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. 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