/. Embryol exp. Morph. Vol. 52, pp. 13-21, 1979
Printed in Great Britain © Company of Biologists Limited 1979
\3
Foetal haemopoiesis during the hepatic period
II. Topographic histology
By ANALIA C. NESSI 1 AND CARLOS E. BOZZINP 3
From the Facultad de Odontologia, Universidad de Buenos Aires,
Republica Argentina
SUMMARY
Serial sections of mouse foetal liver, during the 9th and 16th days of gestation, were
studied.
The erythropoietic process starts in the central area of the growing hepatic lobes, and
subsequently spreads towards the periphery. Each lobe passes successively through an undifferentiated, proliferating and differentiating period, but does not proliferate and differentiate in a homogeneously synchronized way, since the onset of both histogenesis and
erythropoiesis is delayed at the periphery, when compared with the central area. As a consequence, in sections it is possible to find three different zones, namely: marginal, which is
the most undifferentiated; intermediate (transitional); and a central area, which is the most
mature, since it is the first site where erythropoiesis arises. These zones are distinguishable
not only by their cell distribution, but also by the biological behaviour of their elements,
specially in the transitional areas between undifferentiated and proliferating zones.
The hepatic lobes do not start their organogenesis at the same time; but those of them in
which histogenesis is delayed, repeat courses of development similar to those previously
organized. The first interstitial erythropoiesis appears in the central area, after the development of an organized network of sinuses, and in close relation to vascular channels. Dynamic
images of immature blood cells, between interstitial and vascular spaces, suggesting passage
through the endothelium, are often seen. These steps are followed, in a concentric form and
spreading towards the periphery, by each hepatic lobe.
INTRODUCTION
The interaction between septum transversum and hepatic bud results in
'a mass composed of a spongework of parenchyma with the spaces occupied
by a network of sinuses' (Wilson, Groat & Leduc, 1963).
In considering the development of liver erythropoietic function (LEF), one
may wonder whether the haemopoietic cells are homogeneously distributed all
over the growing hepatic lobes showing a randomized arrangement, or if they
follow a determined distribution pattern. The actual question is whether the
1
Author's address: Instituto Argentino de Biologia, Teorica, 32-1824 La Plata (1900),
Argentina.
2
Author's address: Facultad de Odontologia, M.T. de Alvear 2142, 1122 Buenos Aires,
Argentina.
3
Career Investigator, Consejo Nacional de Investigaciones, Cientificas y Tecnicas de la
Republica Argentina.
2
EMB 52
14
A. C. NESSI AND C. E. BOZZINI
erythropoietic cells start their proliferation and differentiation in a'preferential'
area, or if all areas of the developing liver, randomly considered, have capacity
for LEF onset.
Although LEF in 'ex vivo' has been extensively studied (Cole & Paul, 1966;
Silini, Pozzi & Pons, 1967; Rifkind, Chui & Epler, 1969; Orlic, 1970; Bateman
& Cole, 1971), the possibility of a topographic cell distribution has apparently
been neglected. This problem may be approached by means of serial sections
of whole foetal livers and hepatic rudiments and a description, by areas, of the
different kinds of cell populations during the period in which LEF takes place.
MATERIAL AND METHODS
C3H-S mice embryos between the 9th and 16th days of gestation were used.
In calculating the foetal age, the morning after overnight mating on which the
vaginal plug was scored, was counted as day 0. Pregnant females were sacrificed by decapitation and the embryos dissected as previously described (Nessi,
1978). Livers were fixed in Helly's fluid and the samples embedded in paraplast
after routine processes. Serial sections were stained with Benzidine (Merck) by
Pearse's technique (1972), Mayer's Haemalum and Giemsa. The sections were
made either transversal, frontal or oblique. Each section was considered as an
aggregate of three different and concentric areas, namely marginal, intermediate
and central (Fig. 1); except the first and last sections of each specimen (varying
its number proportionally to the actual size of the foetal liver) where neither
intermediate nor central areas could be seen.
The marginal area was located between the boundary of the section and the
first capillary lumen. The intermediate area was taken as a microscope field
inwards, and the remaining zone of the section was considered as central.
An area was called ' undifferentiated' either when it was composed by mesenchyme alone or when LEF was not seen. It was considered 'proliferating'
when proerythroblasts predominated, and 'differentiating' when basophilic
erythroblasts or other more mature erythroid cells were predominant.
RESULTS
The first hepatic rudiment appears as a small diverticulum in the bounding
area between fore and midgut, when the mouse embryo is at the 16-somite stage.
The hepatic-bud cells, which are first located in a small, thickened area of the
endoderm, begin to migrate towards the septum transversum. The branching
of the onphalomesenteric veins begins in the sinuses, which are first seen in the
central part of the future hepatic lobes. Shortly after this, the process spreads
towards the boundaries of the septum transversum. A rich capillary network,
with many immature blood cells, is first seen at the 10th day of gestation. The
interstitial space between the sinusoids and the future 'laminae hepatis' is the
site where LEF develops.
Foetal haemopoiesis during hepatic period. II
15
ginal
emiediate
Central
Fig. 1. A three-dimensional representation of the mouse foetal liver, showing the
different topographic areas.
The central area of each lobe is the first to show interstitial LEF (Fig. 2K),
which, in the early lobes occurs during the 11th day of gestation (Table 1). Jn
the intermediate area LEF appears somewhat delayed (Table 2). Nevertheless,
the area which is the slowest in becoming erythropoietic is the marginal one.
A particular characteristic develops during the following days: both the central
and intermediate zones are either homogeneously proliferating or differentiating
(Tables 1 and 2, Fig. 2A and D), whereas the marginal does not show an
homogeneous phase of LEF (Tables 3 and 4). In fact, this area can be subdivided and classified into two categories. One of them, which morphologically
corresponds to the flat-nosed boundaries (LR), shows LEF already at the 11th
day, although red blood cell production is rather poor (Table 3). The other one,
which corresponds to the angular boundaries (LP), remains undifferentiated
until the 13th day in nearly all of the samples. Most of the LP areas are occupied
16
A. C. NESSI AND C. E. B O Z Z I N I
•JA B | _ \ *
*
C
Fig. 2. For legend see facing page.
17
Foetal haemopoiesis during hepatic period. II
Table 1. Distribution of erythroid cells in the central area between the 11th and 16th days
P
BE
OE
;
PE
i.
i
Int.
Vase.
Int.
Vase.
11th
8-5
±30
12th
7-27
±1-33
1-2
20-4
±1-8
4-39
±01
13 th
±019
3-44
32-2
+ 40
27-5
21-6
+ 1-7
61
+ 0-3
3-76
±0-41
±009 ±2-4
±0-45
Day
14th
2-44
±01
15th
0-27
0-62
004
±00
0
004
±001
018
±00
16-8
0
±0-7
±00.1
16th
21-3
±0-2
0
805
±0-36
Int.
4-43
±0-2
25-6
±30
Int.
0-98
±01
Others
\J 11 Id
o
.
Vase.
6-82
±0-87
R
4-21
±0-61
E
(int)
34-5
65-7
±20
±8-1
8-37
8 01
4-6
4-82
191
60-8
50-4
±0-37
±0-32
±0-2
±0-66
±0-9
±2-6
±2-3
25-6
±1-6
320
±1-5
43-6
±1-8
0
Vase.
35-6
±1-5
3-35
5-64
314
3-59
85-5
39-6
±0-58
±0-81
±0-26
±0-51
±4-6
±3-2
3-23
±014
5-3
92-6
40-5
±3-2
±2-1
0-76
3-72
3-15
±018
±0-53
±0-45
1 -94
707
±0-28
±0-45
0-27
100
+ 004 ± 1 0
0-56
±0-12 ±0-2
7-2
0-37
±006 + 0-1
87-9
41-6
±01
±4-3
92-2
+ 6-2
51-3
+ 4-3
Abbreviations: P, Proerythroblasts; BE, basophilic erythroblasts; PE, polychromatic erythroblasts;
OE, ortochromatic erythroblasts; R, reticulocytes; E, erythrocytes. Others: septum transversum and
endoderm-derived cells plus myeloid and platelet precursors. Vase, vascular channels; Int., interstitial
space. Both interstitial and vascular spaces are considered as 100% each one. ± : 95% confidence from
the study of at least 500 microscope fields for each day and area.
FIGURE 2
Fig. 2. Morphogenesis of early and late hepatic lobes. (A) Central area on the
15th day of gestation. (B) Eleventh day. The marginal area (top) still appears very
undifferentiated, whereas the intermediate one (bottom) already shows immature
blood cells, some of them in a dynamic attitude between interstice and vascular
channels (arrow). (C) Marginal (LR) area on the 14th day. Magnification as in (A).
(D) Panoramic view of a developing hepatic lobe during the 11th day. The intermediate and central areas already show LEF, whereas the marginal one remains
undifferentiated. (E) Dynamic images of young blood cells between the vascular
channels and interstitial space, during the 11th day. It is a transitional area between
undifferentiated and proliferating zones. Some endodermic cells (asterisks) are seen.
Magnification as in (B). (F) Marginal (LP) area during the 14th day. Note the
difference with (C). Magnification as in (A). (G) Panoramic view of a whole embryo
section on the 10th day. In the septum transversum (arrow) only wide-lumen blood
vessels are seen, and interstitial LEF is still absent. Magnification as in (D). (H) LP
area in an early lobe during the 16th day of gestation. Magnification as in (A).
(1) Marginal (left) and intermediate (right) areas at the hilium of the organ, on the
14th day. This cell distribution is still seen in the late lobes during the 16th day.
Magnification as in (A). (J) Septum transversum on the 1.1th day. Neither an
organized vascular network, nor LEF are seen. It is the most primitive framework
of a future hepatic lobe. Magnification as in (A). (K) Late lobe during the 12th day.
Note the immature blood cells in close relation to the sinuses. Magnification as
in (A).
18
A. C. NESSI AND C. E. BOZZINI
Table 2. Distribution of RBC in the intermediate area between the 11th and 16th days
P
BE
PE
Day
Int.
Vase.
Int
Vase.
11th
113
±0-9
100
±1-7
8-27
±1-65
3-62
±019
0-36
±003
005
±00
9-5
±1-3
2-55
±0-32
0-72
13-4
±2-4
35-6
±1-9
29-4
±4-1
24-8
±11
20-7
±1-4
16-6
18-8
±0-5
5-61
±0-23
10-2
±1-5
0-32
±002
0-27
±00
12th
13th
14th
15th
16th
±01
004
±00
0
0
0
±2-0
OE
Int.
Vase.
Int.
Vase.
R
E
5-21
±0-74
11-4
±1-6
18-6
±1-7
26-5
±1-2
35-5
±1-8
30-5
24-7
±1-5
11-2
±3-1
13-5
±3-1
0-57
±005
3-56
±005
0-51
±003
0-21
±005
6-4
±1-3
5-21
±0-8
2-2
±0-7
4-59
±008
11-7
±0-9
612
±0-73
703
±0-62
3-27
±0-48
3-93
±0-81
4-71
±0-69
4-39
±019
13-8
±1-7
22-7
±3-3
10-4
±2-5
14-9
±11
141
±1-2
12-8
±1-2
27-1
±3-3
51-2
±4-5
58-4
±7-6
80-3
±7-4
77-5
±2-8
82-3
±2-6
±4-2
Others
(int.)
68-8
±5-4
390
±3-2
41-2
±61
42-9
±8-5
391
±20
41 1
±1-5
References as in Table 1.
Table 3. Distribution of RBC in the marginal (LR) area between the 11th and 16th days
P
BE
PE
OE
A
Day
Int.
Vase.
Int.
11th
2-62
±0-37
23-4
±2-4
13-5
±2-4
5-37
±0-37
2-57
±0-34
1-42
±003
12-3
±1-3
0-38
±005
617
±0-99
4-34
±0-4
0-65
±002
018
±003
008
±00
18-2
±1-3
33-7
±3-7
24-3
12th
13 th
14th
15th
16th
±2-1
35-1
±4-3
26-3
±0-8
Vase.
21-5
±3-5
7-4
±1-8
6-34
±1-42
4-24
±015
1-30
±002
018
±001
Int.
Vase.
0007 42-3
±00
±40
61
±0-8
10-6
±2-9
18-2
±1-5
28-3
±4-1
410
±1-9
30-6
±3-4
15-3
±1-3
8-49
±015
618
±077
0-87
±003
Int.
Vase.
R
E
0
8-44
±2-57
4-85
±0-94
7-51
±0-96
2-21
±019
2-83
±0-56
10-9
±10
6-65
±0-97
15-7
±1-0
9-92
±104
2-66
±0-58
.16-4
±1-3
18-7
±1-3
8-81
±1-33
35-3
±10
56-8
±4-5
81-3
±5-7
0-22
±007
Ml
±0-26
5-32
±0-87
3-99
±018
8-4
±0-7
70-5
±8-4
69-3
±2-0
Others
(int.)
96-9
±8-2
52-6
±3-2
42-5
±3-3
46-8
±4-1
301
±6-2
22-9
±3-5
References as: in Table 1.
only by mesenchyme, more or less evolved. They show wide-lumen blood
vessels, but these are scarce and do not form an organized network of sinuses.
Only when these undifferentiated areas are invaded by narrow-lumen sinusoids
do they start the proliferative phase of LEF. Nevertheless, LEF takes place in
some of the LP areas about the 12th day of gestation. The peak of proerythroblasts is seen on the 14th day (Table 4) but they do not predominate over other
more mature demes.
19
Foetal haemopoiesis during hepatic period. II
Table 4. Distribution of RBC in the marginal (LP) area between the 11th and 16th days
P
Day
Int.
nth
0
PE
BE
Vase.
Int.
Vase.
Int.
OE
Vase.
30-6
0
44-5
±2-5
±4-2
002 36-5
9-23
0-42
14-5
±00
±0-43 ± 0 0 2 ±2-2
±30
3 93
8-27
617
2-96 18-9
± 0 1 2 ±0-55 ±0-51 ±009 ±1-1
1-67
4-78 13-8
131
16-8
± 0 0 4 ±0-9
±0-63 ±0-5
±0-2
19-7
3-67 30-8
517
1-28
± 0 1 4 ±1-1
±0-93 ±2-3
±1-1
0-52 40-6
0-55 28-5
0-73
± 0 0 5 ±3-1
±006
±002 ±2-4
14-9
0
Int.
Vase.
R
E
0
3-59
±0-67
0
13-2
±3-5
307
±0-29
21
±0-7
8-39
±0-92
9-57
±0-65
2-83
±0-45
8-84
±112
4-22
±0-33
7-17
±1-3
7-41
±0-88
8-45
±018
3-75
±0-17
17-7
±2-1
63-2
±5-5
70-2
±8-5
74-1
±5-1
80-2
±3-4
±2-2
12th
13th
14th
15th
2-56
±0-26
5-17
±0-26
9-41
±0-43
4-53
±0-66
16th
213
±0-34
0-43
±00
2-1
±0-3
2-32
±0-48
5-23
±00
Others
(int.)
1000
970
±1-4
83-2
±10
60-9
±2-2
42-6
±3-5
28-5
±1-8
References as in Table 1.
In the transitional areas between undifferentiated and proliferating phases of
LEF, at the time of change, there are many dynamic images of immature red
blood cells, which are seen between the vascular channels and the interstitial
space (Fig. 2B, I and J).
The hepatic lobes do not develop simultaneously. Therefore, both organogenesis and LEF do not start at the same time in all of them. However, the
erythropoietic pattern followed by each lobe is similar: a central area which
is quickly capillarized. Shortly after the dynamic images appear. Following
this the proliferative phase of LEF starts. This pattern is neither altered nor
destroyed by the position of the sample at the time of sectioning.
From the 14th day onwards, no area of the oldest lobes can be called 'proliferative' since none of them shows a domination of proerythroblasts over the
other more mature erythroid cells. Nevertheless, there are still several places
where proerythroblasts predominate, namely the young lobes, a rim in some of
the LP areas and the hilium of the organ (Fig. 21). A narrow and incomplete
ribbon of proerythroblasts is still seen in several edges of the LP areas during
the 16th day of gestation (Fig. 2H).
DISCUSSION
Interstitial liver erythropoiesis starts in close relation to blood vessels, at
least in the mouse embryo. Therefore, the first area to show a rich capillary
network is also the first in having LEF, and as soon as the surrounding areas
have sinuses they also show erythropoietic activity. Thus, it is reasonable to
assume that there is a direct relationship between the capillarization of a given
20
A. C. NESSI AND C. E. BOZZINI
area and its LEF onset. The bore size of the blood vessels also appears to be
important for LEF onset. We were unable to find a proliferative area surrounding
a wide-lumen channel (WLC) (Fig. 2G and J) whereas narrow-lumen blood
vessels are typical in both proliferating and differentiating zones. These may
have, WLC, but in this case there is a difference in the ratios between vascular
lumen and liver parenchyma. A WLC surrounded by a narrow rim of mesenchyme does not show LEF (Fig. 2G and J). On the contrary, LEF is seen when
a WLC is surrounded by a wide rim of parenchyma.
During adult life, in normal conditions, only circulating substances, and not
red blood cells are allowed to gain a direct access to the Disse's interstitial
space. The numerous dynamic images found in the transitional areas between
undifferentiated and proliferating zones (reported also by Rifkind et al. 1969)
suggest that in foetal life the circulating erythropoietic cells encounter no
obstruction to passage through the sinusoidal fenestrations. Bearing in mind
these facts, we emphasize the obligatory development of an adequate capillary
network, capable of supporting the LEF onset, and we suggest the following
pattern for LEF: a sinusoidal network appears at the central area of the growing
lobes; it spreads towards the intermediate zone, finally reaching the marginal
LR and LP areas. LEF follows the same distribution pattern, and accompanies
capillary growth. Although all hepatic lobes do not start their organogenesis
at the same time, the development of both the sinusoidal network and LEF
follow the above pattern in all of them.
RESUMEN
Se estudiaron cortes seriados de higado fetal de raton, entre los dias 9° y
16° de desarrollo. Se encontraron tres areas distintas (marginal, intermedia y
central) que se diferencian entre si por su distribution celular y por el comportamiento biologico de sus elementos. Cada area pasa, a su vez, por tres
periodos histogeneticos distintos y sucesivos, que son: (1) indiferenciado, (2)
proliferativo y (3) de diferenciacion.
La eritropoyesis intersticial comienza en el area central de los lobulos en
desarrollo, proceso que posteriormente se extiende hacia las porciones perifericas de cada uno de ellos. Todos los lobulos hepaticos no se forman al mismo
tiempo, y los de formation tardia siguen los mismos pasos que los que se
originan primero: un area central que es rapidamente capilarizada y que casi
inmediatamente despues muestra celulas eritroides, tanto en estrecha vecindad
con los canales vasculares, como en actitud dinamica entre vaso e intersticio,
en este caso, sugiriendo un pasaje a traves del endotelio. Este patron se observa
posteriormente en las zonas intermedia y marginal, y en todos los lobulos.
Foetal haemopoiesis during hepatic period. II
21
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BATEMAN,
{Received 3 July 1978, revised 7 December 1978)