J. Embryol. exp. Morph. Vol. 62, pp. 129-138, 1981
Printed in Great Britain © Company of Biologists Limited 1981
129
Erythropoiesis and haemoglobin
ontogeny in the turtle Emys orbicularis L.
By J.VASSE 1 AND D. BEAUPAIN
From the Institut cTEmbryologie du CNRS et du College de France
Nogent-sur-Marne
SUMMARY
The erythropoietic sites and developmental patterns of haemoglobins have been investigated
during ontogeny of Emys orbicularis. The yolk-sac blood islands seem to be the unique
erythropoietic site during most of embryonic life. Bone marrow haemopoiesis is first observed in young turtles aged one year. The cortical haemopoietic layer of the liver appears
involved mainly in granulopoiesis. There is no morphologically well-defined series of primitive
or definitive erythrocytes. Rather there is a gradual shift in size from a mean length of 17-4 /tm
in embryos to 19-9 /im in the adult. However the size of erythrocytes is highly variable at
all stages. Three haemoglobins of adult type and three haemoglobins of embryonic type
have been identified by electrophoretic separation. It seems that one haemoglobin is synthesized during the whole life. Embryonic haemoglobins persist for more than a year after
hatching while the typically adult haemoglobins appear shortly before hatching.
INTRODUCTION
Little is known about the ontogeny of erythropoiesis in reptiles. Morphological data were collected some years ago: Riickert & Mollier (1906) in Lacerta,
Dantschakoff (1916) in Tropidonotus have described the differentiation of blood
islands in the yolk sac, at the time when the first somites are laid down. Thereafter erythropoiesis remains active in the yolk sac during most of embryonic
development. In the turtle Chelydra serpentina, bone-marrow erythropoiesis
sets in shortly before hatching (Jordan & Flippin, 1913). In Lacerta muralis,
diffuse haematopoiesis has been found in the embryonic mesenchyme and in the
bone marrow long before hatching (Schmekel, 1962).
On the other hand, haemoglobin changes during development in that class of
vertebrates have hardly been studied. The existence of embryonic haemoglobins,
distinct from adult ones, has been inferred in Malaclemys centrata (McCutcheon,
1947) and in the garter snake Thamnophis sirtalis (Manwell, 1960; Pough, 1969,
1971) from determinations of oxygen affinity. Separation of haemoglobin
components by electrophoresis has been carried out in the garter snake (Pough,
1971, 1977), in the loggerhead Caretta caretta and the green sea turtle Chelonia
1
Author's address: Institut d'Embryologie du CNRS et du College de France, 49bis,
Avenue de la Belle-Gabrielle, 94130-Nogent-sur-Marne, France.
130
J. VASSE AND D. BEAUPAIN
Erythropoiesis and haemoglobin ontogeny in turtle Emys
131
my das (Isaacks, Harkness & Witham, 1978). Pough has observed a continuous
change in the electrophoretically separable haemoglobin components of garter
snake blood with increasing body size; at birth, most of the haemoglobin
moved as one slow-migrating band; faster migrating fractions appeared
progressively in larger snakes. In the two species of sea turtles studied, Isaacks
et al. (1978) have demonstrated a shift from embryonic to adult haemoglobins
during development.
No previous attempts have been made with reptiles to relate development of
red-cell series, sites of erythropoiesis and sequential synthesis of different
haemoglobins, as has been done in amphibians or higher vertebrates.
MATERIAL AND METHODS
Fresh-water turtles of the species Emys orbicularis L. were collected from the
ponds of the Brenne region, near Chateauroux (France) and eggs incubated as
described previously (Vasse, 1973, 1974). Twenty-seven stages were distinguished during embryonic development. In the present study, the staging was
based on age of the embryo (number of days of incubation at 25 °C). Blood was
studied in the embryos starting from the 33-somite stage (stage 11 obtained
after 12 days of incubation at 25 °C) to hatching (75-80 days of incubation),
in young turtles (seven individuals aged 8 days to 2 years) and in four adult
individuals. Young turtles and adults were raised at a temperature of 20 °C
approximately.
Haemopoietic organs were fixed in Maximow's or Zenker's fluid, embedded in
paraffin, cut into 7-5 /im thick sections and stained by the May-Griinwald
technique. Young embryos have been stained by dimethoxybenzidine embedded
in paraffin, cut into 7-5 ^in-thick sections.
Blood was collected from the embryos by rupturing one of the extraembryonic
vessels, and from young or adult animals by cutting oif the tip of the tail. Smears
and electrophoresis were always performed from the blood of individual animals.
Smears were stained according to the May-Grunwald-Giemsa technique. For
electrophoresis the erythrocytes were collected and washed in isotonic buffer
then lysed in about five volumes of lysis buffer (Brans & Ingram, 1973). Analytical polyacrylamide gel electrophoresis was performed according to the
method of Ornstein and Davis as modified by Moss and Ingram at pH 10-3
(Moss & Ingram, 1968). All haemoglobins extracts were electrophoresed as
Fig. 1, 2. Yolk-sac blood islands of a 14-somite embryo turtle. Fig. 1. Thickening
of the splanchnopleure with basophilic cells (arrows). Fig. 2. Free cells in a newly
formed extraembryonic vessel.
Fig. 3. Demonstration of haemoglobin in erythroid cells following benzidine
staining: b + : benzidine-positive cells; b~: benzidine-negative cells.
Fig. 4. Liver of a young turtle aged two years: granulocytes (arrow) accumulated
around a vessel.
132
J. VASSE AND D. BEAUPAIN
30-
25-
20-
— Embryo St 18
Adult
15-
10j
i
51
12
13
14
15
16
17
18
19
20
21
22
23/mi
Fig. 5. Cell population diagrams showing the change in length of the major axis of
turtle erythrocytes
, stage-18 embryo (35 days of incubation at 25 °C);
adult; Abscissa, lengths (/*m) measured photomicrographically on fixed
smears. Ordinate, % number of erythrocytes in each class.
cyanomethemoglobins. The gels were stained with diaminobenzidine or Coomassie blue.
RESULTS
I. Development of erythropoietic organs
At the 14-somite stage (stage 8: 6 days of incubation at 25 °C) which was the
earliest stage studied, blood islands were present in the splanchnic layer of the
extraembryonic mesoderm, in contact with the endodermal layer (Fig. 1). The
cells in these thickenings had a basophilic cytoplasm. Some were dividing. In
the blood islands, spaces appeared between the cells. Some of these were free
and peripheral cells formed a limiting endothelium (Fig. 2). Between the 17and 25-somite stages (stages 9 and 10: respectively 7 and 9 days of incubation)
extraembryonic vessels connected the yolk sac with the embryo. The cells,
lying free in the vessels, were more elongated than at the primitive stage.
Differentiation stages towards mature erythrocytes were found.
In embryos at stages 11-20 (12-42 days of incubation) in which kidney,
spleen and liver were formed, no haemopoietic activity was observed in these
organs.
At stages 20-25, haemopoiesis appeared in the superficial cortex of the liver.
This haemopoietic layer was granulopoietic rather than erythropoietic. Thus
Erythropoiesis and haemoglobin ontogeny in turtle Emys
8
Fig. 6-8. Erythrocytes of turtle at various stages (smears). Fig. 6. Stage-19 embryo
(38 days of incubation at 25 °C). Fig. 7. Stage-25 embryo (66 days of incubation
at 25 °C). Fig. 8. Adult turtle. G, eosinophil granulocyte; M, mast cell (with
basophilic granules).
133
134
J. VASSE AND D. BEAUPAIN
I
A,
W
St. 11
St. 18
T
St. 25
Fig. 9. Electrophoretic separation of haemoglobins fractions of turtle blood at
various stages: St. 11, Stage-11 embryo (12 days of incubation at 25 °C); St. 18,
Stage-18 embryo (35 days of incubation at 25 °C); St. 25, Stage-25 embryo (66
days of incubation at 25 °C); Ji-J2, Young turtles respectively aged one and two
years; Ad^Ada, Adult individuals.
the yolk sac is the main erythropoietic organ for most of embryonic development. Erythropoiesis occurs in the lumen of blood islands, and granulopoiesis
is extravascular. Some erythroblasts are released in the blood where they pursue
their maturation processes. The benzidine technique has been used to establish
the identity of erythroid cells in presumed erythropoietic sites. Benzidinepositive cells have been observed in the blood islands of yolk sac (Fig. 3) but
not in the embryonic area.
After hatching, bone marrow was found in the leg bones. Granulopoiesis and
erythropoiesis were observed there, and the granulopoietic layer of liver
remained functional in the young turtle (Fig. 4).
II. Morphological evolution of circulating erythrocytes
Turtle erythrocytes are nucleated cells, variable in size. Some appear very
different from the mean population being either small or large (Fig. 5-8).
During early embryonic stages red cells are slightly more rounded than in the
young and the adult where they usually are elongated with an elliptical shape
(Fig. 6-8). The mean length is slightly larger in the adult (Fig. 5). Chromatin is
arranged in a network containing small scattered masses of denser material.
The homogeneously acidophilic cytoplasm is typical of haemoglobin-rich cells.
Immature cells, i.e. basophilic and polychromatophilic erythroblasts, are
frequent in the blood of the embryo. These cells are round or oval with diverse
sizes. Their chromatin network displays larger masses of dense material
than in erythrocytes. Mitoses are frequent, especially in the embryo (Fig. 6).
Erythropoiesis and haemoglobin ontogeny in turtle Emys
135
Granulocytes first seen around stage 14 (21 days of incubation), are very
frequent after stage 25 (Fig. 7-8).
One striking feature of turtle blood is erythrocyte size variations at all stages.
Despite the fact that the adult blood smear differs in general appearance from
the embryo's, it is not possible to identify specific forms typical of embryonic or
adult stages. This progressive evolution is probably due to the co-existence of
maturation stages rather than to the sequential inflow of cells belonging to
different series.
III. Ontogeny of haemoglobins (Fig. 9)
The haemoglobins in the blood of four adult animals have been analysed by
polyacrylamide gel electrophoresis. In three animals (Adx in Figure 9), two
major components Ax and A3 make up respectively 50 to 40 % of the total
haemoglobin content of erythrocytes, while a minor component A2 accounts for
10 %. These three components are also found in the haemolysate of a fourth
adult animal (Ad2), but the proportions of the components are rather different
in this individual, the A2 band representing 30 % of the total haemoglobin.
The erythrocytes of 41 embryos analysed between stage 11 and stage 20 (42
days of incubation at 25 °C) yielded identical haemoglobin patterns, different
from the adult one. Two major embryonic haemoglobins Ex and E3 have
electrophoretic mobilities distinct from that of adult. The Ex band, very wide,
which accounts for 75-80 % of the material is probably composed of several
molecular forms which have very similar electrophoretic mobilities in the
experimental conditions used. A third band, E2, less important, moves to the
same position as A2.
At stage 25 (66 days of incubation at 25 °C), that is shortly before hatching,
the adult bands A1 and A 3 appear clearly.
During a very long period, adult and embryonic haemoglobins coexist in
young turtle blood: one year after hatching, the haernolysates yielded about as
much embryonic as adult haemoglobins (J^. In a 2-j'ear-old individual, the Ex
band had disappeared (J2). In this same animal, Al9 A2 and A3 were present in
approximately equal proportions, thus the haemolysate was very similar to
that of the fourth adult (Ad2) described above. In some adult or embryonic haemolysates, faint supernumerary bands have been found (e l , e2, al5 a2). In view of
the variations also observed in major adult bands, it is likely that these components correspond to individual variations rather than to artifacts.
DISCUSSION
The data reported here show that erythropoiesis in Emys orbicularis evolves
rather differently from that of other classes of vertebrates. The first point is the
importance of the yolk-sac blood islands which seems to be the unique progenitor of erythrocytes during most of embryonic life. We have not been able to
detect with certainty an intraembryonic organ supplementing the yolk sac in
136
J. VASSE AND D. BEAUPAIN
erythropoiesis during embryonic life. The liver has a cortical haemopoietic
layer which appears involved mainly in granulopoiesis. This function goes on
after hatching and still exists in the 2-year-old animal at a time when haemopoiesis is found in bone marrow. It is difficult to observe bone marrow immediately after hatching. This should be related to observations of Salvatorelli,
Gulinati & Anzanel (1973), who could not find bone marrow in young turtles
(Emys orbicular is and Testudo graeca).
The second point is the absence of a morphologically defined series of
primitive erythrocytes. The highly variable size of red cells described previously
in adult Emys (Saint-Girons & Duguy, 1964, Saint-Girons & Saint-Girons,
1969; Saint-Girons, 1970; Duguy, 1967, 1970; Frair, 1977 a) and in other
species as well (Jordan & Flippin, 1913; Frair, 19776) is also observed during
embryonic life, but at no stage does a well defined new recognizable cell line
appear in the peripheral blood. The evolution of embryonic toward adult blood
suggests either a progressive maturation of the cells or a very slow replacement
of the first line by the next ones rather than an abrupt shift in the erythroid
population.
The third point is the finding of adult haemoglobins (Ax and A3) at stage 25,
two weeks before hatching, that is, independently of that event. The change
from embryonic to adult haemoglobin is not correlated with either the replacement of erythrocytes in peripheral blood, or with the appearance of other
erythropoietic site, as observed in other classes of vertebrates. The electrophoretic analysis of adult Emys erythrocyte haemolysates reveals the presence
of three haemoglobins, the proportions of which seem to vary from one individual to the next. The existence of two major and two minor components
has been described in several other turtle species, in particular among the
Emydiid family (Dozy, Reynolds, Still & Huisman, 1964; Sullivan & Riggs,
1967; Dessauer, 1970). During embryonic development of Emys orbicular is we
find at least two components differing from the adult haemoglobins. In all
individuals examined, whether they are embryos, young turtles or adults, one
band is found in position E2 A2. Further biochemical studies are necessary to
find out whether this component found at very different times of life is the same
molecule or not. For comparison, it should be recalled that in the chick a A and a D
globin chains are synthesized throughout life. In man, a foetal haemoglobin,
and in Xenopus a tadpole haemoglobin persist throughout adult life but these
haemoglobins are present only in very small amounts in adults (Jurd & Maclean.
1970).
The fourth point is the slow disappearance of embryonic haemoglobins
observed in Emys in agreement with scattered data from the literature. In
Malaclemys centrata, McCutcheon (1947), studying the blood oxygen affinity
during embryonic, postnatal and adult life, postulated the existence of an
embryonic haemoglobin which would be completely replaced by adult ones
only two years after hatching. In the sea turtles Caretta caretta and Chelonia
Erythropoiesis and haemoglobin ontogeny in turtle Emys
137
mydas, Isaacks et al. (1978) found that adult-type haemoglobin appears late,
indeed after hatching; however the replacement is completed in a few weeks.
On the other hand, Pough's data (1977) based on electrophoretic analysis of
haemoglobins in the snake Thamnophis may reflect the persistence of embryonic
haemoglobins long after hatching. In other classes of vertebrates a comparable
phenomenon has been described only in the toad Bufo bufo (Salvatorelli &
Turpin, 1971). In any case, two sets of haemoglobins, embryonic and adult,
are simultaneously present in the peripheral blood during a relatively long
period of the development of Emys. It remains to determine if each set is
confined to specialized lines or if all the haemoglobins may be synthesized by
the same erythrocyte. In the first case either embryonic erythrocytes are long
lived (the life span of adult erythrocytes of Terrapene Carolina has been estimated
by Altland and Brace (1962) to be 600-800 days) or erythrocytes of embryonic
type are produced after hatching. The presence in circulation of erythrocytes
possessing both embryonic and adult haemoglobins has been recently demonstrated in the chick embryo (Chapman & Tobin, 1979) and in the mouse embryo
(Brotherton, Chui, Gauldie & Patterson, 1979).
This work was supported by CNRS and by Grant no. 79-7-1224 from DGRST. The authors
wish to express their grateful thanks to Dr F. Dieterlen for valuable comment and criticism
during the course of this work.
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(Received 10 April 1980, revised 24 October 1980)