/. Embryo!, exp. Morph. Vol. 30, 1, pp. 83-96, 1973
Printed in Great Britain
83
Differentiation of the mammalian hepatic
primordium in vitro
I. Morphogenesis and the onset of haematopoiesis
By G.R.JOHNSON1 AND R.O.JONES1
From the Department of Zoology, University of Melbourne
SUMMARY
This study was made to determine the nature of tissue contributions to murine hepatic
morphogenesis, the developmental potential in vitro of various hepatic regions prior to and
during definitive liver development, and to accurately determine the time of onset of hepatic
haematopoiesis in vitro. Embryos were precisely staged by somite counts as well as time of
original mating, and tissues were removed from the hepatic area at stages between anterior
intestinal portal (9 days gestation) and definitive foetal liver (11-75-12 days gestation), and
maintained in vitro for up to 4 weeks.
Explants of anterior intestinal portal origin gave rise only to sheets of epithelial cells,
whereas tissues from embryos of 10-11 days gestation, containing the hepatic sacculation of
the ventral wall of the gut, gave rise to sheets of polygonal, frequently binucleate cells and
granule containing cells, both suggestive of hepatic cytodifferentiation. Sinusoidal morphogenesis was also observed. Haematopoiesis was detected in vitro only in explants from 11-5to 12-day embryos (28 to 30+ somites). The blood elements observed consisted of erythroblasts, granulocytes, megakaryocytes as well as macrophages and yolk sac derived erythroblasts. Duct systems, probably representative of the bile system, were also observed to
develop.
Current theories regarding hepatic morphogenesis are considered in the light of these
observations. The inductive and cell seeding concepts of the origin of haematopoietic cells
are discussed in view of the observed precise timing of the onset of hepatic haematopoiesis.
Due to the substantial gestation time-gap between the detection of possible circulating
haematopoietic stem cells (9 days gestation) and the actual onset of hepatic haematopoiesis
(11-5 days gestation), it is suggested that either a suitable microenvironment must first
develop within the hepatic area for haematopoiesis to occur, or that haematopoietic tissue
arises in situ in the hepatic Anlage.
INTRODUCTION
The developmental interactions involved during morphogenesis of the chick
hepatic area have been extensively analysed (Croisille & Le Douarin, 1965). A
similar analysis of the development of the mammalian hepatic area, however,
has not been performed. Rugh (1968) suggests that the murine liver primordium
develops as an endodermal diverticulum from the ventral wall of the gut,
posterior to the lung primordia and anterior to the pancreatic diverticulum.
1
Authors' address: Department of Zoology, University of Melbourne, Parkville, Victoria
3052, Australia.
6-2
84
G. R. JOHNSON"AND R. O. JONES
The hepatic diverticulum extends ventrally and cranially and at 11-25 days
gestation cords of endodermal cells begin to proliferate into the region of the
junction of the vitelline veins and the mesenchyme of the transverse septum
(Rifkind, Chui & Epler, 1969). The current paper, in part, describes the type of
differentiation obtained in vitro from the hepatic area of the mouse at stages
from the anterior intestinal portal of 9 days gestation to the definitive foetal
liver (11-75 days gestation).
It has been firmly established that foetal hepatic haematopoiesis occurs
entirely within the liver tissue, and external to the sinusoids of the vitelline
veins in the mouse (Jones, 1970), the rabbit (Ackerman, Grasso & Knouff,
1961; Sorenson, 1963; McCuskey, 1968), the pig and the human (Ackerman
et al. 1961). Granulocytes, megakaryocytes, possible haematopoietic stem cells
as well as erythroid elements were observed in the mouse foetal liver (Jones,
1970), and observations suggested that the various blood cells entered the foetal
circulation by diapedesis from the liver tissue. Similar observations have been
made in the rabbit (McCuskey, 1968). Rifkind et al. (1969) consider that the
erythroid cells arise by an inductive interaction between the proliferating endodermal cells of the liver cords and the mesenchyme of the transverse septum,
the blood cells being mesenchymal in origin. Moore & Metcalf (1970), on the
other hand, consider that the hepatic haematopoietic cells arise by the migration
of a precursor cell into the liver tissue from the yolk sac, via the vitelline veins.
The current paper correlates the morphogenesis of the hepatic area with the
onset of haematopoiesis in vitro, and describes the development of various
blood elements in association with hepatocyte differentation.
MATERIALS AND METHODS
Animals. Two strains of mice were used for all experiments - an inbred
albino strain (University of Melbourne, Zoology Department) and a C57B
strain (Walter and Eliza Hall Institute for Cancer Research, Melbourne).
Pregnancies were obtained by placing three virgin females with a single male;
midnight following vaginal plug discovery was regarded as the end of the first
day of gestation, the embryo at this time designated a 1-day embryo and the
time of gestation 1 day. Ovulation in mice appears to be independent of the
time of mating, which usually occurs mid-way through the dark phase, but this
can vary by up to 5 h (Snell, Fekete, Hummel & Law, 1940; Braden, 1957).
Furthermore, within a litter, developmental variation can extend over several
somites, particularly during the early stages of gestation (Allen & MacDowell,
1940). Due to these variations embryos were further staged by external features
and somite counts.
Dissection. Pregnant females of appropriate age were rendered unconscious
with CO2 and killed by cervical dislocation. Intact uterine horns were removed
aseptically and immersed in nutrient medium. Embryos were dissected free of
Hepatic morphogenesis in vitro
85
uterine tissue and completely immersed in nutrient medium in 3-2 cm embryological watch glasses.
The embryonic liver between 11-25 and 12 days gestation (26 to 32+ somites)
consists of three lobes and these were removed in the following manner. The
portions of embryo anterior to the otocyst and posterior to the umbilical region
were first removed. The somites and neural tube of the remaining thoracic area
were then dissected away, thus exposing the dorsal surface of the foregut.
Removal of the lateral walls of the coelom and foregut followed, resulting in the
exposure of the ventral wall of the latter. The three liver lobes can then be
clearly observed, and tissue ventral to these, including the heart, was carefully
removed. Removal of the heart was postponed until as late as possible since this
organ allows excellent orientation of the hepatic region. In this manner it is
possible to expose the entire foregut from the level of the pharynx to that of the
developing duodenum (Fig. 1). The liver lobes, either together or separately,
could then be removed from the gut with relative ease, after removal of the heart.
Embryos of 11 days gestation (20-26 somites) do not have three distinct
hepatic lobes but instead a ventral sacculation of the foregut (see Fig. 2). This
was removed with the adjacent septum transversum in a manner similar to that
described above.
in embryos earlier than 10-5 days gestation the foregut is not very extensive,
developing from the posterior movement of the cells of the anterior intestinal
portal. The prospective hepatic area of these embryos was removed by cutting
through the lateral walls of the anterior intestinal portal and thereby isolating
the ventral floor of the foregut.
All micro-dissections were carried out under a Zeiss binocular dissection
microscope (x 40) using cataract knives and watchmaker's forceps.
Culture methods. Eagle's Basal Medium containing 10 % horse serum
(Commonwealth Serum Laboratories, Melbourne) was used throughout. Provided that the horse serum was fresh these proportions gave a final pH of 7-4.
Pieces of tissue were explanted into a modified Rose chamber (Light Co. Ltd.)
with the use of an orally controlled micropipette. Explants were held on to the
cover-glass (Gold Seal Brand, size 43 x 50, thinness 1) by strips of cellulose
dialysis tubing 1 cm wide (Visking Co. size 36/32, flat width 1-47/64), according
to Rose, Pomerat, Shindler & Trunnell (1958).
It was observed that flattening of the explanted tissue by the cellulose strip
was essential, the most extensive outgrowth and differentiation occurring when
the tissue was five cells or less in thickness. Central necrosis was frequently
observed in thicker explants, and occasionally the cellulose strip was mechanically compressed to ensure a flattened tissue.
Assembled chambers were maintained at 37 °C in either a dry-air incubator
or a humidified incubator continuously flushed with 5 % CO2 in air, chambers
being alternated between these to maintain the pH at 7-4. The medium in each
chamber was changed twice a week.
86
l
G. R. JOHNSON AND R. O. JONES
Hepatic morphogenesis in vitro
87
Observations. Observations were made with a Wild M 40 inverted phasecontrast microscope at magnifications between 100 and 500 times. Photomicrographs were obtained with a Zeiss Ikon camera mounted on a Zeiss Standard
R.A. phase-contrast microscope, using Kodak 35 mm Pan F film and automatic
exposure. Time-lapse cinematography was performed with a Wild M 20 phasecontrast microscope, with attached Bolex 16 mm movie camera using Kodak
Plus X Reversal Film 7276.
Histology. Whole embryos between 10 and 12 days gestation were fixed in
either 10 % formalin or formalin acetic acid, and 10 /an sections were stained
with haematoxylin and eosin. The foregut regions of embryos between 11 and 12
days gestation were removed intact, fixed in formalin acetic acid and stained
with borax carmine, and whole mounts were prepared and observed on cavity
slides. Cultures were fixed in Zenker-formalin and stained with Jenner-Giemsa
(Humason, 1962).
RESULTS
Hepatic morphogenesis
Embryos of 9-10 days gestation (7-15 somites) do not show any modification
of the ventral wall of the foregut, and the anterior and posterior intestinal
portals are still some considerable distance apart. However, during the latter
part of the 10th and early 1 lth day of gestation (16-26 somites) a sacculation
FIGURES 1-6
Fig. 1. Lateral view of 32-somite mouse embryo showing light-hand lateral liver
lobe (L), ventral livei lobe in septum transversum (v), gall bladder (g), developing
duodenum (d), sinus venosus (s.v.) and pharynx (p). Formalin fixation; borax
carmine stain. Scale 250/<m.
Fig. 2. Transverse section of 26-somite embryo at the level of the hepatic sacculation
(h) which lies in the septum transversum (s.t.) and is ventro-medial to the paired
vitelline veins (v.v.). The mesothelial thickening (m) that contributes mesenchyme
to the liver Anlage is readily seen. Formalin fixation; haematoxylin and eosin stain.
Scale 50 fim.
Fig. 3. Transverse section of 28-somite embryo at the level of the hepatic sacculation.
The sacculation is becoming separated from the gut by a constriction (c). The lefthand vitelline vein (v.v.) is developing several channels due to cellular invasion.
Formalin fixation; haematoxylin and eosin stain. Scale 50/tm.
Fig. 4. Epithelial cells displaying perinuclear deposition of phase-light droplets and
a relatively clear cytoplasm. Explant of portion of anterior intestinal portal from an
8- to 9-somite embryo, 3 days in vitro. Scale 10 fim.
Fig. 5. Epithelial cells derived from explant of ventral liver lobe and septum transversum of 25-somite embryo, 5 days in vitro. These cells have a more phase-dense
cytoplasm than those derived from earlier explants (Fig. 4), but have fewer phase
light droplets. Scale 10/tm.
Fig. 6. Sinusoid (s) bordered by endothelial cells. The nuclei (n) of these cells protrude slightly into the sinusoidal space and are flanked by cytoplasmic processes (c)
which connect adjacent endothelial cells. Ventral liver lobe explant from a 28-somite
embryo, 2 days in vitro. Scale 10 fim.
88
G. R. JOHNSON AND R. O. JONES
appears in the ventral wall of the foregut (Fig. 2), and at 26 somites the cephalic
margin of this broad based sacculation abuts against the point of fusion of the
two lateral vitelline veins. Caudally the sacculation extends to the level of the
anterior intestinal portal. Between these two landmarks the endodermal sacculation lies ventro-medial to the vitelline veins and in the mesoderm of the
septum transversum. At this stage also (26 somites) a mesothelial contribution
to the hepatic Anlage appears, in the ventral coelomic mesothelium lying against
the dorsal endothelium of the vitelline veins begins to thicken, and endodermal
cells from the ventral sacculation appear to approach this area during the early
part of the 1 lth day of gestation (26-28 somites). By 11-5-11-75 days of gestation
(28-30 somites) the region of each individual vitelline vein has been replaced by
smaller vascular spaces, due probably to endodermal cell proliferation (Fig. 3).
Serial sections suggest a mesothelial as well as this endodermal contribution to
the developing hepatic lateral lobes. Endodermal cells also appear to extend
ventrally, ventro-laterally as well as cranially to infiltrate the mesenchyme
tissues of the septum transversum, near the junction of the vitelline veins, and
so form a ventral hepatic lobe. At this stage the sacculation is beginning to show
signs of separation from the dorsal foregut to produce the definitive hepatic
diverticulum.
Tissue culture
Explants of the ventral wall of the anterior intestinal portal (9-10 days
gestation, 7-15 somites) gave rise to a monolayer of epithelial cells (Fig. 4).
Apart from a perinuclear deposition of phase-light droplets, these cells had a
relatively clear cytoplasm, with a single nucleus with one or two prominent
nucleoli. No blood cells were observed to differentiate.
Explants of the ventral gut wall of the 10- to 11-25-day embryo (16-26
somites), including the sacculation together with its surrounding mesenchyme,
gave rise to sheets of polygonal-shaped cells in a pavement arrangement. These
cells contained fewer, more evenly distributed phase-light droplets compared to
the cells from the anterior intestinal portal explants, and the cytoplasm was
more phase-dark (Fig. 5). Binucleate cells were frequently observed and most
nuclei showed two or more prominent nucleoli.
Granule-containing cells with large rounded nuclei and several nucleoli were
frequently observed in association with the polygonal cells (Fig. 15). Time-lapse
cinematography showed that these cells, though usually arranged in groups,
had an active periphery and were continually forming and reforming connexions
with each other, and they survived together with the polygonal cells, for the
whole duration of the culture period. We suggest that these granule-containing
cells represent a form of hepatic cellular differentiation obtained in vitro (Rose,
Kumegawa & Cattoni, 1968).
Towards the end of the first week in vitro these explants displayed a form of
morphogenesis in cell layers directly adjacent to the cellophane strip, most
cultures having several layers of cells. These structures were usually elongate,
Hepatic morphogenesis in vitro
89
being bounded by long filmentous cells that delineated them from the polygonal
cell sheet (Fig. 6). These long filamentous cells were similar to sinusoidal
endothelial cells observed in vivo (Jones, 1970).
Occasionally a few amoeboid macrophages appeared in some cultures after
1 week in vitro, and after 2 weeks the macrophage population was very extensive. No blood cell types were observed to differentiate.
in explants from embryos of 30 somites (11-5-12 days gestation) or older,
phase-white spacings lined with granule containing cuboidal cells were frequently
observed (Fig. 16). These extended for considerable distances and occasionally
developed a substantial lumen. The cuboidal cells were always sharply demarcated from the surrounding epithelial cells and frequently had thin bands of
cells aligned along their outer surfaces. We suggest that these ducts probably
represent portions of the in vivo bile duct system.
Haematopoiesis in vitro
With the 11-5- to 12-day embryos (28-30+ somites) each lateral lobe and the
ventral lobe were explanted separately. During dissection a dorsal view of the
gut and hepatic area showed that the left lateral lobe was considerably larger
than the right and both appeared as hollow bulges on separation from the gut
wall and the ventral lobe. In the 11-75-day embryo (30 somites) fine circulatory
sinusoids containing blood cells were observed in the lateral lobes during
dissection.
The hepatic ventral lobe of the 11-5-day embryo (28 somites) appeared much
more solid than the lateral lobes, and the rounded outline of the hepatic
diverticulum could be clearly observed deep within the mass of endodermal
cells. In the 11-75- to 12-day embryo (30+ somites) the ventral lobe appeared
much larger than either lateral lobes, and a small rounded structure, probably
the developing gall bladder, could be seen protruding caudally from the main
mass of hepatic tissue (Fig. 1). The ventral lobe and the investing mesenchyme
of the transverse septum were explanted together.
Explants of both lateral lobes and the ventral lobe of the 11-5-day embryo (28
somites) behaved in vitro similarly to the ventral sacculation explants of the
early 11-day embryo (16-26 somites), in that sheets of polygonal cells were
formed as well as the sinusoidal-like structures. However, all explants of the 28somite embryo (11-5 days gestation) showed haematopoiesis within 24 h in vitro,
as did similar explants from older embryos (30+ somites). An ever increasing
population of macrophages was observed throughout the culture period of 3-4
weeks, as well as (i) primitive yolk-sac-derived erythroblasts, (ii) differentiating
adult-type erythroblasts, (iii) granulocytes and (iv) megakaryocytes. Due to the
presence of the vitelline vessels it was impossible to exclude circulating yolk-sac
cells. These yolk-sac-derived erythroblasts could be distinguished from adulttype erythroblasts by their larger diameter and smaller nuclear-cytoplasmic
ratio. Direct morphological comparisons were also made between the two
90
G. R. JOHNSON AND R. O. JONES
8
Hepatic morphogenesis in vitro
91
Table 1
Mean diam
(/tin)
Yolk-sac erythroblasts
Hepatic erythroblasts
Hepatic granulocytes
14-5
9
16
Range
10-18
5-13
12-22
populations of red blood cells, in that embryonic yolk-sac explants were
simultaneously cultured with liver explants (confrontation cultures), and the
differences could then be easily ascertained (Figs. 11 and 12 and Table I).
(i) Erythroid series
The greatest number of these were of the adult-type red blood cells rather
than the yolk-sac-derived blood cells, and of the former the more immature
forms predominated. These were very abundant and were denoted by their high
nucleo-cytoplasmic ratio, rounded shape, peripheral nucleochromatin clumping
and basophilic cytoplasm (Fig. 11). These cells usually appeared in groups and
frequently showed mitotic activity (Fig. 14). Time-lapse studies showed that
they were highly motile with an active cell periphery, and were often observed
to move in streams between adjacent epithelial cells (Fig. 7). Normoblasts were
relatively scarce and mature erythrocytes were observed only very occasionally,
probably due to the prolific macrophage activity (Fig. 9). Usually after 10 days
in vitro erythroid cells had completely disappeared.
(ii) Granulocytes
These cells were recognized by their nuclear shape, which ranged from beanshaped through to doughnut-ringed forms (Fig. 10), the latter not normally
FIGURES
7-12
Fig. 7. Groups of haematopoietic cells arranged between adjacent epithelial cells.
Explant from 36-somite embryo, 4 days in vitro. Zenker-formaltn fixation; JennerGiemsa stain. Scale 50/*m.
Fig. 8. Megakaryocyte (m) with multi-lobed nucleus. The large size of this cell is
apparent when compared to the adjacent erythroblast (<?). Explant from 36-somite
embryo, 3 days in vitro. Zenker-formalinfixation; Jenner-Giemsastain. Scale 20/*m.
Fig. 9. Amoeboid macrophage with large nucleus («), engulfed erythroid cells (e) are
apparent in the cytoplasm. Explant from 36-somite embryo, 3 days in vitro. Zenkerformalin fixation; Jenner-Giemsa stain. Scale 10/tm.
Fig. 10. Granulocyte (g) with lobulated nucleus. Explant from 36-somite embryo,
4 days in vitro. Zenker-formalin fixation; Jenner-Giemsa stain. Scale 10/*m.
Fig. 11. Hepatic erythroblasts (e) obtained from 36-somite embryo 4 days in vitro.
Zenker-formalin fixation; Jenner-Giemsa stain. Scale 10/*m.
Fig. 12. Yolk-sac erythroblasts (y.e.) obtained from 10-somite yolk-s?.c culture 2
days in vitro. Formalin fixation; Jenner-Giemsa stain. Scale 10/«n
G. R. JOHNSON AND R. O. JONES
14
Hepatic morphogenesis in vitro
93
being observed in vivo (Jones, 1970). With Jenner-Gierasa stain numerous
eosinophilic granules could be observed in many of these cells. These granulocytes were usually found in association with erythroid elements but were slightly
larger (see Table 1), being of the order of 16/tm in diameter, and persisted for
at least 12 days in vitro.
(iii) Megakaryocytes
The megakaryocytes were observed irregularly, usually singly and isolated
from other haematopoietic cells. They were easily recognized by their large size
(average diameter 48 /im) and large multi-lobed nucleus (Fig. 8), and the cytoplasm contained many small, evenly distributed phase-dark granules. Megakaryocytes were the least abundant of the blood cell types and displayed a highly
active periphery.
Macrophages were observed in all explants but were abundant only in
cultures derived from embryos of 28 somites or older. Two morphological
forms were apparent - amoeboid and clasmotic (Jacoby, 1964) - and many of
the former type could frequently be observed actively surrounding haematopoietic cells (Fig. 9). Clasmatosing macrophages were observed only in cultures
containing a substantial haematopoietic cell population (Fig. 13). As the
haematopoietic cells decreased in number so did the clasmotic forms of the
macrophages, so that after 3 weeks in vitro only the normal amoeboid forms
could be observed.
After about 3 weeks in vitro explants consisted mostly of epithelial cell sheets,
amoeboid macrophages and duct-like systems, and cultures were usually
terminated after about 4 weeks.
DISCUSSION
Sheets of polygonal cells and granule-containing cells, often arranged in
lobules, have been observed in vitro from the hepatic areas of different animals
by several authors. Rose (1967) and Rose et al. (1968) described such cells in
explants from the chick embryo as well as foetal and newborn mice. Similar
cells have been reported in explants from newborn rats (Watanabe, 1966;
FIGURES
13-16
Fig. 13. Clasmatosing macrophage (/??) with hepatic erythroblasts. Explant of
hepatic ventral lobe from 30-somite embryo, 5 days in vitro. Scale 10/tm.
Fig. 14. Group of hepatic erythroblasts, one of which is in division (d). Explant of
hepatic lateral lobe obtained from 31-somite embryo 3 days in vitro. Scale 10 /tm.
Fig. 15. Cells containing numerous phase-dark granules (g) observed in an explant
from a 26-somite embryo 10 days in vitro. Scale 10/tm.
Fig. 16. Duct (d) lined by cuboidal granular cells which are sharply demarcated from
the adjacent epithelial cells. This type of structure is suggestive of bile duct formation. Explant of hepatic lateral lobe obtained from 12-5-day embryo. Seven days
in vitro. Scale 10 /tm.
94
G. R. JOHNSON AND R. O. JONES
Alexander & Grisham, 1970), adult monkey (Hillis & Bang, 1959) and the
human foetus (Hillis & Bang, 1959, 1962; Guillonzo et al. 1972). That these
cells represent hepatic parenchyma has been suggested by histochemical
analyses (Rose et al. 1968; Alexander & Grisham, 1970), as well as ultrastructural observations (Watanabe, 1966; Rose et al. 1968; Alexander &
Grisham, 1970; Guillonzo et al. 1972). It has also been suggested that the
differences in cytology between the polygonal cells and the granule-containing
cells is probably due to different degrees of cellular proliferation (Williams,
Weisburger & Weisburger, 1971).
Hepatic granule-containing cells in all the above cases were obtained from
explants of differentiated liver tissue. In the present work we have observed these
cells in explants from the developing hepatic site before any parenchymal cells
have appeared (Rifkind et al. 1969) and it would seem that this tissue becomes
determined very early during the 1 lth day of gestation in the mouse. This event
occurs before the onset of hepatic haematopoiesis since explants from embryos
earlier than 28 somites still developed both the sheets of polygonal cells and the
granule-containing cells. Current work is aimed at defining the precise timing of
hepatic determination and the tissue interactions that occur in this region at this
period of development.
The culture method chosen in the present analysis allows some morphogenesis as well as cellular differentiation. Structures resembling sinusoids (Fig. 6)
as well as possible parts of the bile system (Fig. 16) were observed to develop,
although so far it has proven difficult to ascertain the cellular origins of these
structures. The latter could arise from purely endodermal cells or by an inductive
interaction between endodermand splanchnicmesoderm (Lewis, 1912; Doljanski
& Roulet, 1934; Elias, 1955). The appearance of these structures in the
explants however suggests that some aspects of hepatic morphogenesis are
satisfied by this method.
The most interesting observation of this work has been the precise timing of
the onset of haematopoiesis, in that before the 28-somite stage (11-5 days
gestation) haematopoiesis did not occur in vitro. Explants from embryos of 28
somites invariably displayed extensive haematopoiesis given 24 h in vitro for the
explants to spread. Explants of 30 somite embryos showed the presence of
numerous haematopoietic elements immediately on explantation. This clearly
shows that haematopoiesis commences at the 28- to 30-somite stage in the mouse.
Rifkind et al. (1969), on evidence based purely on morphological criteria, consider that cells of the erythroid series originate by an inductive interaction
between the mesenchyme cells of the transverse septum and the hepatic endoderm cells, the erythroid elements differentiating from the former. Sorenson
(1960, 1963) suggests that the hepatic endodermal cells function as a source of at
least ferritin to the developing erythroblasts, particulate matter apparently being
transferred by pinocytosis. It is possible therefore that haematopoiesis is initially
dependent upon the differentiation of the hepatic cells and the later development
Hepatic morphogenesis in vitro
95
of a particular microenvironmental interrelationship necessary for pinocytosis.
It does not follow however that this initial cellular association (Rifkind et al.
1969) is necessarily of a developmentally inductive type.
Moore & Metcalf (1970) have shown that cells capable of instigating blood
colonies in adult hosts are present in the 9-day mouse-embryo yolk sac, and they
suggest that haematopoiesis in the liver site originates by colonization of the
hepatic anlage by yolk-sac haematopoietic stem cells. Moore & Metcalf (1970)
maintained whole embryos of 8 days gestation for 2 days on a Millipore filter
well system. Tn one series of experiments the embryos were maintained intact
and in another the yolk sac was removed, but in both cases development was
normal up to 20 somites. In the embryos without yolk sacs it was observed that
the circulating fluid was acellular, and it was deduced that liver haematopoiesis
develops by colonization of stem cells from the yolk sac via the circulation.
Clearly, however, in both series of experiments, the embryos were not maintained for a sufficient length of time, since haematopoiesis in the liver site does
not become apparent until 11-5 days gestation (28 somites minimum). It must
be assumed therefore that colonization of the hepatic Anlage by circulating yolksac stem cells remains unproven, and consequently the possibility of in situ
hepatic haematopoiesis cannot be ruled out.
In the present study explants taken immediately prior to the 28-somite stage
contain all the tissue types necessary for full hepatic differentiation and haematopoiesis. These include some of the postulated circulating stem cells, and yet
haematopoiesis does not occur until the 28-somite stage. It is possible, however,
that the microenvironment of the hepatic Anlage has been modified by the
conditions pertaining in vitro, although the method does allow sinusoidal
morphogenesis (Fig. 6), as well as the development of possible bile ducts
(Fig. 16). Consequently the possibility remains that hepatic haematopoietic
tissue may arise de novo in a manner similar to that in the yolk sac, or that
prior to the 28-somite stage, a suitable microenvironment has not yet developed
within the hepatic Anlage for the postulated circulating stem cells to instigate
haematopoiesis.
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ELIAS,
{Received 8 November 1972)