ARTICLE |
Correspondence to: Nobuyuki Takakura, Dept. of Molecular Genetics, Faculty of Medicine, Kyoto University, Shogoin Kawaharacho 53, Sakyo-ku 606-01 Japan.
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Summary |
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We investigated the cells that express platelet-derived growth factor receptor (PDGFR
) during mouse embryogenesis. PDGFR
expression has been identified by in situ hybridization or immunohistochemistry using polyclonal antibodies on tissue sections. Because no immunostaining study using whole-mount specimens has been published to date, we established a new monoclonal antibody (MAb), APA5, for this purpose. Our results differed in that APA5 stained only the paraxial mesoderm, whereas other investigators concluded that most if not all mesodermal cells expressed PDGFR
. Moreover, we did not find PDGFR
expression in embryonic erythrocytes, which have been previously suggested to express PDGFR
. On the basis of our present results, we wish to revise the proposed PDGFR
expression as follows. At the pregastrulation stage, PDGFR
is expressed only in primitive endoderm, particularly that in the ectoplacental cone. On gastrulation, it is expressed at high levels in the paraxial mesoderm. This expression continues after its differentiation into the somite. Along with the differentiation and migration of the sclerotome, PDGFR
1 cells begin to become distributed throughout the embryonal mesenchyme. During organogenesis, particularly intense staining is detected in regions of epithelial and mesenchymal interaction, such as the tooth bud and bronchi. In addition to mesodermal derivatives, the developing lens, apical ectodermal ridge, glial precursor, cardiac valves, and choroid plexus express PDGFR
. Our results with whole-mount immunostaining show that PDGFR
is abundantly expressed and may play important roles during embryogenesis. (J Histochem Cytochem 45:883-893, 1997)
Key Words:
PDGFR, mesoderm, somite, embryogenesis, whole-mount immunohistochemistry, mouse
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Introduction |
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Platelet-DERIVED GROWTH FACTOR receptor (PDGFR) is one of two receptors for platelet-derived growth factors (PDGF) and binds to all isoforms of PDGFs, PDGF-AA, PDGF-AB, and PDGF-BB (
in a variety of tissues throughout embryonic development and postnatal life suggest its roles in multiple processes such as gastrulation (
(
expression and defects in various tissues is implicated, this line of study has been hampered by difficulties in precisely localizing expression at higher resolution. Results of whole-mount in situ hybridization of the murine embryo have not been presented, although several studies have used this procedure in tissue sections. No monoclonal antibodies (MAbs) against murine PDGFRs have been produced, although several polyclonal Abs have been raised (
We therefore established an MAb that specifically recognizes PDGFR. Here we describe MAb APA5, which can be used for Western blotting, immunoprecipitation, immunostaining of tissue sections, whole mount immunostaining and the surface staining of living cells. Using this MAb, we investigated the immunolocalization of PDGFR
during murine embryogenesis. Most of the staining profiles obtained here were comparable to those of in situ hybridization analyses (
.
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Materials and Methods |
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Animals
C57BL/6 mice and Wistar rats were purchased from Japan SLC (Shizuoka, Japan). Ph/+ mutant mice were a gift from Dr. L.D. Shultz of the Jackson Laboratory and were maintained in our animal colony for a few generations. Females were paired with males overnight and vaginal plugs were checked in the following morning. The noon when a vaginal plug was detected was designated as day 0.5 of gestation (0.5 day postcoitum, 0.5 dpc).
Production of Anti-murine PDGFR Antibody
The production of anti-murine PDGFR MAb and its characterization by Western immunoblotting were previously described (
(
gene was inserted into CD4Rg (
and the Fc portion of human IgG1 was inserted into pEF-BOS (a gift from S. Nagata, Osaka Bioscience Institute) (
-human(h) IgG1 recombinant fusion protein was purified from the culture supernatant of the transfected COS1 cells using Procep-A (Bioprocessing; Princeton, NJ). The spleen cells from a Wistar rat that was immunized and boosted with this fusion protein were fused with X63.Ag8 cells as described (
-positive BALB/c-3T3 cell line (
Immunohistochemistry
Tissue fixation procedures were basically the same as described (
Whole-mount immunohistostaining was performed according to the protocol described by MAb APA5 (0.5 µg/ml) overnight at 4C, then washed five times in PBSMT each for 1 hr at 4C for the initial three washes and at RT for the final two. The primary antibody was developed by incubating 1 µg/ml HRP-conjugated anti-rat Ig antibody (Biosource; Camarillo, CA) overnight at 4C. After extensive washing with more than five exchanges of PBSMT, including the final 20-min wash in PBST (0.1% Triton X-100 in PBS) at RT, the specimens were soaked in PBST containing 0.05% NiCl2 and 250 µg/ml diaminobenzidine (Dojin Chem; Kumamoto, Japan) for 10-30 min, and hydrogen peroxide was added to 0.01%. The enzymatic reaction was allowed to proceed until the desired color intensity was reached, and the specimens were rinsed three or four times in PBST. Finally, the specimens were dehydrated in 100% methanol and stored at -20C until photographed.
Cell Preparation and Flow Cytometry
The fibroblast cell line BALB/c-3T3 was maintained in DMEM (GIBCO) containing 10% FCS (Bio Whittaker, lot no. 1M1250; Walkersville, MD). Exponentially growing BALB/c-3T3 cells on 10-cm tissue culture plates were harvested by an incubation in 10 mM ethylenediamine tetraacetic acid containing PBS for 10 min at 37C. Cells (5 x 105) were suspended in 50 µl of 0.1% BSA containing Hanks' solution (Nissui; Tokyo, Japan) (HANKS/BSA) and nonspecific binding of the MAb was blocked with 10 µl of normal mouse serum. The cells were then stained with FITC-conjugated APA5 in the presence or absence of either mPDGFR, mPDGFRß (unpublished), or mC-FMS (
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Results |
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Specificity of APA5
The specificity of APA5 was first tested by staining BALB/c-3T3 cells that expressed both PDGFR and PDGFRß. This staining was inhibited by the recombinant hIgG/PDGFR
but not by hIgG/PDGFRß (Figure 1A). APA5 staining was then compared with whole- mount specimens of the Ph/Ph and normal embryos from the same litter at 13.5 days of gestation. The Ph/Ph mouse bears a deletion in the region encoding the PDGFR
gene (
1 BALB/c-3T3 cells in Western blotting, but not from the CYG/SCID7 pro-B cell line (
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Immunolocalization of PDGFR in Presomitic Stages
Figure 2 shows a sectioned specimen of a 6.5 day old (E6.5) embryo before mesoderm induction. APA5 stained both the visceral and the parietal endoderm overlying the extraembryonic ectoderm, but not ectodermal cells. On generation of mesoderm cells in the posterior portion of the egg cylinder (E7.0), the embryonic mesoderm localizing the proximal posterior part of the embryo and ectoplacental cone was stained with APA5 (Figure 3A and Figure 3B). At this stage it was difficult to determine if all nascent mesodermal cells express PDGFR. In the E7.5 embryo (Figure 3C and Figure 3D), however, at this early head fold stage, the proximal lateral and extraembryonic mesoderm, except the allantois, were not stained with APA5, whereas the paraxial mesoderm was strongly positive. The staining in the ectoplacental cone extends as it develops, whereas only faint staining is detectable in the allantois. This staining distribution strongly suggests that not all mesodermal cells express PDGFR
protein in the E7.5 embryo.
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Immunolocalization of PDGFR During Somitogenesis
APA5+ cells were diffused throughout the paraxial region as it started to change into developing somites. Whole-mount immunohistostaining showed that diffuse staining in the anterior region became divided into the outer and inner regions (Figure 3C). The inner APA5+ region invaginated and extended forward under the outer region, and probably develops into head fold mesenchyme. At the same time, the APA5+ region in the trunk started to segregate and develop into the somites (Figure 4A and Figure 4B). Diffuse staining in the head mesenchyme continued. No APA5 staining was found in the neural fold except for the neural crest cells at prorhombomeres A and B (Figure 4A, arrowheads). After formation of the neural tube, APA5 stained the dorsal midline (Figure 4C, arrows), particularly at the head region.
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Immunolocalization of PDGFR During Early Organogenesis
In the E9-10 embryo (Figure 5A and Figure 5B), when only posterior somites were stained by APA5, only the basal part of anterior somites were APA5+. Owing to the formation of vertebrae and intervertebral discs, APA5 staining was concentrated in the latter. At the same stage, PDGFR immunolocalization in the anterior region was seen in the mesenchyme of the most anterior and basal parts of the head, branchial arch, and lens. In the trunk region, staining in the heart and basal part of the fore- and hindlimbs was most conspicuous. Transient expression of PDGFR
was detected also in the apical ectodermal ridge (AER) of the fore- and hindlimbs. In the posterior region, the splanchnic mesenchyme was strongly APA5+.
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Immunolocalization of PDGFR During the Later Stages of Organogenesis
In sagittal sections of E12.5-E16.5 embryos, PDGFR was diffusely immunolocalized in the connective tissue. At this stage, several areas were conspicuously stained. These included the mesenchyme around vertebrae (Figure 6B and Figure 7A), kidney (Figure 6C and Figure 7B), and thymus (Figure 6D and Figure 7C). During renal development, mesenchymal cells within the kidney expressed PDGFR
(Figure 1B and Figure 7B). Also notable was the mesenchyme staining surrounding the urethric tubes (Figure 1B and Figure 6C). The heart was markedly stained in cells covering valves and the pericardium (Figure 6D and Figure 7D). The trachea was also lined by APA5+ mesenchyme (Figure 6D). Within the trachea, stripes of APA5 staining were present in the regions where tracheal cartilage develops (Figure 1B). The developing lung was intensely stained around the developing bronchial tree (Figure 8C and D).
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In the head region, the most intense staining was noted in the mandibular and palate regions (Figure 1B, Figure 6A, and 8A). Although most APA5+ cells were present in the mesenchyme beneath the ectodermal surface, those surrounding the tooth bud were highly positive (Figure 8A). Although weakly stained, repetitive strings ran vertically in the muscle region of the tongue (Figure 8A and Figure 8B).
The nervous system was basically APA5-negative. However, the midportion of the spinal cord was faintly stained. A magnified view of this region in E12.5 embryo shows cells with dendrites (Figure 6E and Figure 7E). Similarly, an array of APA5+ cells was concentrated in the dorsal part of the eye, emanating downwards along with the presumptive optic nerve (Figure 6F and Figure 7F). These APA5+ cells represent glial precursors. Similar APA5+ regions were also seen in the central nervous system. In the cranial region, highly immunoreactive cells were found in the epithelial surface of the choroid plexus (Figure 8E and Figure 8F).
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Discussion |
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The aim of this study was to show the immunolocalization of PDGFR in murine embryos and to compare it with the result of in situ hybridization studies (
Table 1 compares our results with those of in situ hybridization. Basically, most regions that were PDGFR-positive by in situ hybridization were also positive by APA5 staining. However, the two methods gave entirely different results with respect to PDGFR
expression in E7.5 embryos. Studies by
1 from the onset of mesoderm formation during gastrulation. In contrast, whole-mount immunostaining of the E7.5 embryo with APA5 demonstrated that only the mesoderm in the paraxial region was positive, although a few APA5+ cells might present in the proximal lateral and extraembryonic mesoderm. Our results are in contrast to the conclusion by
transcripts and also to that of
antibody. APA5 hardly stained mesodermal layers of the extraembryonic tissues, including blood islands, whereas they were stained by anti-c-Kit MAb (data not shown). The allantois is the only notable APA5+ extraembryonic mesoderm in the gastrulating embryo, although the expression in E7.5 embryo was faint. In contrast, the ectoplacental cone was APA5+ from the pregastrulation stage and the stained area was extended. We concluded that most if not all cells of proximal lateral and extraembryonic mesoderm do not express PDGFR
protein, although the allantois expresses PDGFR
at later stages.
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In addition to PDGFR expression, several studies have suggested that Flk1 expression can be used to mark the nascent mesoderm (
It is difficult to explain the discrepancy in the results obtained by the two methods. Certainly, this discrepancy is due to inherent differences in the level of sensitivity between nucleic acid in situ hybridization and immunohistochemical techniques. Alternatively, in situ hybridization may detect untranslated PDGFR transcripts present in cells that do not express the PDGFR
protein. APA5 can block the binding of PDGFs to PDGFR
, thus recognizing its ligand binding site. Hence, all functional PDGFR
proteins on the cell surface should be detected by APA5, whereas truncated molecules without the ligand binding will not be recognized.
The other plausible possibility is that our whole- mount immunostaining result simply gives a clearer view of PDGFR expression in the whole embryo. In other words, which part of the embryo expresses more or less PDGFR
can be determined easily by whole-mount immunostaining. Thus, although it could be that all mesodermal cells express PDGFR
at a level that is above the detection sensitivity of in situ hybridization, regional differences in its expression level cannot be noted by the in situ hybridization of tissue sections.
It is important to emphasize that there was no significant discrepancy in the PDGFR expression profiles of later embryos determined by these two methods. Much has been discussed regarding the correlation between PDGFR
expression and phenotypes of the Ph/Ph mutant mice, which have a large deletion in the region containing the PDGFR
gene (
is involved in many events of organogenesis.
One important aspect of PDGFR that has not been addressed is its expression in the spinal cord of the E12.5 embryo. PDGFR
has been implicated in the development of glial precursors (
plays a role in this early process of glial cell differentiation. Likewise, the PDGFR
staining in the eye region of the same embryo supports the notion that timely expression of PDGFR
is important for glial cell differentiation and neuronal development.
Accepting the notion that PDGFR plays a diverse role in murine embryogenesis, the most important issue is to determine how it regulates the processes of organogenesis. APA5 is an antagonistic MAb that can be used to inhibit the function of PDGFR
both in vitro and in vivo. Therefore, studies to elucidate the function of PDGFR
in the formation of various organs are now in progress.
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Acknowledgments |
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Supported by grants from the Japanese Ministry of Education, Science and Culture (nos. 07CE2005, 07457085, and 06277103), by a grant from the Ministry of Science and Technology (no. 13073-2125-14), and by a grant from RIKEN.
We would like to thank Dr L. Shultz (The Jackson Laboratory) for providing the Ph/Ph mice.
Received for publication August 12, 1996; accepted December 30, 1996.
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