To analyze the ontogenic emergence of leukocytes during early development, a mouse monoclonal antibody (IgG1), designated as XL-1, was produced against the peritoneal macrophages of adult Xenopus laevis. The XL-1 determinant was expressed on all types of leukocytes, including lymphocytes, granulocytes, thrombocytes and macrophages, but not on erythrocytes of either larvae or adults. Immunohistochemical observations of the hemopoietic organs revealed that the XL-1+ cells with granulocyte and/or macrophage morphology appeared at st.36–37 in the liver, at st.44–45 in the mesonephric and the thymus rudiments, and at st.47 in the spleen. The XL-1 determinant was expressed on the precursor cells of T lymphocytes in the thymus rudiments at st.46–47, on the pre-B cells in the liver rudiments at st.47, and on lymphocytes in the spleen at st.48–49. A few XL-1+ cells were present in the ventral blood island of the st.35/36 embryos, where differentiating erythrocytes had predominated since st.28. XL-1+ cells with a macrophage-like morphology were found in several locations of the mesenchyme in the st.32 embryos, before the establishment of vascularization at st.33/34 and far earlier than the emergence of lymphocytes.

It is generally accepted that various types of blood cells in vertebrates are derived from a common, pluripotent stem cell capable of differentiating into discrete lineages under the control of specific microenvironments. The initial population of hemopoietic stem cells is believed to arise in the yolk sac during embryogenesis (Metcalf and Moore, 1971), although more recent studies of birds emphasize the intraembryonic origin of lymphocytes, monocytes and definitive erythrocytes (Dieterlen-Lievre, 1975). Hemopoietic cell lineage studies both in vitro and in vivo on mammals and birds have been made with a variety of antibodies detecting the surface marker antigens specific to each type and/or subset of hemopoietic cells (Shaw, 1987; Holmes and Morse, 1988). No specific markers have been developed, however, which are both common to, and restricted to, leukocyte-series cells. Thus, the leukocyte-common antigen (L-CA) is expressed on T- and B-lymphocytes, thymocytes, granulocytes and macrophages, together with erythroblastic cells, but not on mature erythrocytes (reviewed b’y Thomas and Lefrançois, 1988).

Recent experiments employing grafts of cytogenetically labeled tissues in Xenopus laevis have established that stem cells of early larval erythrocytes and lymphocytes are localized in the ventral blood island (VBI) mesoderm, while those of their more advanced larval and adult counterparts gather in the dorsolateral plate mesoderm of tailbud embryos (Maéno et al. 1985a; 1985b; Kau and Turpen, 1983; Smith et al. 1989; Flajnik et al. 1984). Of these hemopoietic cells, erythrocytes undergo differentiation in the VBI (Mangia et al. 1970), whereas B- and T-lymphocytes start to express their differentiation markers IgM (Hadji-Azimi et al. 1982) and XT-1 antigen (Nagata, 1985, 1986) after migration to the rudiments of liver and thymus, respectively. Compared with what is known of lymphocytes, however, very few reports have been published about the embryonic origin, and the ontogenic emergence, of non-lymphoid leukocytes such as macrophages, granulocytes and thrombocytes; studies based on the classical criteria of cell identification are an exception (Manning and Horton,-1969, 1982).

The present study was intended to identify the differentiation markers for leukocytes in X. laevis, as clues for studying the ontogenic emergence of leukocytes in this animal. We report here a monoclonal antibody (mAb) named XL-1, which recognizes all Xenopus leukocyte-series cells but not those of the erythrocyte-series. Our immunohistochemical observations employing this mAb demonstrate that the cells that express the XL-1 determinant differentiate in the mesenchyme of embryos chronologically far earlier than has been previously supposed.

Materials

The animals used in this study were outbred (HD-group) and MHC-homozygous J strain individuals (Tochinai and Katagiri, 1975) of Xenopus laevis. Embryos and larvae were reared at 23 °C, and were staged according to the Normal Table of Nieuwkoop and Faber (1967).

Production of monoclonal and polyclonal antibodies

Peritoneal macrophages were collected from the J strain X. laevis by the method described previously (Sekizawa et al. 1984), and were suspended in phosphate-buffered saline (PBS). The viable macrophages (each 0.2–1.0×106 cells) were injected intraperitoneally into BALB/c mice, five times at two-weekly intervals. Four days after the final injection, the spleen cells were removed and fused with P3-X63-Ag8.653 myeloma cells, according to the principle devised by Kohler and Milstein (1975), with minor modifications. Undiluted hybridoma supernatants were screened for the identification of antibodies by immunofluorescence on cells attached to the Terasaki plate or tissue sections as described below. The hybridoma line used in this study was cloned three times by the limiting dilution technique. The anti-larval hemoglobin monoclonal antibody (m Ab) was provided by T. Enami of our laboratory. The polyclonal antibodies to Xenopus IgM were raised in a rabbit by injecting the periodate-treated purified IgM, according to the procedure devised by Mattes and Steiner (1978) and Hadji-Azimi et al. (1982).

Immunohistochemical staining

For screening of antibodies produced by hybridomas, various cells obtained from adult spleen, thymus, peritoneum and peripheral blood were attached to the poly-L-lysin-coated Terasaki plates by centrifugation. The cells were fixed either with 0.25% glutaraldehyde in PBS (10min), absolute methanol (5 min) or 4 % paraformaldehyde in PBS (30 min) at 4°C. Adult white blood cells were collected from the uppermost layer (buffy coat) of the centrifuged peripheral blood, smeared on glass slides and fixed with absolute methanol. Larval peripheral blood was smeared directly onto glass slides and fixed with absolute methanol. For subsequent study of the distribution of leukocytes, 10 individuals at various developmental stages were fixed with an ethanol and acetic acid mixture (1:3) (60min), embedded in Tissue Prep, and serially sectioned at 7 μm. Five individuals each of embryos or larvae for whole-mount preparation were fixed with 4 % paraformaldehyde in PBS overnight at 4 °C.

For indirect immunofluorescent study, fixed cells, sectioned tissues or fixed embryos and larvae were incubated with PBS supplemented with 10 % fetal calf serum (10 % FCS-PBS) for blocking of nonspecific protein binding, and were incubated overnight at 4 °C with undiluted hybridoma culture supernatants or a 1:1000 diluted rabbit antiserum to Xenopus IgM, followed by incubation with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse Ig antibodies or fluorescein isothiocyanate (FITC)-conjugated goat antirabbit Ig antibodies. Control samples were incubated either with 10% FCS-PBS, myeloma culture media or a diluted normal rabbit serum instead of mAbs or antiserum, followed by incubation with the TRITC- or FITC-conjugated reagent. All controls were negative. Observations were performed with a epifluorescence microscope.

Electron microscopy

St.46–47 larvae were fixed with 4% paraformaldehyde in 0.1 M-phosphate buffer (pH7.2), dehydrated in ethanol and acetone, and were embedded in LR-Gold (TAAB Lab., Berkshire) according to the procedure devised by Newman et al. (1982). Ultrathin sections were made with glass knives on a Porter-Blum MT-1 ultramicrotome, and were made to react for 2 h each at room temperature with mAb and the goat antimouse IgG antibodies coupled to colloidal gold particles (20nm, E-Y Labs Inc.). Control samples incubated with only the colloidal gold-conjugated reagent showed distribution of an extremely few gold particles in nonspecific fashion. Along with these series, some specimens were processed by the ordinary method of glutaraldehyde-osmium tetroxide fixation and Epon embedding. Ultrathin sections were stained with uranyl acetate and lead citrate, and were observed with a JEOL JEM-100S electron microscope.

Electrophoresis and immunoblotting

Adult spleens and thymuses were teased and filtered through fine-mesh nylon netting with PBS. Adult peripheral blood suspended in PBS was centrifuged several times to remove the buffy coat-leukocytes. After several washes with PBS, the cells were solubilized with lysis buffer (2% NP-40, 20 mM-Tris–-HCl, pH 8.0, 0.15M-NaCl, 2mM-MgCl2 and 0.1 mM-PMSF), kept on ice for 30 min with occasional vortexing and microcentrifuged. Supernatants of cell lysates and diluted adult serum were mixed with the sample buffer (0.125 M-Tris-HCl, pH6.8, 20% glycerol, 4% SDS and 10% 2-mercaptoethanol) in 1:1 ratio.

SDS-PAGE using 8 % gel was carried out under reducing conditions, according to Laemmli (1970). For Western blotting, the electrophoresed gels were blotted to nitrocellulose sheets (cf. Towbin et al. 1979). The blotted sheets were successively incubated in 1:50 diluted XL-1 and alkaline phosphatase (AP)-conjugated goat anti-mouse IgG, then were processed for AP activity using the developing solution of BCIP and NBT (Bio-Rad Labs, California).

Expression of the XL-1 determinant in leukocytes

The monoclonal antibody (mAb) named XL-1 that was used in the present study reacted with all the leukocyteseries cells including lymphocytes, granulocytes, thrombocytes and macrophages (cf. Hadji-Azimi et al. 1987) from both adult and larval Xenopus laevis but not with erythrocytes (Figs 1, 2). The antibody subclass was determined to be IgG1. The reactivity of leukocytes with XL-1 on the indirect immunofluorescence technique did not differ significantly according to the fixatives used, either by smear or sectioning of the materials. The cytoplasm of leukocytes showed granular staining with various degrees of brightness, although large refractile cytoplasmic granules of eosinophils did not stain. Exactly the same staining pattern was obtained with viable leukocytes. Upon incubation of viable lymphocytes under conditions without NaN3, a capping of the fluorescence was evident, suggesting that the XL-1 determinant is also localized on the surface of leukocytes. The leukocytes from outbred animals were also stained with XL-1 as strongly as those from inbred J frogs. Adult and larval leukocytes from Xenopus borealis were stained in a similar pattern, although more weakly than their X. laevis counterpart.

Figs 1 and 2.

Immunofluorescence (A) and phase-contrast (B) micrographs of methanol-fixed peripheral blood cells from an adult (Fig. 1) and st.52 larva (Fig. 2), showing that all leukocytes but not erythrocytes (arrowheads) are reactive to XL-1 mAb. Fig. 1, buffy coat cells; Fig. 2, smear. Visualized by TRITC-conjugated anti-mouse Ig as a secondary antibody, ne, neutrophil; e, eosinophil; b, basophil; 1, lymphocyte; t, thrombocyte. Bars, 25μm.

Figs 1 and 2.

Immunofluorescence (A) and phase-contrast (B) micrographs of methanol-fixed peripheral blood cells from an adult (Fig. 1) and st.52 larva (Fig. 2), showing that all leukocytes but not erythrocytes (arrowheads) are reactive to XL-1 mAb. Fig. 1, buffy coat cells; Fig. 2, smear. Visualized by TRITC-conjugated anti-mouse Ig as a secondary antibody, ne, neutrophil; e, eosinophil; b, basophil; 1, lymphocyte; t, thrombocyte. Bars, 25μm.

Western blot analyses revealed that XL-1 determinant comprises several molecules ranging from 100 to 300 × Mr, showing both unique and sharing components with each other in splenocytes and thymocytes (Fig. 3). No reactivity was seen at all in erythrocyte lysates. Although no other tissue components showed reactivity against XL-1, the XL-1 determinant was found also in the serum expressing some unique electrophoretic mobilities (Fig. 3). Presumably reflecting this aberrant reactivity, XL-1 showed occasional, but not consistent, immunohistochemical localization in and along the epithelial lining of nephric ducts and along the inner surface of blood vessels in advanced larvae. Except these particular organs, the XL-1 determinant was expressed only on the leukocyte-series cells in Xenopus.

Fig. 3.

Western blot analyses of XL-1 reactive components in lysates from adult splenocytes (a), thymocytes (b) and erythrocytes (c), and serum (d). Arrowheads indicate bands specific for each splenocytes and thymocytes. Mr; relative molecular mass.

Fig. 3.

Western blot analyses of XL-1 reactive components in lysates from adult splenocytes (a), thymocytes (b) and erythrocytes (c), and serum (d). Arrowheads indicate bands specific for each splenocytes and thymocytes. Mr; relative molecular mass.

Ontogenic emergence of XL-1 + cells in early hemopoietic and lymphoid organs

The ontogenic emergence and distribution of XL-1+ cells were examined on sections of various hemopoietic tissues or whole-mount preparations of embryos and larvae. The XL-1+ cells first appeared in the liver rudiments at st.36–37, following the establishment of a blood stream, scattered both in the parenchyma and sinusoids. The positive cells increased in number thereafter and, from st.47 on, some of them could be seen in the perihepatic layer as if lining the organ (Fig. 4).

Figs 4 and 5.

Immunofluorescence (A) and phase-contrast (B) micrographs of a transverse section through the liver (1) of st.47 larva (Fig. 4), and the prospective mesonephric region of st.44–45 larva (Fig. 5), showing localization of XL-1+ cells in the perihepatic areas (Fig. 4A) and the mesenchyme ventral to the dorsal aorta (d) (Fig. 5A). n, notochord; s, somite; i, intestine; v, vena cava; w, Wolffian duct. Bars, 100μm.

Figs 4 and 5.

Immunofluorescence (A) and phase-contrast (B) micrographs of a transverse section through the liver (1) of st.47 larva (Fig. 4), and the prospective mesonephric region of st.44–45 larva (Fig. 5), showing localization of XL-1+ cells in the perihepatic areas (Fig. 4A) and the mesenchyme ventral to the dorsal aorta (d) (Fig. 5A). n, notochord; s, somite; i, intestine; v, vena cava; w, Wolffian duct. Bars, 100μm.

During the succeeding stages, the positive cells were preferentially localized as clusters in the subcapsular area as well as in the blood vessels. The pre-B cells that possessed the cytoplasmic IgM but not any surface Ig (clg+ sig) were first detectable at st.47, but their localization was not identical with that found for the XL-1+ cells described above. Double immunofluorescence staining using antisera to IgM and XL-1 during these stages showed that some of these pre-B cells were also XL-1+ although eventually all B cells (clg+ slg+) were XL-1+ in the later stages. Most of the positive cells at st.47 were identified as non-lymphoid leukocytes such as granulocytes and macrophages. At this stage, larval hemoglobin (LHb)-positive cells (larval erythrocytes) were few in number in the perihepatic layer.

In the mesonephric rudiment, XL-1+ cells appeared first at st.44–45 in the area surrounded by the dorsal aorta, the vena cava and the Wolffian ducts (Fig. 5). By st.47 numerous XL-1+ cells had aggregated in this area, where they underwent extensive mitosis. Electron microscopy of the mesonephric primordia revealed that these XL-1+ cells were granulocytes and macrophages, as defined by their lobulated or bean-shaped nuclei, unique chromatin condensation pattern, numerous cytoplasmic granules, as well as by their distinct pseu dopodia (Figs 6, 7). None of the XL-1+ cell aggregates in the mesonephros possessed IgM prior to st.49.

Fig. 6.

Transmission electron micrograph of the mesenchymal area near the mesonephric primordium ventral to the dorsal aorta (d) in st.47 larva, showing many leukocyte-series cells (arrowheads) with lobulated nuclei. Occasionally observed cells undergoing mitosis (arrow) appear the same-series cells. Bar, 5 μm.

Fig. 6.

Transmission electron micrograph of the mesenchymal area near the mesonephric primordium ventral to the dorsal aorta (d) in st.47 larva, showing many leukocyte-series cells (arrowheads) with lobulated nuclei. Occasionally observed cells undergoing mitosis (arrow) appear the same-series cells. Bar, 5 μm.

Fig. 7.

Immunoelectron micrograph of the cell as shown in Fig. 6, demonstrating localization of XL-1 antigens by colloidal gold particles both on the cell surface (arrowheads) and in the cytoplasm, nu, nucleus. Bar, 1μm.

Fig. 7.

Immunoelectron micrograph of the cell as shown in Fig. 6, demonstrating localization of XL-1 antigens by colloidal gold particles both on the cell surface (arrowheads) and in the cytoplasm, nu, nucleus. Bar, 1μm.

In the thymus rudiments, a small number of XL-1+ cells appeared for the first time at st.44-45. The positive cells were regarded as macrophage/monocyte-series cells as defined by their dendritic shape and a stronger reactivity than the lymphocyte-series cells described below. Two types of XL-1+ cells appeared in the thymus at st.46–47: the large, macrophage-like cells with brightly stained dendritic cytoplasm and the lymphoblasts with weakly stained dots in the thin cytoplasm (Fig. 8). At this stage, the latter cells tended to locate preferentially in the outer region of the thymus (future cortex). The reactivity of these cells increased gradually as they differentiated into lymphocytes, so that by st.49 virtually all thymic lymphocytes were stained uniformly with XL-1. In the thymus of the more advanced stages, as Fig. 9 shows, all thymic lymphocytes and macrophage-like cells were positive, but epithelial cells and myoid cells were entirely negative.

Figs 8–11.

Immunofluorescence (Figs 8A, 9, 10A, 11) and phase-contrast (8B, 10B) micrographs of transverse sections of the thymus (Figs 8, 9) and the spleen (Figs 10, 11) of st.47 (Fig. 8), st.49 (Fig. 10) and st.56 (Figs 9, 11) larvae, showing XL-1+ macrophage-like dendritic cells (arrowheads) and lymphoid cells (arrows). Visualized by TRITC-conjugated anti-mouse Ig. c, cortex; m, medulla; rp, red pulp; wp, white pulp; i, intestine. Bars, 40μm.

Figs 8–11.

Immunofluorescence (Figs 8A, 9, 10A, 11) and phase-contrast (8B, 10B) micrographs of transverse sections of the thymus (Figs 8, 9) and the spleen (Figs 10, 11) of st.47 (Fig. 8), st.49 (Fig. 10) and st.56 (Figs 9, 11) larvae, showing XL-1+ macrophage-like dendritic cells (arrowheads) and lymphoid cells (arrows). Visualized by TRITC-conjugated anti-mouse Ig. c, cortex; m, medulla; rp, red pulp; wp, white pulp; i, intestine. Bars, 40μm.

The spleen rudiments possessed XL-1+ cells for the first time at st.47. Similar to the thymus rudiments at st.47, the positive cells in the spleen at st.48–49 consisted of large, irregularly shaped cells with a macrophage-like morphology and faintly stained lymphocytes (Fig. 10). At st.56 all the lymphocytes and large den-dritic macrophage-like cells were stained well with XL-1 both in white and red pulps (Fig. 11), whereas the cytoplasm of LHb+ cells was always XL-1 negative. The occasional staining with XL-1 of intercellular spaces among erythrocytes in red pulp (Fig. 11) may be ascribable to the exudates from adjacent leukocytes and/or deposition of serum components which were positive to XL-1 (cf. also Fig. 3).

Occurrence of XL-1+ cells in the mesenchyme of embryos and early larvae

Previous studies have indicated that the major population of lymphocytes and erythrocytes in early larvae are derived from the mesodermal cells localized in the ventral blood island (VBI) of st.22 embryos (Maéno et al. 1985a; Smith et al. 1989). Immunofluorescence staining that employs anti-LHb mAb revealed that the LHb+ cells did occur exclusively in the VBI mesoderm as early as st.28. These LHb+ cells were the major constituents of the VBI region during st.31-36/37 (vascularization started in embryos at st.33/34). On the other hand, XL-1 did not react with these hemopoietic series cells in the VBI. Only a few XL-1+ cells could be found scattered along the inner surface of the epidermal cells in VBI at st.33/34. The flattened shape and pseudopodia of these XL-1+ cells differed from those found on the erythroid cells.

XL-1+ cells were found in early embryos outside the hemopoietic regions. From st.32 on the XL-1+ cells were detectable as isolated cells in the mesenchyme in such locations as beneath the epidermis, around the notchord or somites, and above the spinal cord. At the initial stages of their appearance during st.32–35/36, most XL-1+ cells still possessed yolk platelets, and were of either round or highly flattened shape with occasional prominent pseudopodia (Fig. 12C,D). The shape of their nuclei was also variable, either round, indented or lobulated. The number of these cells rose sharply during st.32–36/37, so that they were easily detectable in the whole-mount preparations at later stages as shown in Fig. 12. It is thus clear that, chronologically, these mesenchymal XL-1+ cells appear before the differentiation of any hemopoietic or lymphoid organs.

Fig. 12.

Immunofluorescence micrographs of the whole-mount preparations of st.35/36 embryos, showing distribution of cells reactive to XL-1 (A,C,D) and anti-larval hemoglobin monoclonal antibody (B). Lateral views, anterior to the right. Arrowheads in B indicate the ventral blood island region where at this stage the cells producing larval hemoglobin are boundedly distributed. C and D, higher magnification views of XL-1+ cells in the areas shown in Fig. 12A (C, posterior box; D, anterior box), ey, eye. Bars (A,B), 0-5mm; Bars (C,D), 40μm.

Fig. 12.

Immunofluorescence micrographs of the whole-mount preparations of st.35/36 embryos, showing distribution of cells reactive to XL-1 (A,C,D) and anti-larval hemoglobin monoclonal antibody (B). Lateral views, anterior to the right. Arrowheads in B indicate the ventral blood island region where at this stage the cells producing larval hemoglobin are boundedly distributed. C and D, higher magnification views of XL-1+ cells in the areas shown in Fig. 12A (C, posterior box; D, anterior box), ey, eye. Bars (A,B), 0-5mm; Bars (C,D), 40μm.

The determinant detected by our monoclonal antibody (mAb) XL-1 is similar to the leukocyte-common antigen (L-CA) reported in mammals (reviewed by Thomas and Lefrançois, 1988) and birds (Houssaint et al. 1987) in that it is expressed on all leukocytes but not on mature erythrocytes. However, the XL-1 determinant differs from the L-CA that is also expressed on both immature erythroid cells and leukocyte-series cells. In fact, our present study shows that the XL-1 determinant is not expressed on any erythroid precursor cells in the embryonic ventral blood island (VBI), which is the amphibian counterpart of the avian and mammalian extraembryonic yolk sac. We can therefore regard XL-1 as a unique probe for studying the differentiation of leukocyte-series cells during embryonic and larval development. The L-CA reportedly comprises a family of surface glycoproteins with relative molecular masses between 180 and 240×103, whose expression is variable according to the cell types and stages of hemopoiesis. Similarly, our XL-1 determinant was detected as several different molecular mass entities in splenocytes and thymocytes. It should be mentioned that the XL-1 determinant is not exclusively specific to leukocytes, as observed in the adult serum showing electrophoretic mobilities different from those in leukocytes. Appar-ently this reactivity is responsible for the occasional immunohistochemical localization along the inner wall of the blood vessels and intercellular spaces in splenic red pulps. In this sense, XL-1 also resembles the quail anti-MB1 mAb (Peault et al. 1983; Labastie et al. 1986), which reacts with endothelial cells and plasma components as well as with all mature leukocytes.

Another important issue is the determination of the stages when embyronic or larval lymphoid cells express the antigen(s) detected with XL-1. Previous studies by Nagata (1977) and Tochinai (1980) have demonstrated that lymphoid precursor cells immigrate into the thymus rudiment during the restricted period of st.42-45. The T-cell-specific determinant XT-1 is first expressed on thymocytes at st.48 (Nagata, 1986) when morphologically mature thymus lymphocytes appear for the first time. The present study shows that the earliest XL-1+ cells in the thymus rudiments at st.44–45 are macrophage-like dendritic cells, and that the lymphoidseries cells with XL-1+ can first be observed at st.46–47, followed by an increasing fluorescence intensity until st.48–49. This means that the XL-1 determinant is expressed on the lymphoid precursor (pre-T) cells at st.46–47. Similarly, the XL-1* cells first occurred in the liver at st.47 in the pre-B cells as defined by their coexpression of cytoplasmic IgM (cf. Hadji-Azimi et al. 1982). It should be noticed that these T- and B-lymphocytes in the primary lymphoid organs have emigrated from the VBI of tailbud embryos (Maéno et al. 1985a,b). In view of the present observations with XL-1, it is clear that lymphoid precursor cells, although ‘committed’ to some extent during their migration, start to express the XL-1 determinant as a result of residence in the microenvironments provided by the thymus or the liver rudiments.

In Xenopus larvae, the liver and the mesonephros are known to be the major hemopoietic organs. Manning and Horton (1969, 1982) have shown that the granulopoiesis and lymphopoiesis in these organs initiate from st.49-50 on. The present study reveals that prior to the initiation of liver leukopoiesis at st.47, a cluster of XL-1+ cells, defined electron microscopically as typical granulocytes and macrophages, are first detectable at st.44-45 in the mesonephric primordium. Although a few XL-1+ pre-B cells occur in the liver at st.47, as we note above, most, if not all, perihepatic XL-1+ cells during st.47–49 represent macrophages and granulocytes as well. In comparison with the positioning of their lymphoid precursors, the exact localization of precursor cells for these non-lymphoid leukocytes in early embryos has not been elucidated.

An intriguing finding derived from the present study is that a fairly large number of the XL-1+ cells with macrophage-like morphology were found in the mesenchyme at st.32 and in the blood vessels at st.36–37, prior to the appearance of lymphocytes. Because it is not until st.48 that Xenopus lymphoid immune responses are activated (Horton and Manning, 1972; Kidder et al. 1973), the presence of these leukocytes draws attention to their possible relevance as nonspecific defense mechanisms required for the free-living early tadpoles. The following findings may support this view: Turner (1969) reported that pericardial and peritoneal free macrophages are the first line of defense against intraperitoneally injected particles in the st.48 larvae. Lehman (1953) also observed the ingesting activities of macrophages in the transparent tail fin of the larvae whose stages correspond roughly to st.45-49. Besides their early occurrence in Xenopus, macrophages are reportedly present in the liver of Rana pipiens at Shumway Stage 22, equivalent to st.41 of X. laevis before the onset of hemopoiesis (erythropoiesis, granulopoiesis and lymphopoiesis; Turpen et al. 1979). The classical observations by Metchnikoff (1893) and Clark and Clark (1930) offered evidence for migratory and phagocytic macrophages in the connective tissue of the tail fin of the axolotl, Hyla and Rana embryos immediately after hatching. Although their functional characterization is still insufficient, the present mesenchymal XL-1+ cells found in early tadpoles most likely function in the defense against pathogens. Compared with the study devoted to lymphocytes and erythrocytes, not much attention has hitherto been paid to the embryonic origin of these phagocytic leukocytes. An explanation for the origin of the extremely early-emerging mesenchymal XL-1+ cells may be that prior to the onset of heart beating, the precursor cells emigrate from the VBI to undergo subsequent differentiation into leukocytes. Our experiments including grafting of labeled VBI-mesodermal cells, however, have excluded this emigration which could account for the occurrence of many mesenchymal XL-1+ cells at st.32–35/36 (Ohinata et al. in preparation). It is thus reasonable to postulate a population of XL-1+ cells that do not share the embryonic origin with other hemopoietic cells. Experiments designed to give a definite answer to this question are currently under way.

We wish to thank Mr Y. Takakuwa for his excellent technical assistance in pursuing the electron microscopic study. This study was supported by research grants from the Japanese Ministry of Education (no. 0130432, 63540556).

Clark
,
E. R.
and
Clark
,
E. L.
(
1930
).
Observation on the macrophages of living amphibian larvae
.
Am. J. Anat
.
46
,
91
147
.
Dieterlen-Lievre
,
F.
(
1975
).
On the origin of haematopoietic stem cells in the avian embryo: an experimental approach
.
J. Embryol. exp. Morph
33
,
607
619
.
Flajnik
,
M. F.
,
Horan
,
P. K.
and
Cohen
,
N.
(
1984
).
A flow cytometric analysis of the embryonic origin of lymphocytes in diploid/triploid chimenc Xenopus laevis
.
Devi Biol
.
104
,
247
254
.
Hadji-Azimi
,
I.
,
Coosemans
,
V.
and
Canicatti
,
C.
(
1987
).
Atlas of adult Xenopus laevis hematology
.
Dev. Comp. Immunol
.
11
,
807
874
.
Hadji-Azimi
,
I.
,
Schwager
,
J.
and
Thiebaud
,
CH
. (
1982
).
B-lymphocyte differentiation tn Xenopus laevis larvae
.
Devi Biol
.
90
,
253
258
.
Holmes
,
K. L.
and
Morse
,
H. C.
Ill
(
1988
).
Murine hemopoietic cell surface antigen expression
.
Immunol. Today
9
,
344
350
.
Horton
,
J. D.
and
Manning
,
M. J.
(
1972
).
Response to skin allografts in Xenopus laevis following thymectomy at early stages of lymphoid organ maturation
.
Transplantation
14
,
141
154
.
Houssaint
,
E.
,
Tobin
,
S.
,
Cihak
,
J.
and
Losch
,
U.
(
1987
).
A chicken leukocyte common antigen: biochemical characterization and ontogenetic study
.
Eur. J. Immunol
.
17
,
287
290
.
Kau
,
C. L.
and
Turpén
,
J. B.
(
1983
).
Dual contribution of embryonic ventral blood isLond and dorsal lateral plate mesoderm during ontogeny of hemopoietic cells in Xenopus laevis
.
J. Immunol
.
131
,
2262
2266
.
Kidder
,
G. M.
,
Ruben
,
L. N.
and
Stevens
,
J. M.
(
1973
).
Cytodynamics and ontogeny of the immune response of Xenopus laevis against sheep erythrocytes
.
J. Embryol. exp. Morph
.
29
,
73
85
.
Kohler
,
G.
and
Milstein
,
C.
(
1975
).
Continuous cultures of fused cells secreting antibody of predefined specificity
.
Nature, Lond
256
,
495
497
.
Labastie
,
M. C.
,
Poole
,
T. J.
,
Péault
,
B. M.
and
Le Douarin
,
N. M.
(
1986
).
MB-1, a quail leukocytes-endothelium antigen: Partial characterization of the cell surface and secreted forms in cultured endothelial cells
.
Proc. natn. Acad. Sci. U.S.A
.
83
,
9016
9020
.
Laemmli
,
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
.
Nature, Lond
.
227
,
680
685
.
Lehman
,
H. E.
(
1953
).
Observations of macrophage behavior in the fin of Xenopus larvae
.
Biol. Bull. mar. biol. Lab. Woods Hole
105
,
490
495
.
Maéno
,
M.
,
Tochinai
,
S.
and
Katagiri
,
CH
. (
1985b
).
Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-tnploid or interspecific chimeras
.
Devi Biol
.
110
,
503
508
.
Maéno
,
M.
,
Todate
,
A.
and
Katagiri
,
CH
. (
1985a
).
The localization of precursor cells for larval and adult hemopoietic cells of Xenopus laevis in two regions of embryos
.
Dev. Growth Differ
.
27
,
132
148
.
Mangia
,
F.
,
Procicchiani
,
G.
and
Manelli
,
H.
(
1970
).
On the development of the blood isLond in Xenopus laevis embryos: light and electron microscopic study
.
Acta Embryol. exp
.
1970
,
163
184
.
Manning
,
M. J.
and
Horton
,
J. D.
(
1969
).
Histogenesis of lymphoid organs in larvae of the South African clawed toad, Xenopus laevis (Daudin)
.
J. Embryol. exp Morph
.
22
,
265
277
.
Manning
,
M. J.
and
Horton
,
J. D.
(
1982
).
RES structure and function of the Amphibia
.
In The Reticuloendothelial System 3
(ed.
N.
Cohen
and
M. M.
Sigel
), pp.
423
459
.
New York
:
Plenum Press
.
Mattes
,
M. J.
and
Steiner
,
L. A.
(
1978
).
Antisera to frog immunoglobulins cross-react with a periodate-sensitive cell surface determinant
.
Nature, Lond
.
273
,
761
763
.
Metcalf
,
D.
and
Moore
,
M. A. S.
(
1971
)
Haematopoietic Cells
.
Amsterdam
:
North-Hollond
.
Metchnikoff
,
E.
(
1893
).
The Comparative Pathology of Inflammation
.
London
:
Kegan Paul, Trench, Trubner & Co
.
Nagata
,
S.
(
1977
).
Electron microscopic study on the early histogenesis of thymus in the toad, Xenopus laevis
.
Cell Tissue Res
.
179
,
87
96
.
Nagata
,
S.
(
1985
).
A cell surface marker of thymus-dependent lymphocytes in Xenopus laevis is identifiable by mouse monoclonal antibody
.
Eur. J. Immunol
.
15
,
837
841
.
Nagata
,
S.
(
1986
).
Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an antithymocyte mouse monoclonal antibody
.
Devi Biol
.
114
,
389
394
.
Newman
,
G. R.
,
Jasani
,
B.
and
Williams
,
E. D.
(
1982
).
The preservation of ultrastructure and antigenicity
.
J. Microsc
.
127
,
RP5
RP6
.
Nieuwkoop
,
P. D.
and
Faber
,
J.
(
1967
).
Normal Table of Xenopus laevis (Daudin)
.
Amsterdam
:
North-HolLond
.
Peault
,
B. M.
,
Thiery
,
J. P.
and
Le Douarin
,
N. M.
(
1983
).
Surface marker for hemopoietic and endothelial cell lineages in quail that is defined by a monoclonal antibody
.
Proc. natn. Acad. Sci. U.S.A
.
80
,
2976
2980
.
Sekizawa
,
A.
,
Fujii
,
T.
and
Tochinai
,
S.
(
1984
).
Membrane receptors on Xenopus macrophages for two classes of immunoglobulins (IgM and IgY) and the third complement component (C3)
.
J. Immunol
.
133
,
1431
1435
.
Shaw
,
S.
(
1987
).
Characterization of human leukocyte differentiation antigens
.
Immunol. Today
8
,
1
3
.
Smith
,
P. B.
,
Flajnik
,
M. F.
and
Turpén
,
J. B.
(
1989
).
Experimental analysis of ventral blood isLond hematopoiesis in Xenopus embryonic chimeras
.
Devi Biol
.
131
,
302
312
.
Thomas
,
M. L.
and
Lefrançois
,
L.
(
1988
).
Differential expression of the leucocyte-common antigen family
.
Immunol. Today
9
,
320
326
.
Tochinai
,
S.
(
1980
).
Direct observation of cell migration into Xenopus thymus rudiments through mesenchyme
.
Dev. Comp. Immunol
.
4
,
273
282
.
Tochinai
,
S.
and
Katagiri
,
CH
. (
1975
).
Complete abrogation of immune responses to skin allografts and rabbit erythrocytes in the early thymectomized Xenopus
.
Dev Growth Differ
.
17
,
383
394
.
Towbin
,
H.
,
Staehelin
,
T.
and
Gorgon
,
J.
(
1979
).
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications
.
Proc, natn. Acad. Sci. U.S.A
.
76
,
4350
4354
.
Turner
,
R. J.
(
1969
).
The functional development of the reticuloendothelial system in the toad, Xenopus laevis (Daudin)
.
J. exp. Zool
.
170
,
467
480
.
Turpén
,
J. B.
,
Turpén
,
C. J.
and
Flajnik
,
M.
(
1979
).
Experimental analysis of hematopoietic cell development in the liver of larval Rana pipiens
.
Devi Biol
.
69
,
466
479
.