/ . Embryo!, exp. Morph. Vol. 30, 1, pp. 83-96, 1973 8 3

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Differentiation of the mammalian hepaticprimordium 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 hepaticmorphogenesis, the developmental potential in vitro of various hepatic regions prior to andduring definitive liver development, and to accurately determine the time of onset of hepatichaematopoiesis in vitro. Embryos were precisely staged by somite counts as well as time oforiginal mating, and tissues were removed from the hepatic area at stages between anteriorintestinal portal (9 days gestation) and definitive foetal liver (11-75-12 days gestation), andmaintained 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 ofthe ventral wall of the gut, gave rise to sheets of polygonal, frequently binucleate cells andgranule containing cells, both suggestive of hepatic cytodifferentiation. Sinusoidal morpho-genesis was also observed. Haematopoiesis was detected in vitro only in explants from 11-5-to 12-day embryos (28 to 30+ somites). The blood elements observed consisted of erythro-blasts, granulocytes, megakaryocytes as well as macrophages and yolk sac derived erythro-blasts. Duct systems, probably representative of the bile system, were also observed todevelop.

Current theories regarding hepatic morphogenesis are considered in the light of theseobservations. The inductive and cell seeding concepts of the origin of haematopoietic cellsare 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 circulatinghaematopoietic 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 firstdevelop within the hepatic area for haematopoiesis to occur, or that haematopoietic tissuearises in situ in the hepatic Anlage.

INTRODUCTION

The developmental interactions involved during morphogenesis of the chickhepatic area have been extensively analysed (Croisille & Le Douarin, 1965). Asimilar analysis of the development of the mammalian hepatic area, however,has not been performed. Rugh (1968) suggests that the murine liver primordiumdevelops 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, Victoria3052, Australia.

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84 G. R. JOHNSON"AND R. O. JONES

The hepatic diverticulum extends ventrally and cranially and at 11-25 daysgestation cords of endodermal cells begin to proliferate into the region of thejunction of the vitelline veins and the mesenchyme of the transverse septum(Rifkind, Chui & Epler, 1969). The current paper, in part, describes the type ofdifferentiation obtained in vitro from the hepatic area of the mouse at stagesfrom the anterior intestinal portal of 9 days gestation to the definitive foetalliver (11-75 days gestation).

It has been firmly established that foetal hepatic haematopoiesis occursentirely within the liver tissue, and external to the sinusoids of the vitellineveins in the mouse (Jones, 1970), the rabbit (Ackerman, Grasso & Knouff,1961; Sorenson, 1963; McCuskey, 1968), the pig and the human (Ackermanet al. 1961). Granulocytes, megakaryocytes, possible haematopoietic stem cellsas well as erythroid elements were observed in the mouse foetal liver (Jones,1970), and observations suggested that the various blood cells entered the foetalcirculation by diapedesis from the liver tissue. Similar observations have beenmade in the rabbit (McCuskey, 1968). Rifkind et al. (1969) consider that theerythroid cells arise by an inductive interaction between the proliferating endo-dermal cells of the liver cords and the mesenchyme of the transverse septum,the blood cells being mesenchymal in origin. Moore & Metcalf (1970), on theother hand, consider that the hepatic haematopoietic cells arise by the migrationof 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 theonset of haematopoiesis in vitro, and describes the development of variousblood elements in association with hepatocyte differentation.

MATERIALS AND METHODS

Animals. Two strains of mice were used for all experiments - an inbredalbino strain (University of Melbourne, Zoology Department) and a C57Bstrain (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 firstday of gestation, the embryo at this time designated a 1-day embryo and thetime of gestation 1 day. Ovulation in mice appears to be independent of thetime of mating, which usually occurs mid-way through the dark phase, but thiscan vary by up to 5 h (Snell, Fekete, Hummel & Law, 1940; Braden, 1957).Furthermore, within a litter, developmental variation can extend over severalsomites, particularly during the early stages of gestation (Allen & MacDowell,1940). Due to these variations embryos were further staged by external featuresand somite counts.

Dissection. Pregnant females of appropriate age were rendered unconsciouswith CO2 and killed by cervical dislocation. Intact uterine horns were removedaseptically and immersed in nutrient medium. Embryos were dissected free of

Hepatic morphogenesis in vitro 85uterine tissue and completely immersed in nutrient medium in 3-2 cm embryo-logical 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. Theportions of embryo anterior to the otocyst and posterior to the umbilical regionwere first removed. The somites and neural tube of the remaining thoracic areawere then dissected away, thus exposing the dorsal surface of the foregut.Removal of the lateral walls of the coelom and foregut followed, resulting in theexposure of the ventral wall of the latter. The three liver lobes can then beclearly observed, and tissue ventral to these, including the heart, was carefullyremoved. Removal of the heart was postponed until as late as possible since thisorgan allows excellent orientation of the hepatic region. In this manner it ispossible to expose the entire foregut from the level of the pharynx to that of thedeveloping 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 distincthepatic lobes but instead a ventral sacculation of the foregut (see Fig. 2). Thiswas removed with the adjacent septum transversum in a manner similar to thatdescribed 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 intestinalportal. The prospective hepatic area of these embryos was removed by cuttingthrough the lateral walls of the anterior intestinal portal and thereby isolatingthe ventral floor of the foregut.

All micro-dissections were carried out under a Zeiss binocular dissectionmicroscope (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. Pro-vided 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 thecover-glass (Gold Seal Brand, size 43 x 50, thinness 1) by strips of cellulosedialysis tubing 1 cm wide (Visking Co. size 36/32, flat width 1-47/64), accordingto Rose, Pomerat, Shindler & Trunnell (1958).

It was observed that flattening of the explanted tissue by the cellulose stripwas essential, the most extensive outgrowth and differentiation occurring whenthe tissue was five cells or less in thickness. Central necrosis was frequentlyobserved in thicker explants, and occasionally the cellulose strip was mechanic-ally compressed to ensure a flattened tissue.

Assembled chambers were maintained at 37 °C in either a dry-air incubatoror a humidified incubator continuously flushed with 5 % CO2 in air, chambersbeing alternated between these to maintain the pH at 7-4. The medium in eachchamber was changed twice a week.

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G. R. JOHNSON AND R. O. JONES

Hepatic morphogenesis in vitro 87Observations. Observations were made with a Wild M 40 inverted phase-

contrast microscope at magnifications between 100 and 500 times. Photomicro-graphs were obtained with a Zeiss Ikon camera mounted on a Zeiss StandardR.A. phase-contrast microscope, using Kodak 35 mm Pan F film and automaticexposure. Time-lapse cinematography was performed with a Wild M 20 phase-contrast microscope, with attached Bolex 16 mm movie camera using KodakPlus X Reversal Film 7276.

Histology. Whole embryos between 10 and 12 days gestation were fixed ineither 10 % formalin or formalin acetic acid, and 10 /an sections were stainedwith haematoxylin and eosin. The foregut regions of embryos between 11 and 12days gestation were removed intact, fixed in formalin acetic acid and stainedwith borax carmine, and whole mounts were prepared and observed on cavityslides. 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 modificationof the ventral wall of the foregut, and the anterior and posterior intestinalportals are still some considerable distance apart. However, during the latterpart 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 liverlobe (L), ventral livei lobe in septum transversum (v), gall bladder (g), developingduodenum (d), sinus venosus (s.v.) and pharynx (p). Formalin fixation; boraxcarmine 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 pairedvitelline veins (v.v.). The mesothelial thickening (m) that contributes mesenchymeto 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 left-hand 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 anda relatively clear cytoplasm. Explant of portion of anterior intestinal portal from an8- to 9-somite embryo, 3 days in vitro. Scale 10 fim.Fig. 5. Epithelial cells derived from explant of ventral liver lobe and septum trans-versum of 25-somite embryo, 5 days in vitro. These cells have a more phase-densecytoplasm than those derived from earlier explants (Fig. 4), but have fewer phaselight droplets. Scale 10/tm.Fig. 6. Sinusoid (s) bordered by endothelial cells. The nuclei (n) of these cells pro-trude slightly into the sinusoidal space and are flanked by cytoplasmic processes (c)which connect adjacent endothelial cells. Ventral liver lobe explant from a 28-somiteembryo, 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 cephalicmargin of this broad based sacculation abuts against the point of fusion of thetwo lateral vitelline veins. Caudally the sacculation extends to the level of theanterior intestinal portal. Between these two landmarks the endodermal saccu-lation lies ventro-medial to the vitelline veins and in the mesoderm of theseptum transversum. At this stage also (26 somites) a mesothelial contributionto the hepatic Anlage appears, in the ventral coelomic mesothelium lying againstthe dorsal endothelium of the vitelline veins begins to thicken, and endodermalcells from the ventral sacculation appear to approach this area during the earlypart 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 bysmaller vascular spaces, due probably to endodermal cell proliferation (Fig. 3).Serial sections suggest a mesothelial as well as this endodermal contribution tothe developing hepatic lateral lobes. Endodermal cells also appear to extendventrally, ventro-laterally as well as cranially to infiltrate the mesenchymetissues of the septum transversum, near the junction of the vitelline veins, andso form a ventral hepatic lobe. At this stage the sacculation is beginning to showsigns of separation from the dorsal foregut to produce the definitive hepaticdiverticulum.

Tissue culture

Explants of the ventral wall of the anterior intestinal portal (9-10 daysgestation, 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 arelatively clear cytoplasm, with a single nucleus with one or two prominentnucleoli. No blood cells were observed to differentiate.

Explants of the ventral gut wall of the 10- to 11-25-day embryo (16-26somites), including the sacculation together with its surrounding mesenchyme,gave rise to sheets of polygonal-shaped cells in a pavement arrangement. Thesecells contained fewer, more evenly distributed phase-light droplets compared tothe cells from the anterior intestinal portal explants, and the cytoplasm wasmore phase-dark (Fig. 5). Binucleate cells were frequently observed and mostnuclei showed two or more prominent nucleoli.

Granule-containing cells with large rounded nuclei and several nucleoli werefrequently observed in association with the polygonal cells (Fig. 15). Time-lapsecinematography showed that these cells, though usually arranged in groups,had an active periphery and were continually forming and reforming connexionswith each other, and they survived together with the polygonal cells, for thewhole duration of the culture period. We suggest that these granule-containingcells 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 ofmorphogenesis in cell layers directly adjacent to the cellophane strip, mostcultures having several layers of cells. These structures were usually elongate,

Hepatic morphogenesis in vitro 89being bounded by long filmentous cells that delineated them from the polygonalcell sheet (Fig. 6). These long filamentous cells were similar to sinusoidalendothelial cells observed in vivo (Jones, 1970).

Occasionally a few amoeboid macrophages appeared in some cultures after1 week in vitro, and after 2 weeks the macrophage population was very exten-sive. 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 frequentlyobserved (Fig. 16). These extended for considerable distances and occasionallydeveloped a substantial lumen. The cuboidal cells were always sharply demar-cated from the surrounding epithelial cells and frequently had thin bands ofcells aligned along their outer surfaces. We suggest that these ducts probablyrepresent 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 theventral lobe were explanted separately. During dissection a dorsal view of thegut and hepatic area showed that the left lateral lobe was considerably largerthan the right and both appeared as hollow bulges on separation from the gutwall and the ventral lobe. In the 11-75-day embryo (30 somites) fine circulatorysinusoids containing blood cells were observed in the lateral lobes duringdissection.

The hepatic ventral lobe of the 11-5-day embryo (28 somites) appeared muchmore solid than the lateral lobes, and the rounded outline of the hepaticdiverticulum could be clearly observed deep within the mass of endodermalcells. In the 11-75- to 12-day embryo (30+ somites) the ventral lobe appearedmuch larger than either lateral lobes, and a small rounded structure, probablythe developing gall bladder, could be seen protruding caudally from the mainmass of hepatic tissue (Fig. 1). The ventral lobe and the investing mesenchymeof the transverse septum were explanted together.

Explants of both lateral lobes and the ventral lobe of the 11-5-day embryo (28somites) behaved in vitro similarly to the ventral sacculation explants of theearly 11-day embryo (16-26 somites), in that sheets of polygonal cells wereformed as well as the sinusoidal-like structures. However, all explants of the 28-somite embryo (11-5 days gestation) showed haematopoiesis within 24 h in vitro,as did similar explants from older embryos (30+ somites). An ever increasingpopulation of macrophages was observed throughout the culture period of 3-4weeks, as well as (i) primitive yolk-sac-derived erythroblasts, (ii) differentiatingadult-type erythroblasts, (iii) granulocytes and (iv) megakaryocytes. Due to thepresence of the vitelline vessels it was impossible to exclude circulating yolk-saccells. These yolk-sac-derived erythroblasts could be distinguished from adult-type erythroblasts by their larger diameter and smaller nuclear-cytoplasmicratio. Direct morphological comparisons were also made between the two

90 G. R. JOHNSON AND R. O. JONES

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Hepatic morphogenesis in vitro 91

Table 1

Mean diam Range(/tin)

Yolk-sac erythroblasts 14-5 10-18Hepatic erythroblasts 9 5-13Hepatic granulocytes 16 12-22

populations of red blood cells, in that embryonic yolk-sac explants weresimultaneously cultured with liver explants (confrontation cultures), and thedifferences 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 ratherthan the yolk-sac-derived blood cells, and of the former the more immatureforms predominated. These were very abundant and were denoted by their highnucleo-cytoplasmic ratio, rounded shape, peripheral nucleochromatin clumpingand basophilic cytoplasm (Fig. 11). These cells usually appeared in groups andfrequently showed mitotic activity (Fig. 14). Time-lapse studies showed thatthey were highly motile with an active cell periphery, and were often observedto move in streams between adjacent epithelial cells (Fig. 7). Normoblasts wererelatively scarce and mature erythrocytes were observed only very occasionally,probably due to the prolific macrophage activity (Fig. 9). Usually after 10 daysin vitro erythroid cells had completely disappeared.

(ii) Granulocytes

These cells were recognized by their nuclear shape, which ranged from bean-shaped 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; Jenner-Giemsa stain. Scale 50/*m.

Fig. 8. Megakaryocyte (m) with multi-lobed nucleus. The large size of this cell isapparent when compared to the adjacent erythroblast (<?). Explant from 36-somiteembryo, 3 days in vitro. Zenker-formalinfixation; Jenner-Giemsastain. Scale 20/*m.

Fig. 9. Amoeboid macrophage with large nucleus («), engulfed erythroid cells (e) areapparent in the cytoplasm. Explant from 36-somite embryo, 3 days in vitro. Zenker-formalin 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 2days in vitro. Formalin fixation; Jenner-Giemsa stain. Scale 10/«n

G. R. JOHNSON AND R. O. JONES

14

Hepatic morphogenesis in vitro 93being observed in vivo (Jones, 1970). With Jenner-Gierasa stain numerouseosinophilic granules could be observed in many of these cells. These granulo-cytes were usually found in association with erythroid elements but were slightlylarger (see Table 1), being of the order of 16/tm in diameter, and persisted forat least 12 days in vitro.

(iii) Megakaryocytes

The megakaryocytes were observed irregularly, usually singly and isolatedfrom other haematopoietic cells. They were easily recognized by their large size(average diameter 48 /im) and large multi-lobed nucleus (Fig. 8), and the cyto-plasm contained many small, evenly distributed phase-dark granules. Mega-karyocytes were the least abundant of the blood cell types and displayed a highlyactive periphery.

Macrophages were observed in all explants but were abundant only incultures derived from embryos of 28 somites or older. Two morphologicalforms were apparent - amoeboid and clasmotic (Jacoby, 1964) - and many ofthe former type could frequently be observed actively surrounding haemato-poietic cells (Fig. 9). Clasmatosing macrophages were observed only in culturescontaining a substantial haematopoietic cell population (Fig. 13). As thehaematopoietic cells decreased in number so did the clasmotic forms of themacrophages, so that after 3 weeks in vitro only the normal amoeboid formscould be observed.

After about 3 weeks in vitro explants consisted mostly of epithelial cell sheets,amoeboid macrophages and duct-like systems, and cultures were usuallyterminated after about 4 weeks.

DISCUSSION

Sheets of polygonal cells and granule-containing cells, often arranged inlobules, have been observed in vitro from the hepatic areas of different animalsby several authors. Rose (1967) and Rose et al. (1968) described such cells inexplants from the chick embryo as well as foetal and newborn mice. Similarcells have been reported in explants from newborn rats (Watanabe, 1966;

FIGURES 13-16

Fig. 13. Clasmatosing macrophage (/??) with hepatic erythroblasts. Explant ofhepatic 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 ofhepatic 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 explantfrom a 26-somite embryo 10 days in vitro. Scale 10/tm.Fig. 16. Duct (d) lined by cuboidal granular cells which are sharply demarcated fromthe adjacent epithelial cells. This type of structure is suggestive of bile duct forma-tion. Explant of hepatic lateral lobe obtained from 12-5-day embryo. Seven daysin vitro. Scale 10 /tm.

94 G. R. JOHNSON AND R. O. JONES

Alexander & Grisham, 1970), adult monkey (Hillis & Bang, 1959) and thehuman foetus (Hillis & Bang, 1959, 1962; Guillonzo et al. 1972). That thesecells represent hepatic parenchyma has been suggested by histochemicalanalyses (Rose et al. 1968; Alexander & Grisham, 1970), as well as ultra-structural observations (Watanabe, 1966; Rose et al. 1968; Alexander &Grisham, 1970; Guillonzo et al. 1972). It has also been suggested that thedifferences in cytology between the polygonal cells and the granule-containingcells is probably due to different degrees of cellular proliferation (Williams,Weisburger & Weisburger, 1971).

Hepatic granule-containing cells in all the above cases were obtained fromexplants of differentiated liver tissue. In the present work we have observed thesecells in explants from the developing hepatic site before any parenchymal cellshave appeared (Rifkind et al. 1969) and it would seem that this tissue becomesdetermined very early during the 1 lth day of gestation in the mouse. This eventoccurs before the onset of hepatic haematopoiesis since explants from embryosearlier than 28 somites still developed both the sheets of polygonal cells and thegranule-containing cells. Current work is aimed at defining the precise timing ofhepatic determination and the tissue interactions that occur in this region at thisperiod of development.

The culture method chosen in the present analysis allows some morpho-genesis 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 thesestructures. The latter could arise from purely endodermal cells or by an inductiveinteraction between endodermand splanchnicmesoderm (Lewis, 1912; Doljanski& Roulet, 1934; Elias, 1955). The appearance of these structures in theexplants however suggests that some aspects of hepatic morphogenesis aresatisfied by this method.

The most interesting observation of this work has been the precise timing ofthe onset of haematopoiesis, in that before the 28-somite stage (11-5 daysgestation) haematopoiesis did not occur in vitro. Explants from embryos of 28somites invariably displayed extensive haematopoiesis given 24 h in vitro for theexplants to spread. Explants of 30 somite embryos showed the presence ofnumerous haematopoietic elements immediately on explantation. This clearlyshows that haematopoiesis commences at the 28- to 30-somite stage in the mouse.Rifkind et al. (1969), on evidence based purely on morphological criteria, con-sider that cells of the erythroid series originate by an inductive interactionbetween the mesenchyme cells of the transverse septum and the hepatic endo-derm cells, the erythroid elements differentiating from the former. Sorenson(1960, 1963) suggests that the hepatic endodermal cells function as a source of atleast ferritin to the developing erythroblasts, particulate matter apparently beingtransferred by pinocytosis. It is possible therefore that haematopoiesis is initiallydependent 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 bloodcolonies in adult hosts are present in the 9-day mouse-embryo yolk sac, and theysuggest that haematopoiesis in the liver site originates by colonization of thehepatic anlage by yolk-sac haematopoietic stem cells. Moore & Metcalf (1970)maintained whole embryos of 8 days gestation for 2 days on a Millipore filterwell system. Tn one series of experiments the embryos were maintained intactand in another the yolk sac was removed, but in both cases development wasnormal up to 20 somites. In the embryos without yolk sacs it was observed thatthe circulating fluid was acellular, and it was deduced that liver haematopoiesisdevelops by colonization of stem cells from the yolk sac via the circulation.Clearly, however, in both series of experiments, the embryos were not main-tained for a sufficient length of time, since haematopoiesis in the liver site doesnot become apparent until 11-5 days gestation (28 somites minimum). It mustbe assumed therefore that colonization of the hepatic Anlage by circulating yolk-sac stem cells remains unproven, and consequently the possibility of in situhepatic haematopoiesis cannot be ruled out.

In the present study explants taken immediately prior to the 28-somite stagecontain all the tissue types necessary for full hepatic differentiation and haema-topoiesis. These include some of the postulated circulating stem cells, and yethaematopoiesis does not occur until the 28-somite stage. It is possible, however,that the microenvironment of the hepatic Anlage has been modified by theconditions pertaining in vitro, although the method does allow sinusoidalmorphogenesis (Fig. 6), as well as the development of possible bile ducts(Fig. 16). Consequently the possibility remains that hepatic haematopoietictissue may arise de novo in a manner similar to that in the yolk sac, or thatprior to the 28-somite stage, a suitable microenvironment has not yet developedwithin the hepatic Anlage for the postulated circulating stem cells to instigatehaematopoiesis.

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{Received 8 November 1972)