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Embryonic and fetal hemopoiesis in the mongolian gerbil (Meriones unguiculatus).

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Embryonic and Fetal Hemopoiesis in the Mongolian
Gerbil (Meriones unguiculatus)
Department ofAnatomica1Sciences, School of Medicine, State University of New York at
Buffalo,Buffalo, New York I4214
A study of the development of hemopoiesis in the Mongolian gerbil (Meriones unguiculatus) was conducted in order to determine the temporal
sequence, the organs involved and the cytology of blood cell formation in this species. Hemopoiesis in the intrauterine life of the gerbil can be divided into four
phases based on the site of blood cell formation: (1) the vitelline phase, (2) the
hepatic phase, including thymic histogenesis, (3)the splenic phase and (4) the
medullary phase, with the development of secondary lymphoid tissues. At the
onset of each of these phases a blast-like cell was identifiable in each
hemopoietic organ which, because of its morphology and its presumed multipotentiality was classified as a "lymphoid cell." In the yolk sac phase (gestational
day 12) two generations of erythrocytes, a primitive and a definitive, are formed.
The liver is by day 15erythropoietic and megakaryopoietic, but later, a few granulocytes are also found in its extravascular compartment. The thymus is exclusively lymphopoietic from the appearance of its earliest cells on day 15. Splenic
hemopoiesis is initiated with the presence of lymphoid cells (day 20) followed
later by the appearance of morphologically identifiable blood cell lines. Early
normoblastic and granulocytic activity begins in the marrow cavities on day 23,
though the marrow is not considered to be a source of circulating blood cells during fetal life. Lymph node histogenesis occurs during the last four days of gestation, first in the cervical region and then in other parts of the body. The finding
of undifferentiated lymphoid cells in all organs at the initiation of hemopoiesis
and in the peripheral blood throughout gestation is discussed in light of the migratory theory of hemopoiesis.
Over the past 15 years, the Mongolian ger- its origin in the early embryonic yolk sac to
bil has been shown to be a useful experimen- the establishment of medullary hemopoiesis.
tal animal in a variety of research areas. Some aspects of organogenesis in this species
Many distinctive physiologic characteristics are also presented for temporal and spatial
such as its resistance to X-irradiation (Chang orientation as this information is not present
et al., ,641, its sex-related hematologic dimor- in the literature.
phism (Ruhren, '65; Mays, '69; Dillon and
Glomski, '75a) and its unusual lipid metaboA total of 93 embryos and fetuses were used
lism (Roscoe and Fahrenbach, '62) have stimulated continued study of this species. Though in this study of hemopoiesis in the Mongolian
some data regarding the peripheral blood of gerbil (Meriones unguiculatus). In addition,
the gerbil are present in the literature postnatal studies were conducted on 60 new(Ruhren, '65; Mays, '69;Dillon and Glomski, borns and infants through five weeks of age.
'75a,b; Smith et al., '76) hemopoiesis,especial- Several adults were also examined for the
ly during intrauterine life, has not been inves- presence of splenic hemopoiesis. Table 1 presents the numbers of animals examined for
The present study addresses itself to the de- each age studied.
Received Apr. 4, '77. Accepted July 20. "77.
velopment of hemopoiesis in the gerbil from
Distribution of prenatal and postnatal gerbils studied
Method of
timing gestation
PPE **
W M ***
12 (7,5) *
11 (5,6)*
New Born
14 (3,4,4,3)
2 (1.1)*
10 (4.6) *
2 (1,l)*
8 (1,7) *
7 (1wk)
14 (2 wk)
21 (3wk)
28 (4wk)
36 (5wk)
‘More than one litter studied.
** PPE, postpartum estrus.
*** WM,witneseed mating.
Gerbils were housed in clear plastic cages
and fed a laboratory rodent diet supplemented
by sunflower seeds. Food and water were providedad libitum.Animals were checked every
morning and evening for new litters and evidence of estrous activity.
Gestational ages of embryos and fetuses
were determined in one of two ways. One
method was the estimation of fertilization
time based on the occurrence of postpartum
estrus and limited male-female contact during this period. Using this approach, on the
morning that newborns were discovered they
were removed immediately and the male and
female allowed to remain together for a n additional 24 hours. It was assumed that mating
and fertilization would occur sometime during the late evening hours of this period (this
rodent has documented nocturnal mating
habits). The evening after separation of the
male from the female was considered the end
of gestational day 1. The other method
involved witnessed matings between monogamously paired gerbils. Matings were seen to
occur invariably between the hours of 5:OO
P.M. and 1O:OO P.M. whether or not during postpartum estrus. The activity of the male
mounting the female was considered a witnessed mating and this time was recorded as
the beginning of the first day of gestation.
Embryos and fetuses were then collected in
the late afternoon or early evening a t 24-hour
intervals based on timing by either method.
Table 1 indicates which method was used for
each prenatal study.
Pregnant females were anesthetized with
ether prior to laparotomy. Uterine blood vessels were clamped and the uterus was removed in toto.For younger embryos the uterus
was cut into segments and fixed in Helly’s
fluid without disturbing the embryo or its
membranes. Older fetuses and their membranes (days 17-20) were removed from the
uterus and immersed in the fixative. In the
case of fetuses from the last four days of
gestation (days 21-24) the integument was removed and the fetus was sectioned transversely or mid-sagittally before immersion in
fixative. In the case of sagittally cut embryos
only the left halves were processed for study.
In no case were tissues decalcified before processing. Selected organs from postnatal animals were removed and fixed individually for
histologic examination. All tissues were fixed
in Helly’s solution for four to eight hours,
washed in running tap water overnight, dehydrated in a graded series of ethanol and
cleared for embedding in paraffin. A minimum of three embryos or fetuses from each
day of gestation were serially sectioned a t 6-8
pm for histological study. One serially sectioned individual from each gestational day as
well as random sections from postnatal organs
were subjected to the Nucleal-PAS method of
van Duijn (‘56) with Orange G as a counterstain. All other sectioned material was stained
by the hematoxylin-eosin-azure I1 method of
Maximow (McClung, ’50).
Selected dry film imprints of organs, blood
and bone marrow smears from members of
each litter were also prepared when feasible.
These were stained with a May-GriinwaldGiemsa mixture. All sections, smears and
imprints were examined by light microscopy
for evidence of hemopoietic activity.
In the text the term “primitive erythroblast” has been restricted to the early population of erythroid cells which originate in the
blood islands of the yolk sac and whose morphology is distinct from the erythroid precursors seen in later gestation and adult life. This
latter group has been referred to as the “definitive normoblastic” generation. This distinction is made to emphasize the point that
these appear to be two morphologically separate erythroid populations.
Embryonic hemopoiesis in the Mongolian
gerbil can be divided into four phases based on
the chronologic appearance of blood forming
activity. A considerable overlap occurs, however, in the duration of some of these phases.
The phases are (1)the vitelline, (2) the hepatic, including the development of the thymus,
(3) the splenic, and (4) the medullary, including the development of secondary lymphoid
1. Vitelline phase
The previtelline period covers the interval
from implantation of the blastocyst on gestational day 8 to the end of day 11.Formation of
the embryonic membranes in early gestation
is quite similar to that of the mouse and other
laboratory rodents (e.g., rat and guinea pig) in
that the embryo lies within an “inverted”
yolk sac. During days 9 and 10 the ectoplacental cone and egg cylinder as well as the yolk
sac entoderm and Reichert’s membrane are
formed. By day 11, when all three germ layers
are present, the presomite embryo assumes a
coiled, lordotic posture with its craniocaudal
axis at a right angle to the uterine horn. The
vitelline splanchnopleure is formed when
primitive mesenchyme migrates onto the yolk
sac entoderm. These mesenchymal cells apparently give rise to the first blood forming
cells of the embryo. In one 11-day embryo a
knot of mesenchyme was present on the yolk
sac. Owing to the presence of recognizable
blood islands on day 12, this mesodermal knot
was interpreted as an angioblastic cord, the
immediate precursor of a blood island, However, strictly speaking there was no evidence
of actual hemopoiesis a t this time in any of
the 11-day embryos examined.
The gerbil embryo of gestational day 12 is
characterized by a coiled, lordotic profile, a
partially formed neural tube and the absence
of pharyngeal arches. Hemopoiesis is manifested for the first time on this day in the
form of prominent blood islands within the
yolk sac mesoderm (figs. 1, 2). The cells of the
blood islands are distinctly separate from the
entoderm and do not possess the yolk granules
characteristic of this layer. Flattened endothelium incompletely surrounds some foci of
blood cells. Narrow intercellular spaces are
present in the blood islands which may be an
indication of the lumenization of the future
vitelline vessels. Cellular necrosis apparently
does not contribute to the formation of the
vascular lumina in blood islands. The empty
heart tubes also present a t this time indicate
that circulation in the embryo has not yet
been established.
The cells of the blood islands can be classified into two varieties based on information
obtained from yolk sac smears. Most cells are
very large (diameter approximately 20 km)
(fig. 31, oval shaped cells which possess varying amounts of cytoplasmic basophilia, a delicate, reticular chromatin pattern and prominent nucleoli. These are considered to be the
primitive blood cells (of Dantschakoff). Other
cells possessing extreme cytoplasmic basophilia, somewhat smaller diameter (approximately 15-18 pm) and a more clumped nuclear pattern with or without a visible nucleoli are also seen in the blood islands. These
are the primitive erythroblasts (fig. 41, the
first erythroid cells of the embryo. Transitional stages between the primitive blood cells
and primitive basophilic erythroblasts are
also noticeable in these preparations. The
primitive generation erythroblasts evidently
proliferate, a t first, by differentiation from
the primitive blood cells, and later, by the
commonly observed mitosis of circulating
erythroid cells. Judging from the lack of cytoplasmic eosinophilia, appreciable levels of
hemoglobin are considered to be absent from
primitive erythroblasts a t this stage.
On the thirteenth day of gestation the gerbil undergoes a radical reversal of its lordotic
posture to the C-shaped profile characteristic
of mammalian embryos. Primitive erythroblasts are present in the atria and ventricles
of its coiled heart rudiment indicating that
circulation has been established between yolk
sac and embryo. The allantoic stalk has begun
fusion with the chorion so that the developing
chorioallantoic placenta is connected to the
embryo by a recognizable umbilical rudiment.
Primitive erythroblasts of yolk sac origin are
now present in the small vessels of this structure. No evidence of placental hemopoiesis,
other than the mitosis of circulating erythroid cells is observable in the material studied. The neural tube is closed except for the
posterior neuropore. Otic vesicles are noted
and pharyngeal pouches I and I1 are now present with their associated grooves. Bilateral
nephrogenic ridges are situated in the posterior body wall.
On dry smear preparations of the 13-day
yolk sac primitive basophilic erythroblasts
(diameter 15-18 pm) are clearly the predominant circulating cells. They possess a more
condensed nuclear pattern than the primitive
erythroblasts of day 12. Some cells demonstrate moderate cytoplasmic eosinophilia indicating the likelihood of hemoglobin synthesis at this stage. Some primitive erythroblasts are binucleate or in the process of
mitotic division. In addition, undifferentiated
cells roughly the same diameter as primitive
erythroblasts are observed. The latter are
similarly identified in sectioned material primarily in the vitelline vasculature, but also
throughout the rest of the circulation. They
are smaller than the primitive blood cells of
the yolk sac blood islands but are otherwise of
similar morphology (i.e., delicate nuclear pattern, prominent nucleoli, variable amounts of
cytoplasmic basophilia). The morphology of
these undifferentiated, presumably multipotential, cells has prompted the use of the term
“lymphoid cell” to describe them. Cells of a
form intermediate between these cells and
primitive erythroblasts are not present.
The 14-day-old gerbil embryo assumes an
even more “fetal” posture than its 13-day
counterpart as the primary curvature of the
back is even more pronounced. Lateral outgrowths of the body wall represent the first
traces of forelimb development. The hyoid
arch is sharply delineated and Rathke’s pouch
is now forming in the roof of the oral cavity.
Lung diverticula are observed as small outgrowths from the foregut while the bloodfilled heart, as would be anticipated, is much
larger than on previous days. The first loop of
the small intestine has now formed.
By this time a second generation of
erythroid cells is being formed in the yolk sac.
Whereas previously, only primitive erythroblasts were formed here, now, definitive generation normoblasts are also recognizable in
vitelline smears (figs. 5, 6). These cells are
identifiable by their close resemblance to the
normoblast of the adult animal. As seen in
yolk sac smears in their most immature form
(pronormoblast) the cells of the definitive
series are smaller (diameter 12-15 pm vs. 1518 Fm) and have a more clumped chromatin
pattern than equally immature primitive
erythroblasts. These differences become more
pronounced as each lineage matures. Some
basophilic normoblasts continue to be observed in yolk sac smears on the fifteenth day.
Intermediate stages between the definitive
normoblasts and the so-called lymphoid cells
are identifiable. Granulocytes and megakaryocytes, however, are never observed t,o form
in the yolk sac of the gerbil.
Late in the yolk sac phase clumps of
basophilic cells are found adherent to the ventral luminal wall of the abdominal aorta (fig.
10). These persist until the fifteenth day of
intrauterine life, after which they are no
longer present. Their relationship to hemopoietic activity is as yet unknown.
2. Hepatic phape and formation of
the thymus
As a phase of hemopoiesis, the hepatic period extends from the fourteenth day of gestation until the third week of postnatal life.
During the early part of this phase several
changes in the physical appearance of the gerbil are evident. By day 15 the eye primordia
become visible on the surface of the head and
all the pharyngeal arches have formed. Subsequently, obliteration of the pharyngeal
grooves is initiated so that all are absent by
the seventeenth day except for parts of the
first groove which will give rise to the external auditory meatus. The fore- and hindlimb
buds are distinct flipper-shaped appendages
by day 15; individualization of the digits commences on day 17. Peripheral nerves, sensory
ganglia, mesonephroi and their ducts appear
early in the hepatic phase (approximately the
fifteenth day of gestation) while the gonadal
primordia and metanephric kidney can be
visualized on day 16. For the most part development in the gerbil fetus from gestational
day 18 onward follows a pattern of rapid
growth and refined histologic changes. Grossly, however, nearly all organ systems have
been laid down a t least in rudimentary form.
The liver is without doubt the most massive
organ of hemopoiesis during intrauterine life,
7580% of the cell population being hemopoietic by the nineteenth and twentieth days
of gestation (fig. 11).The development of the
liver begins with the appearance on day 13 of
a prominent septum transversum just caudal
to the heart bulge at the anterior intestinal
portal. It consists of a loose reticulum of undifferentiated mesenchyme and some small
thin-walled vessels which contain circulating
primitive erythroblasts. The hepatic diverticulum, a ventral outgrowth of the midgut,
protrudes a short distance into the septum
transversum. Lymphoid cells can be seen in
dry film imprints of the septum transversum
of 14-day gerbil embryos (figs. 7, 8). These
cells are similar in appearance to the lymphoid cells seen in 14- and 15-day yolk sac
smears, and possess a leptochromatic nucleus,
prominent nucleoli and varying amounts of
cytoplasmic basophilia. By the fifteenth day
the liver is definitely a hemopoietic organ. Its
enormous enlargment over the previous day is
attributable to the massive number of erythroid precursors, (plus a smaller number of
megakaryocytes) formed at this time and
throughout the rest of gestation. Furthermore, until the first week after birth when
medullary hemopoiesis is firmly established,
the liver is apparently the chief source of the
circulating elements of the blood. At a n early
stage, hepatic normoblasts are deeply basophilic cells, while the less numerous megakaryocytes are identifiable by their massive
size, polyploid, lobulated nuclei and their
PAS-positive cytoplasm even in their immature stages. Hemopoiesis appears to occur extravascularly in direct contact with the liver
parenchyma, although some developing normoblasts of indefinite origin are present within the hepatic sinusoids. Beginning on day 15
macrophages with ingested PAS-positive material, remnants of erythrocytic cytoplasm
and extruded nuclei are regularly seen in the
liver (fig. 12). By day 19 a few developing
granulocytes are also identifiable in the extravascular compartment although their
numbers never exceed 1-2%of the nucleated
cells in this organ.
The thymic anlagen arise bilaterally from
the ventral aspect of the third (and possibly
fourth) pharyngeal pouches. In most 15-day
embryos examined the thymic primordia are
still attached to the pharyngeal pouches and
are not as yet lymphoid. In one slightly more
developed 15-day embryo (fig. 9), however, the
thymus was found to be detached from the
pharynx. A vestigial lumen, though reduced
in size, was still present in this gland. Small
round basophilic cells were observed among
the thymic epithelial cells in this specimen.
These possessed a more basophilic cytoplasm
and more condensed chromatin than the surrounding entodermal cells. Although circulation was not yet established to the thymus,
lymphoid cells similar to those within this
gland were found in an adjacent vessel. By
the sixteenth day of gestation, basophilic
lymphoid cells readily distinguishable from
the surrounding epithelium are regularly
seen in the thymus, in its connective tissue
capsule (mostly ventrally and medially) and
in the surrounding loose connective tissue.
Lymphocytes within the thymus continue to
increase in number throughout the rest of
gestation. Trabeculation and lobulation are
features of this gland by day 20, while corticomedullary differentiation is not evident until
day 23.
3. Splenic phase
Splenic hemopoiesis commences on the
nineteenth or twentieth day of fetal life. However, traces of its mesenchymally derived
rudiment may be observed as early as day 16
or 17. The spleen of the gerbil arises in a manner identical to that of other rodents as a
thickening on the surface of the left dorsal
mesogastrium. On succeeding days i t elongates and is adherent to the greater curvature
of the stomach. Vascularization begins on day
18 or 19 and by the twentieth day lymphoid
cells can be identified in spaces within the
splenic parenchyma (fig. 13). Over the next
few days definitive normoblasts, granulocytes, megakaryocytes as well as lymphocytes
can be identified in this organ, though never
to the extent observed in the liver. Normoblasts and granulocytes continue to be formed
here until the end of the third week after
birth, while megakaryocytes and lymphocytes
are present in this organ throughout adulthood. White pulp becomes apparent late in the
first week of extrauterine life.
4. Medullary phase and the development of
secondary lymphoid tissues
Mesenchymal condensations can be seen on
the sixteenth day of gestation indicating the
locations of the future pectoral girdle, verte-
brae and ribs. By the end of the next gestational day precartilage is present in these
areas and also in the pelvic girdle and basioccipital. Chondrification begins in the upper
and lower extremities on the eighteenth day
(this is more apparent proximally than distally in each limb). This process extends to the
temporal bones, nasal septum, ribs and distal
parts of each extremity by the next day while
membranous ossification of the frontal bones,
maxillae, mandible and clavicles has also been
initiated. On day 19, bony collars are present
around some long bones (humerus, scapula,
radius and ulna). Invasion of their cartilaginous primordia by vascular buds begins on
day 20 leading to the formation of the nonhemopoietic “primary marrow cavities.” At
the same time a primary marrow reticulum is
present in the clavicles. Bony collars are present around the ribs and ilia though vascular
invasion of these primordia will not occur
until the next day (day 21). Greatly enlarged
primary marrow cavities are present in most
bones on days 21 and 22.The cell population of
these cavities is sparse, consisting of osteoblasts, osteoclasts and reticulum cells with
long slender processes and abundant intercellular matrix. It is only on day 22 that a few
basophilic lymphoid cells are first seen extravascularly in the marrow. This is considered to be the first instance of hemopoietic
activity of secondary or definitive marrow.
These lymphoid cells are quite similar morphologically to the cells seen previously in the
circulation and in the primordia of other blood
forming organs a t the inception of hemopoiesis. However, transitional stages between the
lymphoid cells and the reticulum cells of
primary marrow cavities are not evident.
Lymphoid cells are first found in the long
bones of the upper and lower limbs (fig. 16),
the scapulae, ribs and frontal bones of the
skull. The clavicles continue to be devoid of
hemopoietic activity until day 24. A considerable number of well differentiated granulocytes can be seen extravascularly in most
marrow cavities on days 23 and 24 (fig. 15).
By the end of gestation definitive marrow is
established throughout most of the vertebrae,
ribs and long bones except for the distal-most
portions of each limb which are still cartilag
inous. Hemopoietic cells (pronormoblasts and
cells too immature to be classified) are especially concentrated in the frontal and maxillary bones of the skull. By the end of the first
week of postnatal life the bone marrow ap-
pears to be the major source of erythroid and
myeloid elements of the blood.
During the last four days of gestation secondary lymphoid tissues begin their development. Coalescence of lymphatic sacs in the
cervical and mediastinal regions give rise to
the first lymphatic plexuses. Invagination of
adjacent mesenchyme into these plexuses results in the formation of early lymph nodes
here on day 21 and elsewhere on succeeding
days. A few lymphocytes and granulocytes are
found within the meshes of early lymph nodes
as well a s in the surrounding loose connective
tissue. Lymphocytic mitotic activity is absent
until late in nodal development. Small capillaries filled with circulating blood cells are
seen to invade most lymph nodes by day 22.
Germinal centers were never observed in fetal
material. Aggregates of lymphocytes in the
intestinal mucosa were seen in some of the 23and 24-day prenatal tissues, though lymphoid
nodules and Peyer’s patches are absent until
after birth. Tonsillar crypts are found in the
oropharyngeal walls on the last day of gestation (day 24) with only a few lymphocytes and
granulocytes present in the underlying connective tissue.
Peripheral blood
Marked alterations in the peripheral blood
picture of t h e Mongolian gerbil occur
throughout embryogenesis. These reflect the
various phases of hemopoiesis described for
this rodent. In fact, characteristic changes in
the blood picture of the gerbil embryo allow
ready estimation of the day of gestation. By
day 13 circulation between embryo and its
yolk sac has been established as indicated by
the presence of basophilic and early polychromatophilic primitive erythroblasts as well as
a few basophilic lymphoid cells in the heart
tubes and peripheral blood vessels. Definitive
normoblasts, characterized by their smaller
size and “adult-like” morphology appear in
the circulation on day 14. Until the nineteenth day of gestation, both erythroid cell
lines undergo mitosis while circulating. It is
interesting to note that even relatively mature primitive polychromatophilic erythroblasts are capable of mitosis (fig. 5). Definitive normoblasts increase in number from the
time of their first appearance until they eventually supplant the primitive generation as
the only red cells in circulation. However, as
late as day 23 (2 days before birth), a few
primitive erythroblasts can still be seen in the
Development of hernopoiesi.8-comparative chronology
Time of initiation (in days)
Yolk sac erythropoiesis
Liver hemopoiesis
Liver granulopoiesis
Splenic rudiment appears
Splenic erythropoiesis
Splenic lymphopoiesis
Medullary hemopoiesia
Pharyngeal pouch formation
Epithelial thymic rudiment
Large lymphocytes in thymus
Lymph node lymphopoiesis
Circulating small lymphocytes
End of yolk sac phase
End of hepatic hemopoiesis
End of splenic phase
Mouse *
Birth **
2nd week
* From Metcalf, D. and M. A. S. Moore,Haernopoietic C e l b London, North-HollandPublishing Company. 1971, p. 173.
** From A m y , L. B., Dewlopmentul AM~OITSY,
Seventh ed. revised. Philadelphia, W. B. S a d e r n bmpany, 1976.
peripheral blood. Both primitive and definitive generation red cells lose their nuclei by
extrusion, though Howell-Jolly bodies are
sometimes found in young erythrocytes. Subsequent to nuclear elimination, erythrocytes
of both generations typically contain cytoplasmic basophilic stippling (fig. 14) which
has been recently characterized as ribonuclease-digestible material and is presumably
of ribosomal origin (Smith et al., '76). Significant numbers of granulocytes and platelets as
well as small lymphocytes are not present in
the circulation until the last two days before
birth, though large lymphoid cells can be seen
in the circulation throughout gestation
(fig. 14).
collected and dated by the present methods.
The size-age and external morphology studies
of Bagwell and Leavitt ('74), the sequence
of placental development as described by
Fischer and Floyd ('72) and the development
of dentition described by Hiatt ('74) correlate
well with the findings of the present investigation. These observations would also indicate that the time sequence of embryogenesis
in the gerbil is predictable and that embryos
and fetuses of desired developmental stages
can be routinely obtained. It should be noted
that, unlike the mouse or rat, littermates of
the Mongolian gerbil show remarkably similar levels of development. That these consistent results can be obtained may foretell a
wider use of this animal in future normal and
The present study was designed to collect abnormal developmental studies.
Blood cell formation in the Mongolian gerembryos, fetuses, newborns and infants of
known ages in order to delineate the sequence bil is, with few exceptions, similar to that of
of events in the establishment of hemopoiesis other rodents. Table 2 summarizes our findfrom its earliest stages to its definitive state. ings and compares the chronology of hemopoiT w o methods of gestational dating were em- esis in the gerbil with that of the laboratory
ployed in this study, namely, witnessed mat- mouse and of man.
Unlike the mouse and rat but similar to the
ing and limited male-female exposure during
post-partum estrus. Several criteria were ap- guinea pig and man, two morphologically displied to the set of embryos and fetuses thus tinct generations of erythroid cells are procollected and indicate that either method is duced in the yolk sac of the gerbil. The first of
reasonably accurate. First, it was obsewed these, the primitive generation, appears by
that no two litters from consecutive days were gestational day 12. It appears that in most
at similar stages of development. Second, sev- mammalian embryos primitive generation red
eral developmental studies conducted by cells circulate in the nucleated condition until
other investigators on the gerbil can be cor- senescence. However, in the gerbil, as in man,
roborated fully by the embryos and fetuses a t least some of them extrude their nuclei and
circulate as anucleate plastids. Barker (‘68)
has shown in mice that, in addition to their
morphologic characteristics, these embryonic
cells also produce a distinct hemoglobin not
found in the red cells of adult animals.
Whether this is also true of gerbils remains to
be established.
The second, or definitive generation, appears by day 14. This cell line is of a slightly
larger size, but otherwise quite similar to
adult, bone marrow-derived normoblasts.
According to Maximow, (‘24) the few primitive blood cells of the yolk sac blood islands
which do not give rise to primitive erythroblasts thereupon lose this capability. He
termed these cells “large lymphocytes” (hemocytoblasts). It appears from the results of
the present study that such large lymphocytes are present in the gerbil yolk sac vasculature and could reasonably be a source for
the secondary or definitive red cells seen here.
Granulocytes and megakaryocytes, on the
other hand, were not observed to form in this
fetal membrane.
Examination of dry film imprints of the hepatic rudiment revealed round cells resembling the lymphoid cells previously observed
in the yolk sac. It is possible that these cells
are the precursors of the future hepatic blood
cells. I t was formerly held by most investigators that hepatic hemopoietic cells arise from
septum transversum mesenchyme. Brynmour-Thomas and Yoffey (‘61, ’64), Stohlman
(‘70)and Yoffey (‘i’l), however, felt that morphologic evidence favored development of
hemopoietic cells from hepatic entodermal
precursors. A theory postulated by van der
Stricht (1891) which has recently received
strong support is the migratory theory of
hemopoiesis. Moore and Metcalf (‘70)have
presented experimental evidence supporting
this concept and have claimed that the yolk
sac is the only source of de nouo hemopoietic
stem cells. These cells are said to migrate and
colonize the liver as well as the other blood
forming tissues of the embryo.
In the gerbil invasion of the thymic rudiments by lymphocyte precursors must occur
very quickly since these anlagen appear by
day 15 and are invariably lymphocytic by day
16. It appears that lymphocyte precursors
probably do not arise from the surrounding
mesenchymal cells as no intermediate stages
between the two are found. It is more likely
that they arise from a hemopoietic organ
whose development precedes thymic lympho-
genesis. As the liver is barely an organ of
hemopoiesis on day 15, circulating cells of
yolk sac origin would seem by process of
elimination to be the first source of colonizing
cells. Later, the liver may contribute some
lymphocytic precursors to this organ, a view
consistent with the work of Taylor (‘65) who
demonstrated that cells in the fetal mouse
liver have the capacity for repopulating the
thymus of a n irradiated subject. If the
thymus is colonized by a n exogenous cell population it is likely that these cells are highly
motile as there is no circulation to the thymus
during the initial stages of thymic lymphogenesis. In contrast to the finding by Taylor
and Skinner (‘76)of significant hemopoiesis
in the human fetal thymus, the thymus of the
Mongolian gerbil is exclusively lymphopoietic.
As circulating blood cells can be seen within
the splenic rudiment by day 18, it is assumed
that the circulatory system could convey colonizing cells to this organ. The immediate
source of the first hemopoietic cells of the gerbil spleen is thought to be the liver, a concept
in accord with the migratory theory expounded by Metcalf a n d Moore (’71).
Erythropoiesis and granulopoiesis occur in
the spleen until the third week of poRtnatal
life but never to the extent seen in the liver.
Megakaryopoiesis, prominent in the fetal
spleen, persists throughout the adult life of
the gerbil as in mice and rats.
Bone marrow is probably not an organ
which produces blood cells for the fetus. The
emergence of circulating medullary erythroid
or myeloid cells is most likely restricted to
some time after birth since hemopoietic cells
are not present in the marrow cavities until
two days before birth, (day 22). Again the
morphological evidence in this study (i.e., absence of intermediate forms between primary
marrow cells and recognizable hemic elements) suggests that colonization by cells
from another source, most likely the liver, is
responsible for the inception of medullary
hemopoiesis. There are no published reports
concerning the presence of significant numbers of mature granulocytes in the marrow
cavity a t the onset of hemopoiesis as was observed in the present study (days 23 and 24).
Perhaps some trophic influences or (more
likely) discontinuities in the sinusoidal lining
allowed these accumulations to occur. That
these cells did not arise in situ is suggested by
the absence of very immature granulocytic
precursors as well as the insufficient time
available for the latter to mature into s e g
mented cells. The only conceivable sources of
these mature granulocytes are the liver and
The results of the present investigation do
not completely support any one theory of
hemopoiesis. However, certain observations
as a result of this examination of gerbil embryos and fetuses lead us to favor the theory
that stem cells of yolk sac origin do colonize
hemopoietic organs as suggested by Metcalf
and Moore ('71) and others. First, a cell resembling a lymphocyte (hemocytoblast? 1 was
found in the yolk sac vasculature on day 13
and then again in every organ a t the onset of
hemopoiesis (liver, day 14; thymus, days 1516; spleen, day 20; bone marrow, day 22). Second, other than in the yolk sac, no cellular
morphologic intermediates between the parenchymal or stromal cells and developing
blood cells were observed in any organ a t the
onset of hemopoiesis. Third, lymphoid cells
were present in the liver throughout gestation, a t times when the spleen and bone marrow were being populated by blood cells.
Fourth, lymphoid cells were observed in yolk
sac smears of the gerbil embryo on days 13
and 14, though no lymphopoietic tissue is
present until the thymus is formed on days
15-16.This apparent dichotomy was noted by
Jones ("73) who found 2%lymphocytes in his
liver specimens of mice a t a time when he
could not identify a lymphopoietic organ in
the same animals. Last, the formation of definitive normoblasts in the vitelline vessels on
day 14 and in the liver on day 15 suggests that
this common stem cell exists in both sites.
Conclusive morphologic evidence for the migratory theory of hemopoiesis, however,
awaits exact identification of the hemopoietic
stem cell and demonstration of its passage
into the extravascular spaces of liver, thymus,
spleen and bone marrow.
Bagwell, J. N., and W. W. Leavitt 1974 Prenatal size-age
relationships and external morphology in the Mongolian
gerbil (Meriones unguiculatus). Am. J. Anat., 140:
Barker, J. E. 1968 Development of the m o w hemopoietic system. I. Types of hemoglobin produced in the
embryonic yolk sac and liver. Dev. Biol., 18: 14-29.
Brynmour-Thomas,D., and J. M. Yoffey 1961 The origin of
the hepatic haemocytoblast. J. Anat., 96: 605.
1964 Human foetal haematopoiesis. 11. Haematopoiesis in the human foetus. Br. J. Haematol., 10:
Chang, M. C., D. M. Hunt and C. Turbyfill 1964 High resistance of Mongolian gerbils to irradiation. Nature, 203:
Dillon, W. G., andC. A. Glomski 1975a The Mongolian gerbil: Qualitative and quantitative aspects of the cellular
blood picture. Lab. Animals, 9: 283-287.
1975b Erythrocyte survival in the Mongolian
gerbil. J. Nucl. Med., 16: 682-684.
Fischer, T. V., and A. D. Floyd 1972 Placental development
in the Mongolian gerbil (Meriones unguiculatus). 11.
From the establishment of the labyrinth to term. Am. J.
Anat., 134: 321-335.
Hiatt, J. L., L.P. Gartner and D. V. Provenza 1974 Molar
development i n t h e Mongolian gerbil, (Meriones
unguiculatus). Am. J. Anat., 141: 1-22.
Jones, R. 0. 1970 Ultrastructural analysis of hepatic
hematopiesis in the fetal mouse. J. Anat., 107: 301-314.
Maximow, A. 1924 Relation of blood cells to connective
tissue and endothelium. Phys. Rev., 4: 633-563.
Mays, A. 1969 Baseline hematological and blood biochemical parameters of the Mongolian gerbil (Meriones
unguiculatus). Lab. Animal Care, 19: 838-842.
McClung, C. E. 1950 Handbook of microscopic technique. R. M. Jones, ed. New York, Hafner Publishing Co.
Metcalf, D., and M. A. S. Moore 1971 Haemopoietic cells.
London, North-Holland Publishing Company.
Moore, M. A. S., and D. Metcalf 1970 Ontogeny of the
haemopoietic system: yolk sac origin of in uiuo and in
uitro colony forming cells in the developing mouee embryo. Br. J. Haematol., 18: 279-294.
Roscoe, H. G., and M. J. Fahrenbach 1962 Cholesterol
metabolism in the gerbil. Proc. SOC.Exp. Biol. Med., 110:
Ruhren, R. 1965 Normal values for hemoglobin eoncentration and cellular elements in the blood of Mongolian
gerbil. Lab. Animal Care, 16: 313-319.
Smith, R. A., E. A. Termer and C. A. Glomski 1976
Erythrocyte basophilic stippling in the Mongolian gerbil.
Lab. Animals, 10: 379-383.
Stohlman, F. 1970 Fetal hemopoiesis. In: Regulation of
Hematopoiesis. Chap. 21. A. S . Gordon, ed. Educational
Division of Meredith Corporation, New York, AppletonCenturyCrofta, pp. 471-486.
Taylor, R. B. 1965 Pluripotential stem cells in the
mouse embryo liver. Br. J. Exp. Path., 46: 376-383.
Taylor, R. B., and J. M.Skinner 1976 Evidence for significant hematopoiesis in the human thymus. Bl-47:
van der Stricht, 0. 1891 Le d6velloppement du sang
dans le foie embryonnaire. Arch. de Biol., 11: 19.As cited
by Metcalf, D. and M.A. 5. Moore 1971 Haemopoietic
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van Duijn, P. 1956 A histochemical specific thionin-SO1
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1 Cross section of 12-day-oldgerbil embryo in situ. The fetal membranes are indicated:
yolk sac (ys), amnion (am),allantois (al), Reichert’s membrane (rm) and yolk sac cavity (yc). Note decidua adjacent to Reichert’s membrane. Arrows indicate yolk sac b l d
islands. Hematoxylin eosin azure 11. X 60.
2 Yolk sac blood islands from embryo in figure 1.The foamy, vacuolated cells represent
yolk sac entcderm laden with yolk granules. Hematoxylin eosin azure 11. x 1,100.
Richard A. Smith and Chester A. Glomski
3 Twelve-day yolk sac dry film smear. Two primitive blood cells are demonstrated. MayGriinwald-Giemsa. X 1,500.
4 Twelve-day yolk sac dry film smear. Three early primitive erythroblasts are shown.
May-Griinwald-Giemsa. X 1,500.
5 Fifteen-day yolk sac dry film smear. Four late (polychromatophilic) primitive erythroblasts are shown. One cell is in mitosis. May-Griinwald-Giemsa. X 1,500.
6 Fifteen-day yolk sac dry film smear. A basophilic definitive normoblast is surrounded
by four polychromatophilic primitive generation erythroblasts. May-Griinwald-Giemsa. X 1,500.
Richard A. Smith and Chester A. Glomski
7 Fourteen-day dry film imprint of septum transversum. Several circulating primitive
erythroblasts surrounding a lymphoid cell. May-Grunwald-Giemsa. X 1,500.
8 Fourteen-day dry film imprint of septum transversum. An example of a lymphoid
cell (center) and circulating erythroblasts. May-Grunwald-Giemsa. X 1,500.
9 Thymic anlage from a 15-day gerbil embryo. Note the small basophilic cells which appear to be colonizing this organ. The clear area in the upper field (arrow)is the
pharynx. Hematoxylin eosin azure 11. X 600.
10 Fifteen-day gerbil embryo. This cross section demonstrates a n aggregation of
basophilic cells (arrow) adherent to the ventral wall of the aorta a t the level of the
mesonephros. Hematoxylin eosin azure 11. X 280.
Richard A. Smith and Chester A. Glomski
11 Liver from a 19-day gerbil fetus. Note portal area (top center) and central vein (lower
right). The numerous dark (basophilic) cells are developing definitive normoblasts.
A megakaryocyte is present in this field (arrow). Hematoxylin eosin azure 11. X 380.
12 Dry film imprint of liver from a n 18-day fetus. A macrophage containing numerous
phagocytosed erythrocyte nuclei. Note typically foamy cytoplasm and adjacent
maturing definitive normoblasts. May-Griinwald-Giemsa. X 1,500.
13 Splenic anlage from a 19-day-old gerbil fetus. Note small basophilic cells which appear to be colonizing this organ. Hematoxylin eosin azure 11. X 380.
Richard A. Smith and Chester A. Glomski
14 Peripheral blood smear from a n 18-day-oldgerbil fetus. A lymphoid cell is seen in the
upper center of this field. Note the stippled erythrocytes and mature, nucleated,
primitive erythroblasts also seen in the circulation at this time. May-GrunwaldGiemsa. x 1,500.
15 Bone marrow smear of a 24-day-old gerbil fetus. A lymphoid cell (center) is identified. Note the mature granulocytes also in the field. May-Grunwald-Giemsa.
x 1,500.
16 Developing bone (scapula) from a 24-day fetus. Small extravascular foci (arrows) of
hemopoiesis are seen in the upper field. Hematoxylin eosin azure 11. X 380.
Richard A. Smith and Chester A. Glomski
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hemopoiesis, mongolian, meriones, embryonic, unguiculata, fetal, gerbil
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