/. Embryol. exp. Morph. Vol. 25, 1, pp. 97-113, 1971
Printed in Great Britain
97
The origin and movements
of the hepato genie cells in the chick embryo as
determined by radioauto graphic mapping
By GLENN C. ROSENQU1ST 1
From the Department of Pediatrics, The Johns Hopkins Hospital
SUMMARY
The origin of the prehepatic cells was determined by tracing the movements of [3H]thymidinelabelled grafts excised from medium-streak to 4-somite stage chick embryos and transplanted
to the epiblast, streak and endoderm-mesoderm layer of similarly staged recipient embryos.
Although exact definition of prehepatic areas was not possible because of the small number
of grafts placed at each developmental stage, the study showed in general that at the mediumstreak stage, the prehepatic endoderm cells are in the anterior third of the primitive streak;
they shortly begin to migrate anteriorly and laterally into the endoderm layer ventral to the
precardiac areas of mesoderm. They are in the yolk-sac endoderm at the 2-4-somite stage,
and by the 15-17-somite stage are clustered at the anterior intestinal portal. At the 26-somite
to early limb-bud stages, the anterior and posterior liver diverticula have formed from these
endoderm cells, and some of the branches of the diverticula may have reached the prehepatic
mesenchyme, where the two tissues have begun to form cords and sinuses.
At the medium-streak stage, the prehepatic mesoderm is located slightly more than halfway
from the anterior to the posterior end of the primitive streak. From this position it migrates
anteriorly and laterally into the lateral plate mesoderm, and from the head-process to the
2-4-somite stage it is situated posterior to the prehepatic endoderm and posterior and lateral
to the heart-forming portion of the splanchnic layer. By the 15-17-somite stage the prehepatic
mesoderm has reached a position in the splanchnic layer of mesoderm which forms the dorsolateral wall of the sinus venosus. By the 26-somite to early limb-bud stage the hepatic diverticula have joined with the hepatic mesenchyme to form the rudimentary cords and sinuses
of the liver.
INTRODUCTION
The liver in the chick embryo begins its development at the 22-24-somite
stage, when the more anterior of two diverticula sprouts from the thickened
endoderm which forms the lip of the anterior intestinal portal, and grows into
the posterior wall of the sinus venosus. Branches of the anterior and posterior
diverticula combine with folds of splanchnic mesoderm (the dorsolateral boundaries of the sinus venosus) to form a latticework of proliferating cords and sinuses
which by 4-5 days of incubation extends from the sinus venosus into the lateral
mesocardium.
Previous attempts to locate the hepatogenic regions of the chick embryo at the
1
Author's address: The Department of Pediatrics, The Johns Hopkins Hospital, Baltimore, Maryland 21205, U.S.A.
7
EMB 25
98
G. C. ROSENQUIST
A
B
(
...E
i
... F
i
Origin of hepatogenic cells
99
definitive-streak to head-fold stages utilized the technique of transplanting
fragments cut from donor embryos into the chorioallantoic membranes of host
embryos, where differentiation of the fragments was observed after 7-9 additional days of incubation. Using this method of culture, Willier & Rawles
(1931) and Hunt (1931) first noted that hepatic tissue may differentiate from fullthickness (epiblast and hypoblast) grafts removed from definitive-streak and
head-process stage embryos.
Rudnick (1932) investigated the potential for liver formation of anterior
versus posterior and lateral versus medial blastoderm fragments, noting that at
all stages between definitive-streak and head-fold (Fig. 1 A, D, G) liver cords
and sinuses were present in all fragments cut from the anterior end of the area
pellucida, with the exception of the anteromedial fragment of the head-fold
stage; hepatic tissue was not present in fragments cut posterior to the anterior
end of the primitive streak. Rudnick suggested two reasons for the absence of
liver potential in the anterior median fragment: either there was a single hepatic
Anlage of potency which extended across the anterior midline at the definitivestreak and head-process stages, but which separated into bilateral centres at the
head-fold stage, or there were separate but closely spaced centres of liver potential
at the definitive-streak and head-process stages, which separated further at the
head-fold stage. Hunt (1932) found liver in the same horizontal levels of the
definitive-streak stage embryo as had Rudnick (Fig. IB); Rawles' (1936) results
at the head-process stage also were similar to those of Rudnick except that the
former investigator did not find potential liver in the median fragment (Fig. 1E).
Hunt (1937a, b) separated blastoderms into lower (mesentoderm) and upper
(mesectoderm) layers and transplanted the fragments separately into chorioallantoic membranes. He demonstrated that prior to the definitive-streak stage
the foregut endoderm (including the potential liver) was in the mesectoderm
fragment, and possibly was confined to the primitive streak; after the definitiveStreak stage, however, foregut material was found in the mesentoderm layer
as well, suggesting that the endoderm which forms the foregut and its diverticula
originates in the primitive streak and moves into the endoderm layer (Fig. 1C, F).
Although recent radioautographic mapping studies confirmed Hunt's findings
(Rosenquist 1964, 1965, 1966; Modak, 1965; Nicolet, 1965; Gallera & Nicolet,
1969), they did not resolve the other problems raised by the previous mapping
studies. They could not explain the shift of liver potency from median to lateral
fragments between the definitive-streak and head-fold stages (noted by Rudnick,
Fig. 1. Shaded regions in Fig. A-G represent prehepatic material identified by
previous investigators using grafts to chorioallantoic membrane. (A, D and G),
Rudnick (1932). (B) Hunt (1932). (C and F) Hunt (1937a). (E) Rawles (1936).
(H) Map of presumptive liver endoderm (hatched area) and mesoderm (shaded
area) as described by LeDouarin (1964).
Stages: A-C, definitive streak; D-F, head-process; G, head-fold; H, 9-15somites.
7-2
100
G. C. ROSENQUIST
1932; Rawles, 1936) and the persistence of liver potential in mesectoderm fragments at the head-process stage (noted by Hunt, 1937a).
LeDouarin (1964) attempted to locate the prehepatic endoderm and mesoderm at various stages by separating the endoderm and mesoderm layers and
recombining them in vivo and in vitro, and by implanting carbon particles in the
mesoderm layer to mark the portions which may contribute to the definitive
liver after 8 days of incubation. Using these methods, she determined that
during the 9-15-somite stages the prehepatic endoderm was located at the anterior
intestinal portal, while the prehepatic mesenchyme extended as far posteriorly
as the 15th pair of somites, and as far laterally as the lateral margins of the area
pellucida (Fig. 1H).
Although these previous mapping studies resulted in a general knowledge of
the location of the hepatogenic cells at the various stages, they had certain disadvantages. The chorioallantoic membrane grafts of Rudnick, Hunt and Rawles
tested potential hepatic cells without defining a precise organ-forming region,
and did not separate prehepatic endoderm from mesoderm. The area of presumptive prehepatic mesenchyme outlined by LeDouarin is so large that it
includes cells which other mapping studies have indicated are destined for axial,
paraxial and extra-embryonic mesoderm (Rosenquist, 1966), nephros, (Rosenquist, 19706), heart (Rosenquist, 1966, 1970 a; Rosenquist & DeHaan, 1966;
Stalsberg & DeHaan, 1969) and lung (Rosenquist, 1970c).
Using radioautographic analysis, the present investigation traces the movements of [3H]thymidine-labelled transplants from their original positions in the
prehepatic regions of the endoderm and mesoderm layers and the primitive
streak of recipient embryos into the hepatic endoderm and mesoderm of these
embryos. Although the small number of grafts placed prevents exact definition
of the prehepatic areas, these regions are more precisely defined than they were
previously, and the endoderm and mesoderm layers are treated separately.
MATERIALS AND METHODS
The methods of staging and preparation of recipient and [3H]thymidinelabelled donor embryos and of transplantation and radioautographic analysis of
the grafts were identical to those described in previous publications (Rosenquist,
1966, 1970 a, b) and the description will not be repeated here.
As in previous investigations which traced the migration of organ-forming
regions from early stages, the movements of the hepatic regions in the present
material had to be followed stepwise, since none of the host embryos which
had received grafts at the earliest stages developed all the way to the early limbbud stage. For example, one graft transplanted to the primitive streak at the
medium-streak stage (embryo 4, Fig. 2 A) migrated into the lateral plate mesoderm at the head-process stage (embryo 4*, Fig. 2B). A graft transplanted to the
same position in the lateral plate mesoderm (embryo 50, Fig. 2B) formed part
Origin of hepatogenic cells
101
of the hepatic mesenchyme at the early limb-bud stage (embryo 50*, Fig. 2F).
Consequently it is assumed that part of the graft in embryo 4 would have formed
hepatic mesenchyme if the embryo had developed to the early limb-bud stage.
Hepatic endoderm was identified in serial sections of the recipient embryos in
the following way: in the younger specimens of the 26-somite to early limb-bud
stage group, the anterior liver diverticulum was a thickened area of endoderm
at the anterior end of the hepatic groove; it was usually found along the ventral
wall of the foregut but in the youngest specimens it was occasionally along the
lip of the anterior intestinal portal. In slightly older specimens, the anterior
liver diverticulum had sprouted a bud; this diverticulum grew into the sinus
venosus, ventral to the splanchnic folds which formed the dorsal boundary of
the sinus venosus, and anteriorly and laterally toward the hepatic mesenchyme
in more lateral parts of the right and left splanchnic folds. Although the anterior
diverticulum was located somewhat dorsal as well as anterior to the posterior
diverticulum, it will be referred to simply as the 'anterior liver diverticulum' in
the subsequent discussion.
The posterior liver diverticulum was a more diffuse structure than was the
anterior diverticulum. In the younger host embryos it consisted of a thickening
of the endoderm to the right of the midline on the yolk-sac surface, either slightly
anterior or just posterior to the anterior intestinal portal. In some cases the diverticulum was a single sprout, but in others it appeared to consist of several pouches,
which were shorter and narrower than were the buds of the anterior liver
diverticulum, and which had grown into the right omphalomesenteric vein. This
thickened portion of the yolk-sac endoderm is termed the right half of the
posterior liver diverticulum (RtPLD) in the text, Table and Figures; it is not
clear whether the right posterior liver diverticulum is comparable to the right
liver diverticulum referred to by Goette (1867).
Although there was no posterior liver diverticulum on the left side of the
younger host specimens, there was always a slight thickening of the yolk-sac
endoderm directly opposite the right half of the posterior liver diverticulum
(slightly posterior to but near the anterior intestinal portal and adjacent to the
hypotrophic left omphalomesenteric vein). This region is termed the left half of
the posterior liver diverticulum (LtPLD) in the text, Table and Figures.
In one recipient embryo the posterior liver diverticulum was a single flattened
cord which had grown from the midline of the lip of the anterior intestinal portal.
The present investigation suggests that this midline cord formed by consolidation of the right and left halves of the posterior liver diverticulum. It is therefore
referred to as the median posterior liver diverticulum (MPLD) in the text,
Table and Figures.
Hepatic mesoderm was identified in serial sections of the recipient embryos in
the following way: in specimens in which the endodermal liver diverticulum had
reached the splanchnic layer of mesoderm in the wall of the sinus venosus,
early sinuses had formed and some of the labelled mesoderm cells had surrounded
102
G. C. ROSENQUIST
the endodermal cords. Such mesoderm is termed 'hepatic mesenchyme' in the
text, Table and Figures. In recipient embryos fixed at earlier stages, the endodermal diverticulum had not as yet reached the dorsolateral wall of the sinus
venosus, and liver tissue had not yet begun to form. In these embryos the portion
of the wall of the sinus venosus which was in the same position as the hepatic
portion of the older embryos, and which would thus have formed liver if the
embryo had developed further, was termed hepatic mesenchyme. In eleven of the
embryos, labelled mesoderm cells from the transplant were found clinging to
the anterior liver diverticulum which was growing into the sinus venosus
(Fig. 4C); these were also termed hepatic mesenchyme.
In none of the recipient embryos were labelled hepatic endoderm and mesoderm found adjacent to each other. This rinding is consistent with that of previous
studies, which showed that the endodermal and mesodermal components of
hypoblast grafts migrate separately.
Throughout the text and figures an asterisk (*) after the embryo number
indicates that the position shown is that of the graft after its migration in the host
embryo. If the transplant contributed labelled cells to the hepatic mesoderm as
well as to endoderm, it may be shown both at the right and at the left when it
appears in Fig. 2.
RESULTS
Prehepatic endoderm. (Grafts which contributed labelled cells to prehepatic
endoderm are shown at the right in each stage of Fig. 2.)
At a medium-streak stage the prehepatic endoderm cells were in the anterior
part of the primitive streak, as illustrated by the position of the transplants in
embryos 1 and 2 (Table 1, Fig. 2 A), which had migrated into areas that included
the prehepatic endoderm at the 4-somite stage (Fig. 2D).
By the head-process stage the endoderm cells that would contribute to the
liver diverticula had moved away from the anterior end of the streak, either by
active migration in an anterior direction, or due to the posterior regression of
the streak. Embryos 5-11 (Table 1, Fig. 2B) were fixed before they developed
hepatic diverticula, but a previous study (Rosenquist, 1966) has indicated that
these endoderm cells are destined to form the medial portion of the lip of the
anterior intestinal portal at the 15-17-somite stage (labelled cells in embryos 5,
6 and 9 did reach this area, Fig. 2E). Kingsbury, Alexanderson & Kornstein,
(1956) have noted that the anterior hepatic diverticulum may arise from this
region as early as the 16-somite stage, and LeDouarin (1964) has shown that
endoderm cells which form the lip of the anterior intestinal portal at the
9-15-somite stage are highly specific for liver.
The posterior portion of each of the above grafts would be the first part of the
transplant to be inverted at the anterior intestinal portal during the invagination
of the foregut (Rosenquist, 1966); therefore the posterior end of the prehepatic
part of the graft would be expected to form the anterior part of the hepatic
Origin of hepatogenic cells
103
A
(ms)
3. 4
54-56
E .
(15-17s)
F
(26s-eH>)
gut
•.
> 5 \ 6* •
19*. 37*
-20*
38*
39*
aip
,f
•>-
~-mpld: 47*
__gut
^ It pld: (23*-28*. 42*. 44*-46*, 48*, 49*
32*. 40*-50*
60*-64*
59'
npld: 12*, 32* /
(15*, 29*. 40*. 41* ,43*)
aid: 12*, 15*, 23*-25 * (26*-29*)
x\ .
Fig. 2. Diagrams (ventral views) showing the position of the [3H]thymidine-labelled
grafts which contributed to prehepatic endoderm (shaded areas, right side of each
figure) and mesoderm (shaded areas, left side of each figure). Abbreviations:
aip, anterior intestinal portal; aid, anterior liver diverticulum; elb, early limb-bud
stage; ///, head-fold stage; hp, head-process stage; Itpld, left half of posterior liver
diverticulum; mpld, median posterior liver diverticulum; ms, medium-streak stage;
rtpld, right half of posterior liver diverticulum; s, somite.
An asterisk (*) after the embryo number indicates that the position shown is that
of the hepatic part of the graft after its migration in the host embryo. Since some
grafts contributed to both hepatic endoderm and mesoderm, and some entered both
the anterior and posterior diverticula, each graft may be shown in two positions in
the stage at which it was placed, and in two positions in the stage at which it was
fixed.
104
G. C. ROSENQUIST
Table 1. Position of labelled cells in recipient embryos
carrying [*H]thymidine-labelled grafts*
Embryo
number
1
2
I3
{4
5
6
7
8
9
flO
"111
(12
13
[14
(15
16
17
118
19
(20
21
[22
23
(24
25
26
27
V28
[29
30
131
32
33
34
35
36
37
38
39
(40
41
(42
43
44
45
46
(47
{48
49
Stage
grafted
h. Incubated |
Stage
fixed
MS
MS
MS
MS
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
28
27
16
8
24
25
4
7
25
4
10
66
4
11
53
25
4
7
25
24
7
9
48
48
64
64
•90
48
36
5
7
72
7
4
5
25
3S
4S
HP
HP
HF
HF
HF
HF
HF
HF
HF
HF
25
25
24
82
66
56
66
66
15S
15S
17S
3S
3S
2S
4S
4S
66
66
78
66
72
16S
17S
HF
4S
16S
HF
4S
26S-ELBJ
HF
4S
26S-ELB
17S
HF
4S
17S
15S
HF
4S
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
HF
4S
26S-ELB
4S
HF
HF
16S
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
Position of labelled cells
En anterolateral to St
En anterolateral to St
En, M anterolateral to St
En, M anterolateral to St
YS and ventral G En; LP M
YS and ventral G En; LP M
En, M anterolateral to St
En, M anterolateral to St
YS and ventral G En; LP M
En, M anterolateral to St
En, M anterolateral to St
YS and ventral G En (ALD, RtPLD); LP M
En, M anterolateral to St
En, M anterolateral to St
YS and ventral G En (ALD, RtPLD§); LP M
YS and ventral G En; LP M
En, M anterolateral to St
En, M anterolateral to St
YS and ventral G En; LP M
YS and ventral G En; LP M
En, M anterolateral to St
En, M anterolateral to St
YS and ventral G En (ALD, LtPLD§); LP M
YS and ventral G En (ALD, LtPLD§); LP M
YS and ventral G En (ALD, LtPLD§); LP M
YS and ventral G En (ALD§, LtPLD§); LP M
YS and ventral G En (ALD§, LtPLD§); LP M
YS and ventral G En (ALD§, LtPLD§); LP M
YS and ventral G En (ALD§, RtPLD§); LP M
En, M anterolateral to St
En, M anterolateral to St
YS En (RtPLD); LP M (HM)
En, M anterolateral to St
En, M anterolateral to St
En, M anterolateral to St
YSEn;LPM
YS and ventral G En; LP M
YS and ventral G En; LP M
YS and ventral G En; LP M
YS En (RtPLD§); LP M (HM)
YS En (RtPLD§); LP M (HM)
YS En (LtPLD§); LP M (HM)
YS En (RtPLD§); LP M (HM)
YS En (LtPLD§); LP M (HM)
YS En (LtPLD§); LP M (HM)
YS and ventral G En (LtPLD§); LP M (HM)
YS and ventral G En (MPLD); LP M (HM)
YS En (LtPLD§); LP M (HM)
YS En (LtPLD§); LP M (HM)
Origin of hepatogenic cells
105
Table 1 (cont.)
Embryo
number
50
[51
52
[53
(54
55
[56
157
58
[59
60
61
J62
\63
64
Stage h. Incugrafted bated f
HP
HP
HP
HP
HP
HP
HP
HP
HP
HP
HF
HF
HF
HF
3S
46
4
10
29
5
5
27
4
11
26
98
90
58
72
78
Stage
fixed
26S-ELB
HF
4S
16S
HF
4S
16S
HF
4S
16S
26S-ELB
26S-ELB
26S-ELB
26S-ELB
26S-ELB
Position of labelled cells
YSEn;LPM(HM)
En, M anterolateral to St
En, M anterolateral to St
YSEn;LPM
En, M anterolateral to St
En, M anterolateral to St
YSEn;LPM
En, M anterolateral to St
En, M anterolateral to St
YSEn;LPM
YSEn;LPM(HM)
YSEn;LPM(HM)
YS En; LP M (HM)
YSEn;LPM(HM)
LP M (HM)||
* Key to abbreviations: ALD, anterior liver diverticulum; ELB, early limb-bud stage; En, nonhepatic endoderm; G, gut; HF, head-fold stage; HM, hepatic mesenchyme; HP, head-process stage;
LP, lateral plate; LtPLD, left posterior liver diverticulum; M, non-hepatic mesoderm; MPLD, median
posterior liver diverticulum; MS, medium-streak stage; RtPLD, right posterior liver diverticulum; S,
somite; St, streak; YS, yolk sac.
t Hours of incubation after placement of the graft.
J The 26S-ELB stage is equivalent to stages 16-18 of Hamburger & Hamilton (1951).
§ Presumptive liver diverticulum.
|| This transplant contained only mesoderm and therefore did not contribute to the endoderm layer.
groove and consequently part of the anterior diverticulum. The anterior portion
of each graft would be the last part of the transplant to be inverted at the anterior
intestinal portal during gut formation, and therefore the anterior end of the
prehepatic part of the graft would be expected to contribute to the posterior
part of the hepatic groove and later to the posterior diverticulum (Fig. 3).
The migration of the grafts in embryos 12—31 (Table 1, Fig. 2B) demonstrated that at the head-process stage the prehepatic endoderm cells lateral to the
head-process and the anterior end of the streak are destined for either anterior or
posterior diverticula. The transplants in embryos 12*, 15* and 23*-29* contributed labelled cells to both diverticula (Figs. 2 F ; 4A, B); the other embryos
were fixed before diverticula had formed, and the locations of the grafts in these
embryos show the intermediate positions of the grafts in embryos 12, 15 and
23-29. For example, the position of the labelled cells in embryos 14*, 16*,
18*-20*, 22* and 31* indicates that if the transplants in embryos 12, 15 and
23-29 had been fixed at the 2-4 or 15-17-somite stage they would have contributed to slightly more lateral parts of the lip of the anterior intestinal portal than
had the grafts in embryos 5, 6, 8, 9 and 11, and also to adjacent portions of the
yolk sac and ventral foregut (Fig. 2D, E).
106
G. C. ROSENQUIST
Since the grafts in embryos 12-31 were lateral rather than anterior to the
future head fold at the head-process stage it is concluded that the lateral side of
each graft would be the last part of the transplant to be inverted into the ventral
wall of the gut (Rosenquist, 1966), and would therefore be expected to contribute
to the posterior liver diverticulum and to be adjacent to the posterior portion of
the hepatic groove (Fig. 3). In contrast, the medial side of each transplant would
be the first portion of the transplant to be invaginated at the anterior intestinal
Fig. 3. Diagram illustrating the position of the prehepatic endoderm at the head
process stage (ventral view). The present investigation suggests that the prehepatic
endoderm (shaded areas labelled aid (anterior liver diverticulum) and pld (posterior
liver diverticulum)) is located in a horseshoe-shaped region anterior and lateral to the
primitive streak, overlapping the outer border of the crescent-shaped area which
will be compressed and invaginated into the ventral gut by the 16-somite stage. The
positions given for cells which will contribute to trachea, bronchi, thyroid, stoma
and dorsal gut are interpretations from Rudnick (1932) and Rosenquist (1966,1970 c).
Origin of hepatogenic cells
107
portal and therefore would be expected to be destined for the anterior liver
diverticulum.
The area of endoderm lateral to the middle of the streak at the head-process
stage contributed cells to the posterior diverticulum only (embryo 32, Table 1,
Fig. 2B, F), or would have done so if the host had been allowed to develop
further (embryos 33*—36*, Fig. 2C-E). It is not clear why these transplants did
not form anterior diverticulum in addition to posterior diverticulum. Although
we can be reasonably certain that all of the labelled Jcells were visible in the radioautographed sections, it is possible that some labelled cells were present in the
anterior diverticulum but could not be seen on the radioautographs because of
dilution of the label from extensive cell division during development.
By the head-fold stage the endoderm cells which would contribute to the liver
diverticula had moved farther anteriorly in relation to the anterior end of the
streak, to a position anterior, lateral or posterolateral to the developing head
fold (embryos 7*, 10*, 13*, 17*, 21*, 30*, 34*, 35* and 37-44, Table 1, Fig. 2C).
The transplants in embryos 7* and 10* showed the position at the head-fold stage
of cells which later would contribute to the midline lip of the anterior intestinal
portal, while the transplants in embryos 13*, 17*, 21*, 30*, 35* and 37-44
indicated the position at the head-fold stage of cells which would form more
lateral parts of the lip of the anterior intestinal portal. The transplants which
would have contributed only to the posterior liver diverticulum (embryos 34*,
40-44, Table 1, Fig. 2C) were positioned posterior (and possibly lateral) to those
which might form portions of both anterior and posterior liver diverticula
(embryos 7*, 10*, 13*, 17*, 21*, 30*, 35* and 37-39, Table 1, Fig. 2C).
At the 2-4-somite stage the endoderm cells which would form both anterior
and posterior diverticula had moved farther anteriorly on the yolk-sac surface,
where they extended anterior, lateral and posterior to the anterior intestinal
portal (embryos 1*, 2*, 8*, 11*, 14*, 18*, 22* and 31*, Table 1, Fig. 2D);
the cells which formed or would have formed portions of the posterior liver
diverticulum only (embryos 33* and 45-49, Table 1, Fig. 2D) had also migrated
anteriorly into a position lateral and posterior to the somites.
At the 15-17-somite stage, the endoderm which would form the hepatic
diverticula had been compressed at the lip of the anterior intestinal portal and
the adjacent yolk-sac surface just prior to invagination into the gut (embryos
5*, 6*, 9*, 16*, 19*, 20* and 36*-39*, Table 1, Fig. 2E).
At the 26-somite to early limb-bud stage (stages 16-18 of Hamburger &
Hamilton, 1951), the cells which had formed or would form the liver diverticula
were further compressed into the area which had formed or would form the
hepatic groove, along the ventral wall of the duodenum. The cells which contributed to the anterior liver diverticulum had reached their definitive position in
the endoderm at the base of that structure on either the right or the left (embryos
12*, 15*, 23* and 24*, Table 1, Figs. 2F; 4A, B), or in the base and migrating
cords (embryo 25*, Table 1, Fig. 2F), or were in a position to do so had the
108
G. C. ROSENQUIST
ys
en
pld
sinus venosus
sinus
venosus
Origin of hepatogenic cells
109
host embryos developed further (embryos 26*-29*, Table 1, Fig. 2F). When the
posterior liver diverticulum was present at the midline (as in embryo 47*),
labelled cells were found in the base but not in the tip of the diverticulum.
The transplants originating on the right side of the embryo formed portions of
the right posterior liver diverticulum (embryos 12* and 32*) or were in a position
to do so had the host embryos developed further (embryos 15*, 29*, 40*, 41*
and 43*, Table 1, Fig. 2F).
The transplants in embryos 23*-28*, 42*, 44*-46*, 48* and 49* (Table 1,
Fig. 2F) originated on the left side of the embryo, and after incubation appeared
on the yolk-sac surface to the left of the midline, slightly anterior or slightly
posterior to the lip of the anterior intestinal portal and underlying the hypoplastic left omphalomesenteric vein. They were thus directly opposite the right
posterior diverticulum; for this reason these cells were believed to constitute the
left half of the posterior liver diverticulum. The labelled cells in embryo 47*
likewise originated on the left side of the blastoderm (Table 1, Fig. 2D) and
were developmentally no different from cells arising from the transplants in
embryos 23*-28*, 42*, 44*-46*, 48* and 49*. It is therefore presumed that
cells of the left posterior diverticulum would contribute to the definitive the
(midline) posterior diverticulum at a later stage (when both right and left
posterior diverticula are further compressed at the midline of the anterior
intestinal portal), as had those of embryo 47*.
Prehepatic mesoderm. (Grafts which contributed labelled cells to prehepatic
mesoderm are shown at the left in each stage of Fig. 2.)
At the medium-streak stage the mesoderm which would contribute to the
liver was located slightly more than halfway from the anterior to the posterior
end of the streak (embryos 3 and 4, Table 1, Fig. 2 A), where it was associated
closely with mesoderm which would form the posterior end of the heart.
Between the medium-streak and head-process stages, the prehepatic mesoderm migrated anteriorly and laterally into the lateral plate mesoderm (embryos
3*, 4*, 32-36 and 50-59, Table 1, Fig. 2B). At the head-process stage it was situated in bilaterally symmetrical areas on each side of the embryo, posterior to the
Fig. 4. (A, B) Cross-sections (A, x 200; B, x 600) showing labelled cells (black dots
over nuclei) from a graft placed originally in the endoderm-mesoderm layer of a headprocess stage embryo (embryo 15, Fig. 2.B). The labelled endoderm (arrow) in the
graft has migrated from its original position near the anterolateral margin of the area
pellucida into the base of the anterior liver diverticulum {aid) which has sprouted
from the ventral wall of the duodenum (d) (embryo 15 *, Fig. 2F). In this embryo, the
unlabelled posterior liver diverticulum (pld) has begun to grow into the sinus venosus
from the yolk-sac layer of endoderm (ys en).
(C) Cross-section (x 600) showing labelled cells (black dots over nuclei) from a
graft placed originally in the endoderm-mesoderm layer of a 3-somite stage recipient
embryo (embryo 45, Fig. 2D, F). Some of the labelled mesoderm in the graft has
contributed to the hepatic mesenchyme (Jim) in the splanchnic layer of the lateral
plate mesoderm, and has invested a branch of the anterior liver diverticulum {aid).
110
G. C. ROSENQUIST
heart-forming region(s) (as mapped by Rosenquist & DeHaan, 1966; Stalsberg
& De Haan, 1969).
At the head-fold stage the presumptive liver mesoderm cells were in the lateral
plate mesoderm lateral and posterolateral to the anterior end of the streak, i.e.
in the splanchnic layer posterior and lateral to the heart-forming regions (embryos 35*, 40-44, 51*, 57* and 60-63, Table 1, Fig. 2C).
At the 2-4-somite stage the prehepatic mesoderm was located in bilaterally
symmetrical areas lateral to the somites (embryos 33* and 55*) and between the
last somite which had developed and the anterior portion of the streak (embryos
45-49, 52*, 58* and 64, Table 1, Fig. 2D).
By the 15-17-somite stage, the prospective liver mesoderm was in the right
and left splanchnic layers of the lateral plate surrounding the anterior intestinal
portal (embryos 36*, 53*, 56* and 59*, Table 1, Fig. 2E).
By the 26 somite to early limb-bud stage (stages 16-18 of Hamburger &
Hamilton, 1951), mesoderm cells originating in the transplants in embryos 32*,
40*-50* and 60*-64* (Table 1, Figs. 2F, 4C) had migrated into the sinus
venosus where they contributed to the splanchnic layer of mesoderm which had
begun to form liver in all embryos in which the anterior diverticulum had contacted this mesenchyme. From the present material it was not possible to separate
the mesenchyme which was destined to be contacted by the anterior liver
diverticulum from that which was destined to be contacted by the posterior
liver diverticulum, as the posterior diverticulum had not reached the hepatic
mesenchyme in any of these embryos. Nor was it possible to state whether
hepatic mesenchyme from the right and left splanchnic folds would have
formed the right and left lobes of the liver, respectively, since all the embryos
were fixed prior to formation of these lobes.
The boundary in the splanchnic mesoderm of the sinus venosus between
cells which will form the posterior end of the heart and those which will develop
into liver is not entirely clear. Among the transplants made at the head-process
stage, only the grafts in embryos 32 and 50 (Table 1, Fig. 2B, F) actually
developed long enough to contribute to the hepatic mesenchyme. Furthermore,
the anterior liver diverticulum had not completed its anterolateral migration into
the sinus venosus in either of these embryos. For these reasons, the definitive
boundary of the cells which would form liver and the posterior boundary of the
preheart cells could not be determined.
DISCUSSION
Although part of each of the 64 transplants reported here lay in the hepatogenic region, the number of embryos investigated was relatively small, and each
transplant contained cells other than hepatogenic cells (as indicated in Table 1).
Therefore the positions of the transplants at each stage in Fig. 2 suggest the
location of the hepatogenic cells at that stage, but do not define it precisely.
Origin of hepatogenic cells
111
The mapping of the hepatogenic regions is based upon the following assumptions: (1) that previous studies have established the general position of the endoderm and mesoderm cells of the chick blastoderm without mapping every part
of each layer at each stage (Rosenquist, 1966). Consequently a small number of
transplants, carefully placed, can demonstrate the position of more specific
portions of the yolk-sac and gut endoderm and the lateral plate mesoderm, such
as the prehepatic areas. (2) That identical graft positions in different embryos at
the same stage are homologous even if the embryos were incubated for different
lengths of time. (3) That maps of presumptive organ-forming regions of the
embryo are valid even if areas in the recipient embryos other than the area to be
mapped contain labelled cells. Although this was in most cases due to the large
size of the grafts, which may have overlapped several adjacent organ-forming
regions, some scattering of epiblast cells during gastrulation cannot always be
excluded as a possible source of error, although such scattering apparently did
not occur in the present group of embryos.
The areas identified as being hepatogenic in the present investigation correspond fairly well with the areas assigned to liver by Rudnick (1932), Hunt
(1932) and Rawles (1936), using chorioallantoic membrane methods of mapping
(Fig. 1 A, B, D, E, G). The work of Hunt (1937 a; Fig. 1C, F) is also confirmed,
since the present study assigns a position in the streak at the medium-streak
stage to the prehepatic endoderm. The problem of the shift of prehepatic
endoderm potency from a medial and lateral position at the definitive-streak
stage to two lateral areas at the head-fold stage is not settled with the present
investigation. A previous study (Rosenquist, 1966) which mapped the endoderm of the head-process stage embryo indicated that a portion of the endoderm in the medial fragments of Rudnick (1932) and Rawles (1936) is compressed
at the midline of the anterior intestinal portal by the 15-17-somite stage, and thus
is liver-specific (LeDouarin, 1964). It is still unclear why this fragment did not
form liver in chorioallantoic membrane cultures.
The horseshoe-shaped area of presumptive hepatic endoderm at the headprocess stage (shown in Fig. 3) bears a marked resemblance to the heart-forming
region of mesoderm at the head-process stage as mapped by Rosenquist &
DeHaan (1966). The proximity of the two regions explains the close association
of liver and heart which was found by investigators who used chorioallantoic
membrane grafts to map these regions (Willier & Rawles, 1931; Rudnick, 1932;
Hunt, 1931, 1932, 1931a; Rawles, 1936). The region of prehepatic endoderm
found at the 2-4-somite stage (Fig. 2D) extends farther laterally and posteriorly
than the areas outlined by the experiments of LeDouarin (1964), since LeDouarin
did not find any prehepatic endoderm posterior to the level of the first somite.
Similarly, the present study indicates that the area of prehepatic endoderm at the
15-17-somite stage probably also extends farther laterally and posteriorly from
the anterior intestinal portal than was indicated by LeDouarin (Figs. 1H, 2E).
The reasons for these differences are not clear.
112
G. C. ROSENQUIST
The present study indicates that LeDouarin's observation that the prehepatic
mesoderm occupies a posterior position in the embryo in relation to the prehepatic endoderm throughout the early developmental period is correct. However, the posterior and lateral margins which LeDouarin assigned to the presumptive hepatic mesenchyme are more extensive than are those mapped by the
present techniques.
In future research it may be possible to transplant smaller grafts and to
incubate the recipient embryos for longer periods, so that the cells which will
form more specific portions of the hepatic endoderm and mesenchyme may be
located.
RESUME
Uorigine et les mouvements des cellules hepatiques dans Vembryon de
poulet, demon tres par le marquage radioautographique
L'origine des cellules hepatiques presomptives a ete mise en evidence en suivant les mouvements de greffes marquees a la [3H]thymidine, excisees dans des embryons aux stades s'etendant de la ligne primitive moyenne jusqu'a celui de 4 somites, et greffees dans l'epiblaste, la
ligne primitive et la couche endo-mesodermique d'embryons hotes de stade correspondant.
Malgre qu'une definition exacte des aires presomptives du foie n'ait pas ete possible en
egard au nombre limite de greffes faites pour chaque stade, l'etude a montre de fac.on generate
que les cellules endodermiques du foie presomptif se situent dans le 1/3 anterieur de la ligne
primitive; elles commencent ensuite rapidement a emigrer en avant et sur les cotes dans la
couche endodermique qui se trouve ventralement par rapport aux aires precardiaques du
mesoderme. Elles se trouvent dans l'endoderme et la vesicule ombilicale au stade de 2-4
somites; et au stade de 15-17 somites elles se tassent dans la porte intestinale anterieure. Aux
stades s'etendant des 26 somites aux debuts du bourgeon de membre, les diverticules anterieur
et posterieur du foie sont formes a partir de ces cellules endodermiques, et quelques ramifications de ces diverticules peuvent avoir atteintle mesenchyme pre-hepatique, ou les deux tissus
commencent a constituer des cordons et des sinus.
Au stade de la ligne primitive moyenne, le mesoderme presomptif du foie est situe un peu
en dec.a de la mi-distance entre les extremites anterieure et posterieure de la ligne primitive.
De cette position, il migre vers l'avant et vers le dehors dans le mesoderme de la lame laterale,
et depuis le stade du prolongement cephalique jusqu'a celui de 2-4 somites, il est situe en
arriere de l'endoblaste presomptif du foie et en arriere et lateralement de la partie cardiogene
de la couche splanchnique. Au stade a 15-17 somites le mesoderme presomptif du foie a
atteint la partie de la couche splanchnique du mesoderme qui constitue la paroi dorsolaterale du sinus veineux.
Au stade des 26 somites jusqu'a ceux de bourgeon de membre les diverticules hepatiques se
sont joints au mesenchyme hepatique pour former les cordons encore rudimentaires et les
sinus du foie.
This investigation was supported by USPHS research grants HE 10191 and K3 HE 20074
from the National Heart Institute. The author wishes to thank James D. Ebert for his continued interest in this research, and Soame D. Christianson for help in the preparation of the
manuscript. Special thanks go to Dorothea Rudnick for helpful criticisms of the manuscript.
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