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Localization of fibronectin in megakaryocytes of fetal liver.

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THE ANATOMICAL RECORD 223:90-94 (1989)
Localization of Fibronectin in Megakaryocytes of
Fetal Liver
DALE E. BOCKMAN AND ADARSH K. GULATI
Department of Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000
ABSTRACT
Antibodies specific for fibronectin were utilized to determine the
sites of localization in the liver during development. The livers of fetal rats from
each of gestation days 11-19, and from days 1 and 8 postpartum, were studied by
fluorescence microscopy. Fibronectin was localized predominantly in megakaryocytes and megakaryocyte precursors, and to a lesser extent in the extracellular
matrix surrounding blood vessels and between hepatocytes and sinusoids. The cytoplasm of megakaryocytes and their precursors displayed bright fluorescence but
their nuclei were negative for fibronectin. Hepatocytes had negative or faint fluorescence. Megakaryocytes were present in the liver from day 12, and were numerous
from day 13 through most of the rest of gestation. The relative numbers of megakaryocytes decreased in later gestation; at 8 days postpartum only a few were
observed per section. Hepatic megakaryocytes appeared before megakaryocytes were
established in spleen and bone marrow. The early and persistent high levels of
fibronectin in hepatic megakaryocytes, in the absence of comparable localization
within hepatocytes, leads us to the hypothesis that megakaryocytes are important
in establishing circulating fibronectin levels in the fetus. Similarly, bone marrow
megakaryocytes may contribute to circulating fibronectin in the adult.
Fibronectin is a large glycoprotein that is a normal
component of blood, where its concentration approximates 300 pg/ml. It also is found on the surface of many
cells and as a component of the extracellular matrix.
Fibronectin has binding sites for several substances,
including factor XIII, heparin, fibrinogen, and fibrin,
which may provide a means for carrying out its important functions in hemostasis (see review by Hynes and
Yamada, 1982). Plasma fibronectin and cellular fibronectin, although similar, are not identical molecules.
It is generally accepted that the liver is the source for
plasma fibronectin. Hepatocytes in culture synthesize
and secrete fibronectin moss et al., 1979; Tamkun and
Hynes, 1983). Fibronectin has been localized in hepatocytes immunocytochemically (Clement et al., 1984). Owens and Cimino (1982) studied the synthesis of
fibronectin in the isolated perfused rat liver and concluded that the liver produces enough to account for its
levels in plasma.
However, studies on fibronectin in the liver have been
confined to the adult so far. There have been reported
increases in the levels of plasma fibronectin correlated
with ontological development (Sochorova et al., 1983).
The fetal liver is considerably different from the adult
liver. In the fetal liver, many kinds of blood cells are
differentiating in intimate association with differentiating hepatocytes (Jones, 1970; Medlock and Haar, 1983).
In the present investigation we have studied the liver
from the time it is first present as a distinct organ in
the fetus until early postpartum stages. During this
time we observed that fibronectin was prominent in
hepatic megakaryocytes rather than hepatocytes. Localization in hepatic megakaryocytes was similar to that
0 1989 ALAN R. LISS, INC.
found in the megakaryocytes of spleen and bone marrow. These observations raise interesting questions concerning the role of megakaryocytes as a source of
circulating fibronectin in fetal liver and in adult bone
marrow.
MATERIALS AND METHODS
Sixteen Sprague-Dawley rats were mated, and the day
on which sperm were found by vaginal lavage was taken
a s day 0. At intervals beginning with gestation day 11,
fetuses were removed from the uterus after the mother
was anesthetized with ether and sacrificed by cervical
dislocation. Older fetuses were decapitated before their
liver and spleen were removed. Two rats were allowed
to deliver, and their litters were processed at day 1 or
day 8 postpartum. Tissue was processed for immunocytochemical staining by a technique based on that described by Sainte-Marie (1962). Whole fetuses or separate
organs were fixed in acid alcohol (95 ml of 95% ethanol
plus 5 ml of glacial acetic acid) at O'C, dehydrated
through two changes each of cold absolute ethanol and
cold xylene, and embedded in paraffin. Sections 10 pm
thick were mounted on glass slides. After removal of
paraffin, they were incubated with the appropriate reagents to localize fibronectin as previously described
(Gulati et al., 1982). The sections were mounted in glycerol and viewed by epifluorescence.
In addition to fetal or postnatal organs, adult bone
marrow was prepared for localization of fibronectin. The
Received December 21, 1987; accepted April 28, 1988.
FIBRONECTIN IN LIVER MEGAKARYOCYTES
Fig. 1 . Liver from rat fetus on gestation day 12. The periphery shows
fluorescent cells, indicating the presence of fibronectin. The central
inset is from a section stained with hematoxylin and eosin; the arrows
indicate megakaryocytes and megakaryocyte precursors. Fluorescence
micrograph. ~ 5 0 0inset,
;
~800.
91
Fig. 2. Fluorescence micrograph of liver from gestation day 13. Megakaryocytes are numerous. Their cytoplasm is positive, whereas the
nuclei, frequently multilobed, are negative, ~ 7 0 0 .
Fig. 4. Gestation day 19. The pattern is maintained in later stages of
Fig. 3. At gestation day 16, the organ is taking on a more definitive
organization. Parenchymal cells are negative; fibronectin is localized fetal development, with scattered megakaryocytes positive, and hepatocytes negative. ~ 2 5 0 .
at the periphery of cellular cords, and in megakaryocytes. X 125.
ends of the femur were removed, and the marrow was
expelled by the injection through the marrow cavity of
fixative solution. Subsequent preparation followed the
procedures described for fetal tissue.
Two primary antisera were used for fibronectin localization. The first was prepared in rabbit, fibronectin
from rat plasma, isolated by the technique described by
Weiss and Reddi (1981),being used as antigen. The second was commercially available antiserum to human
plasma fibronectin, also prepared in rabbit (Bethesda
Research Laboratory, Bethesda, MD). Primary antisera
were applied a t a concentration of 20 pg/ml in phosphate-buffered saline (PBS). Similar results were obtained with both primary antisera. The secondary
antiserum that was used was goat anti-rabbit IgG, conjugated with fluorescein isothiocyanate (E-Y Labs, San
Mateo, CAI. The secondary antiserum was applied at a
concentration of 50 pg/ml(1:20 dilution of the commercially supplied product of 1 mg/ml). Extensive washing
with PBS followed each antibody incubation.
92
D.E. BOCKMAN AND A.K. GULATI
Fig. 7. Fluorescence micrograph of spleen from rat one day after
birth. Note the localization of fibronectin in megakaryocytes like those
in Figures 1-5. Diffuse staining of the extracellular matrix also is
present. X500.
Fig. 5. Higher magnification from gestation day 19, showing the
positive cytoplasm in the megakaryocytes, and negative multilobed
nuclei. ~ 5 2 5 .
Fig. 6. Section of fetal liver stained with hematoxylin and eosin. The
size and organization of the megakaryocytes can be compared with the
intervening hepatocytes and hemopoietic cells, which are negative (in
fluorescence micrographs) for fibronectin. x 1,000.
Controls for specificity of localization were used for
both primary and secondary antisera. As a control for
the primary antiserum, preimmune serum from the
same rabbit was substituted for the immune serum. For
the secondary antiserum, PBS was substituted in the
staining process. The specificity of reactivity was confirmed.
In order to verify the morphology of the organs and
cells, sections were stained with hematoxylin and eosin
either after or instead of staining
- for immunolocalization.
RESULTS
Cross sections through the fetal body revealed the
developing liver which as early as gestation day 12
contained large cells that displayed distinct fluorescence, indicating the presence of fibronectin (Figs. 1-5).
These cells stood out because they were the only cells
distinctly positive in the organ. The concentration of
fluorescent cells increased rapidly during early embryonic development, then was maintained a t a high level
during most of the remainder of gestation. Concentration then decreased until only a few were present by 8
days after birth. The only other area in the liver showing distinct fluorescence was at the border of cords or
rows of cells forming the parenchyma (Figs. 1-5). The
smaller hematopoietic cells, which would include cells
in the erythrocytic and granulocytic series, were negative. Hepatocytes were negative or only weakly positive.
Megakaryocytes were not stained in control preparations (figure not shown).
The morphology of the fluorescent cells allowed their
identification as megakaryocytes and megakaryocyte
precursors. Megakaryocytes were obvious because of
their large size and their multilobed nuclei in the fluorescence micrographs; their precursors also were large,
but nuclei varied from single through lobed or double.
Their indentification was confirmed when the sections
were stained with hematoxylin and eosin (Figs. 1,6).All
megakaryocytes and the described precursors were positive for fibronectin localization. Staining with hematoxylin and eosin after staining for fluorescence confirmed
that the positive cells were megakaryocytes and megakaryocyte precursors.
Spleen and bone marrow were not present in the earliest fetuses studied. However, when the spleen was
removed from late fetal and postnatal rats and studied,
it contained megakaryocytes that reacted identically to
those described in the liver (Fig. 7). Similar fibronectincontaining megakaryocytes were observed in adult bone
marrow (Fig. 8). Their morphology, as revealed by hematoxvlin and eosin staining (Fig.. 9). was similar to
that of the fibronectin-contain<ng cells of the liver.
93
FIBRONECTIN IN LIVER MEGAKARYOCYTES
Fig. 8. Localization of fibronectin in adult rat bone marrow. Compare
the positive megakaryocytes with those in Figures 1-5. X325.
Fig. 9. Adult rat bone marrow stained with hematoxylin and eosin.
Compare megakaryocytes with those in Figure 6. X475.
DISCUSSION
Megakaryocytes are a normal component of fetal liver,
and of spleen and bone marrow (Jones, 1970). They are
present in fetal liver before spleen and bone marrow are
formed. Bone marrow becomes the major repository of
megakaryocytes in the adult; their retention in spleen
is species dependent. Normal adult liver has no megakaryocytes.
The presence of fibronectin in bone marrow megakaryocytes has been well documented (Rabellino et al.,
1981; Emura et al., 1983; Reilly et al., 1985). Platelets,
which are a circulating product of megakaryocytes, likewise contain fibronectin (Niewiarowska et al., 1984; Plow
et al., 1979). More than 3 pg of fibronectin is contained
in lo9 platelets (Zucker et al., 1979).
The finding that fibronectin localization in hepatic
megakaryocytes and megakaryocyte precursors is much
more striking than in hepatocytes raises interesting
questions concerning the origins of circulating fibronectin in the fetus and adult.
Plasma fibronectin and cellular fibronectin are not
identical molecules. Although there is a single gene for
fibronectin, it has recently become clear that alternative
RNA splicing produces variability in the nature of the
molecule that may be produced (Yamada et al., 1985;
Gutman and Kornblihtt, 1987). Combination of all the
possible patterns of splicing in the three regions of variability that have been described to date could theoretically generate up to 20 distinct fibronectin polypeptides
from the single gene (Gutman and Kornblihtt, 1987).
Plasma fibronectin is missing a sequence that is contained in cellular fibronectin. There are resulting differences in isoelectric points and in biological activity
(Hynes and Yamada, 1982).
Plow and co-workers (1979) have indicated that, by
radioimmunoassay, platelet fibronectin is immunochemically indistinguishable from plasma fibronectin.
Formal identification of platelet fibronectin as plasma
fibronectin has not been made by isoelectric focusing, by
means of monoclonal antibodies, which can be used to
distinguish cellular fibronectins (Atherton and Hynes,
19811, or by using molecular probes for the pertinent
sequences of mRNA. It seems reasonable, however, that
the fibronectin in platelets is of the plasma type. Platelets circulate in the plasma and release soluble products
from their a-granules, where fibronectin is localized
(Wencel-Drake et al., 1984, 1985).
The fetal liver is interposed in the circulatory system
of the fetus. The presence of a considerable population
of fibronectin-containing megakaryocytes and megakaryocyte precursors in the early fetal liver leads us to
the hypothesis that they are important in providing
fibronectin in the circulation. Furthermore, bone marrow megakaryocytes in the adult, through platelets and/
or directly, may contribute to plasma fibronectin. The
contribution of fibronectin by megakaryocytes in fetal
liver does not necessarily have to be entirely substitutive for fibronectin produced by hepatocytes; megakaryocytes and hepatocytes cou1.d produce fibronectin
simultaneously. The techniques are available to determine if megakaryocytes provide a significant contribution to the circulating fibronectin pool.
ACKNOWLEDGMENTS
We are grateful for the technical assistance of Ms.
Brenda Headrick and Ms. Penny Roon, and to Ms. Connie Benson for typing the manuscript.
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