the assimilation of lumina within
terminal branches of the hepatic diverticula and the
interconnection of small spaces which arise independently between the hepatocytes. From the onset to the
peak of hemopoiesis, bile canaliculi unquestionably
form by the union of intercellular spaces. There is no
evidence indicating that canaliculus formation precedes the establishment of apicolateral junctions between the hepatocytes (Stephens and Bils, 19671, although these junctions are undoubtedly remodelled
during cell rearrangement.
“Dark” cells, characterized by abundant cisternae of
the granular endoplasmic reticulum, predominate in
the avian liver. Association of the cisternae with the
mitochondria may facilitate cell metabolism (Stephens
and Bils, 19671, with mitochondria providing the energy for protein synthesis by the bound ribosomes. The
562
G.K. WONG AND M.J. CAVEY
Figs. 6-10.
HEPATIC DEVELOPMENT IN THE CHICKEN EMBRYO
563
Figs. 6-14. Cytology of embryonic hepatocytes a t Stages 30 (Fig. 6),32/33(Fig. 7), 34 (Fig. 81,35(Fig.
9), 36 (Fig. lo), 37 (Fig. 111, 38 (Fig. 12), 39 (Fig. 131,and 40 (Fig. 14).A Hematoxylin and eosin. B
Periodic acid-Schiff reaction and hematoxylin. X 560.Bars, 50 pm.
“light” cells of the avian liver are ultrastructurally distinct from the “dark” cells (Sandstrom and Westman,
1971; Fancsi, 1982). “Light” cells contain vesicular cisternae of the agranular endoplasmic reticulum. Agranular ER is a prevalent organelle in cells which specialize for lipid metabolism or steroid hormone synthesis
and is implicated in the processes of glycogenesis and
glycogenolysis. The cisternal membranes in hepato-
cytes also contain the enzymes which produce the lipid
moieties for lipoproteins and the enzymes for detoxification reactions (Cardell, 1977).
The prevalence of agranular ER in the “light” cells
and granular ER in the “dark” cells indicates a fundamental difference in cell function. One must not forget,
however, that the 2 forms of endoplasmic reticulum can
interconvert through the addition or loss of ribosomes
Figs. 15-23. Ultrastructure of embryonic hepatocytes at Stages 30 (Fig. 151, 32/33 (Fig. 16), 34 (Fig.
17), 35 (Fig. 18), 36 (Fig. 19), 37 (Fig. 20), 38 (Fig. 21),39 (Fig. 22), and 40 (Fig. 23). bc, bile canaliculus;
gl, glycogen granules; Id, lipid droplet; nu, nucleus. x 7,650. Bars, 1 pm.
HEPATIC DEVELOPMENT I N T H E CHICKEN EMBRYO
565
Fig. 24. Distribution of cell variants during hepatic development. Hepatocytes were scored for the
presence or absence of glycogen and lipid.
(Stephens and Bils, 1967; Cardell, 1977). In differentiating hepatocytes of the bird, cisternae tend to flatten
during the addition of ribosomes. Interconversion and
cisternal modifications may account for some discrepancies in the ultrastructural descriptions of differentiating cells, and the metabolic condition of cells at the
time of fixation may also affect ultrastructural appearance.
In the mammalian liver, Ganote and Moses (1968)
concluded that the “light” cells were just one of many
artifacts resulting from fixation by the immersion
method. These artifacts, including vesiculation of the
endoplasmic reticulum, modifications to mitochondria1
size and shape, and excessive clumping of the chromatin in the nucleus, were attributed to the concentration
of glutaraldehyde, elapsed time in the fixative, and size
of the tissue blocks. Fixation by perfusion was rated
superior t o fixation by immersion. In our study, which
utilized fixation by immersion, there were no significant differences between the mitochondria or the nuclei in the “dark” and “light” cells. Other workers have
concluded that these 2 cell types occur naturally in the
liver and that injury or pathology can alter their proportions (Steiner and Baglio, 1963; Herdson et al.,
1964; Ghidoni, 1967). It has also been suggested that
“light” and “dark” cells represent different stages of
hydration (Herdson et al., 1964) and that “dark” cytoplasm is an indicator of imminent cell death (Ghidoni,
1967). There is an obvious need to reconcile the many
disparate views on the significance of these 2 parenchymal cell types.
Stages 29-31 encompass several important metabolic landmarks in the avian liver, including the onsets
of bile secretion, glycogen synthesis, and specialized
hepatic functions (Sandstrom and Westman, 1971).
Small vesicles among the glycogen granules have been
reported here and in previous studies (Karrer, 1960b,
1961; Stephens and Bils, 1967; Sandstrom and Westman, 1971).The small vesicles might contain glycogen
precursors or the enzymes for glycogenesis (Karrer,
1960a,b). Such suggestions recognize the involvement
of the Golgi apparatus in carbohydrate metabolism and
enzyme production (Rothman, 1981). Golgi vesicles
would deliver these products to the cytoplasmic areas
of glycogen accumulation o r utilization. Stephens and
Bils (1967),on the other hand, contend that the vesicles
arise as terminal blebs from cisternae of the granular
ER. Vesiculation from the ends of cisternae, however,
566
G.K. WONG AND M.J. CAVEY
Fig. 25. Changes in cell size during hepatic development. The areas of vertically sectioned hepatocytes
(cross-hatched bars) were measured, and the volumes were calculated from measurements of cell perimeter (solid squares) and cell area (solid triangles) and by axial rotation of linear equations (solid circles).
was seldom observed in our study. Based upon a fuller
appreciation of the structure and function of endoplasmic reticulum, vesiculation is now thought to be restricted to specific sites, the “transitional elements.”
Transport vesicles from these sites ferry newly synthesized proteins and lipids to the Golgi apparatus for further processing and sorting (Rothman, 1981).
Karrer (1960a, 1961) speculated about involvement
of the Golgi apparatus in glycogenesis. His premise
was based the tandem appearance of the Golgi apparatus and glycogen deposits in the cytoplasm at Stage 29,
the resemblance of vesicles among the glycogen granules to Golgi vesicles, and the proximity of the Golgi
apparatus to some cytoplasmic glycogen pockets. Karrer (1960b)did concede that positioning of the glycogen
pockets near the Golgi apparatus could be coincidental.
Electron-dense material in the Golgi apparatus is an
indicator of activity during early organogenesis, but
such activity need not necessarily relate to glycogenesis.
Romanoff (1960) noted a glycogen decrease in hepatocytes from Stage 35 to 38, but Stephens and Bils
(1967) observed a continual increase in glycogen across
Stages 29,34,36, and 39. By including more embryonic
stages, thus sampling the liver at shorter intervals, we
found no evidence for a glycogen decrease in cells dur-
ing Stages 36 to 40. In his assays of the liver, Lee
(1951)also noted that the amount of glycogen increases
gradually, but not in a linear fashion as implied by
Stephens and Bils (1967). While hepatocytes engage in
glycogenesis during differentiation, the frequencies of
glycogen-containing cells fluctuate widely in early hemopoiesis (Stages 30 to 351, indicating that glycogen
accumulation is progressive but discontinuous. This
trend can be rationalized if one considers that hepatocytes are utilizing, as well as amassing, glycogen during differentiation, a suggestion also made by Lee
(1951). Mitotic activity in early hemopoiesis might, for
example, require glycogenolysis.
Using 10 pm paraffin sections stained with hematoxylin and eosin, Medda et al. (1977) assessed changes
in hepatocyte size during differentiation by measuring
diameters and calculating cross-sectional areas. Noting that liver growth hinges on cell proliferation, increases in cytoplasmic volume, and increases in cytoplasmic mass owing to synthetic activity, these
workers defined three phases of cell growth: an increase in size from Stage 24 to 34 attributed to proliferation, volumetric increases, and synthetic activity; a
relatively constant size from Stage 34 to 40 related to
synthetic activity and proliferation; and another increase in size from Stage 40 to 45 ascribed to synthetic
HEPATIC DEVELOPMENT IN THE CHICKEN EMBRYO
activity and volumetric increases. A period of constant
cell size between Stages 30 and 40, corresponding to
the second phase of cell growth, was not observed in our
study. The decrease in hepatocyte size during early hemopoiesis could be a reflection of proliferative activity
in that mitoses may occur at faster rates, allowing less
time for cell growth between divisions. Slower mitotic
rates between Stages 35 and 38 would afford the cells
more time for growth. This explanation would better
account for the mitotic indices given by Medda et al.
(1977).
The hepatic cords and sinusoids represent major elements of the “hemopoietic environment” of the embryonic liver. Bankston and Pino (19801, in their close examination of the fetal liver of the mammal, observed a
subtle transformation of endothelial cells in the sinusoidal capillaries, from fenestrated forms with diaphragms to fenestrated forms without diaphragms.
Both sinusoidal linings, it was shown, were permeable
to carbon particles, and both lacked basal laminae. A
sinusoidal transformation also occurs in the embryonic
liver of the bird, but it involves a change at the epithelial, rather than the cellular, level of organization. The
sinusoidal lining, initially a continuous layer of endothelial cells and Kupffer cells, becomes discontinuous
through the development of intercellular gaps. Discontinuities appear concurrently in the enveloping basal
lamina. Since sinusoidal configuration has a n influence on cell migration from hemopoietic clusters to the
sinusoidal lumina, it will be considered again in our
companion paper, along with hemopoietic roles played
by the lining cells (Wong and Cavey, 1992).
ACKNOWLEDGMENTS
This investigation was supported by Research Operating Grant No. OGP0000484 from the Natural Sciences and Engineering Research Council of Canada.
We acknowledge the invaluable advice of Professor Edward C. Yeung, Department of Biological Sciences of
The University of Calgary, for preparation of glycol
methacrylate-embedded specimens.
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