ARTICLE |
Correspondence to: Nicolai Miosge, Abteilung Histologie, Kreuzbergring 36, 37075 Goettingen, Germany.
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Summary |
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Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229237, 2000)
Key Words: mouse embryo Day 7, nidogen-1, laminin-1, collagen Type IV, immunogold histochemistry, in situ hybridization
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Introduction |
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Basement membranes and their main components, laminin-1, collagen Type IV, and nidogen-1, fulfill many cell biological functions (
Nidogen-1 (150 kD), also referred to as entactin, is a small rod-shaped molecule with three globular domains (G1, G2, G3) connected by a flexible link and a rod (1III4 on the laminin
1-chain (
Only a few ultrastructural studies have been performed thus far, and these were in adult tissues (
The early mouse embryo Day 7 is a suitable model for the investigation of basement membrane formation. The first well structured inner embryonic basement membrane appears between the endo- and ectoderm at a site in the embryo proper at which a mesoderm is not yet present (
Therefore, we localized nidogen-1 and its binding partners, laminin-1 and collagen Type IV, in a fully developed basement membrane, a disintegrated membrane, and a membrane that was just starting to develop. Moreover, we identified the cells that express nidogen-1 at this developmental stage.
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Materials and Methods |
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Animals
Female NMRI mice were kept on a normal day/night cycle and received Altromin commercial food and water ad libitum. The day on which at 1100 hr a vaginal plug was detected after a mating period of 3 hr was designated as Day 0 of gestation.
Tissue Processing
On Day 7 of gestation, the pregnant animals were sacrificed by cervical dislocation and the embryos were removed from the uterus. For light microscopy, 10 specimens were fixed in 4% paraformaldehyde in phosphate buffer, dehydrated, and embedded in paraffin (
Paraffin sections (5 µm) and semithin (1 µm) and ultrathin (0.08 µm) sections were cut according to procedures previously described in detail (
Sources of Antibodies
An affinity-purified monoclonal antibody JF4 (1-chain in immunoblots. Ammonium sulfate precipitation resulted in IgG which was diluted in PBS to a concentration of 1 mg/ml (
In Situ Hybridization at the Light and Electron Microscopic Levels
An Acc I/Bgl II fragment of mouse nidogen-1 (X14480;
Semithin sections for the final light microscopic silver enhancement procedures were pretreated in Lugol's solution and 4% sodium thiosulfate, then incubated with the hybridization solution (50% formamide, 5 x SSC, 0.1 mg/µl yeast tRNA) for 16 hr at 50C. The RNA concentration for nidogen-1 was 100 ng digoxigenin-labeled antisense probe in 100 µl hybridization solution per section. In control sections, an equivalently labeled amount of sense RNA was used instead of antisense RNA. Posthybridization treatment included washes with 1 x SSC (twice for 15 min at 50C), 1 x SSC (twice for 15 min), 0.1 x SSC (four times for 15 min), both at 60C, and finally PBS for 15 min at room temperature (RT). Specimens were then incubated with a 1:60 dilution of anti-DIG (sheep IgG; Quartett) gold-labeled antibody in PBS for 1 hr at RT (
For electron microscopic in situ hybridization, the grids were pretreated with PBS for 15 min at RT and then incubated with the hybridization solution for 16 hr at 45C. The RNA concentration for nidogen-1 was 200 ng digoxigenin-labeled antisense probe in 20 µl hybridization solution per section. Posthybridization treatment was the same as described above. Specimens were then incubated for 40 min with a 1:60 dilution of gold-labeled anti-DIG antibody at RT (
Light Microscopic Immunohistochemistry
Sections were deparaffinized and rehydrated, and endogenous peroxidase was blocked. They were then treated with 0.01% protease XXIV (Sigma; Deisenhofen, Germany) for 5 min at RT. Each step was followed by a rinse in PBS. Primary antibodies (anti-laminin-1 and anti-nidogen-1 diluted 1:50 in PBS; anti-collagen Type IV 1:100) were incubated for 1 hr at RT. Bridge antibody (anti-rabbit or anti-rat swine IgG; Dakopatts, Hamburg, Germany) was applied (diluted 1:50 in PBS), followed by the PAP complex (diluted 1:150 in PBS), each for 30 min at RT. Finally, the sections were treated with 3,3'-diaminobenzidine and counterstained with hematoxylin. Negative controls were performed by applying normal rabbit or rat serum instead of the primary antibodies, and these remained unstained.
Electron Microscopic Immunohistochemistry
Sixteen-nanometer gold particles were prepared according to
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Results |
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Light Microscopic Immunohistochemistry for Nidogen-1, Laminin-1, and Collagen Type IV
All three components were detected in the mouse embryo Day 7 in basement membrane zones of the maternal blood vessels, Reichert's membrane, extraembryonic, and basement membrane zones of the embryo proper, shown here for collagen Type IV (Figure 1A). At a higher magnification of all three germ layers, linear staining for collagen Type IV was seen in basement membrane zones between the ectoderm and the mesoderm and less intensely between the mesoderm and the entoderm (Figure 1B). A similar staining pattern was also found for nidogen-1 (Figure 1C) and laminin-1 (Figure 1D).
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Ultrastructural Localization of Nidogen-1, Laminin-1, and Collagen Type IV
For the localization of the proteins in the basement membranes, the ultrastructural method was chosen to allow an unequivocal identification of deposits in these structures. Again, staining for nidogen-1 was seen in Reichert's membrane (Figure 2A) still surrounding the mouse embryo at this early developmental stage.
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In the extraembryonic part of the egg cylinder, a basement membrane had developed between the extraembryonic ectoderm and the yolk sac cells. Staining for nidogen-1 revealed diffusely distributed gold labeling over the entire extraembryonic basement membranes (Figure 2B). Within the embryo proper, basement membrane development had started in the embryonic part of the egg cylinder. At this stage of development, the embryonic basement membrane was characterized by the appearance of electron-dense extracellular matrix between the ectodermal and endodermal cell layers, giving the impression of a thick basement membrane that was positive for nidogen-1 (Figure 2C). In the caudal (primitive streak) and the lateral regions of the transition zone between the extraembryonic and the embryonic part of the egg cylinder, the mesodermal layer had started to develop between the ectoderm and the endoderm. In this area, the basement membrane, which is later disrupted by mesodermal cells that migrate from the embryonic ectoderm into a cavity formed between the ectodermal and endodermal layer (mesodermal space), was also stained for nidogen-1. In contrast, the basement membrane remnants of the disrupted basement membrane carried along the surface of the migrating mesodermal cells (
Staining for laminin-1 was seen in Reichert's membrane (Figure 2F) and in all of the basement membranes described above, shown here for the basement membrane between the endo- and ectoderm (Figure 2G) and the ecto- and mesoderm (Figure 2H). Laminin-1 was detected in the basement membrane starting to develop adjacent to the endoderm. The same staining pattern was found for collagen Type IV. Staining was again seen in Reichert's membrane (Figure 2I) and in the basement membranes of the embryo proper, shown here for the ectodermal basement membrane (Figure 2J).
Nidogen-1 mRNA Expression and Localization
To reveal which cells in the early mouse embryo produce nidogen-1, we performed in situ hybridization at the light and electron microscopic levels.
At the light microscopic level, nidogen-1 mRNA was found in the mouse embryo Day 7 in the cytoplasm of the cells of the mesoderm (Figure 3A). As expected, the nuclei of the cells were not stained. Furthermore, the cytoplasm of the cells of the other two germ layers, the endoderm and the ectoderm, exhibited labeling for nidogen-1 mRNA (Figure 3B). In addition, the cytoplasm of the parietal endoderm cells secreting Reichert's membrane were positive for nidogen-1 mRNA (Figure 3B). The control hybridizations with sense probes for nidogen-1 revealed no labeling in any of the described structures (Figure 3C).
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Electron microscopic in situ hybridization confirmed the staining of the cytoplasm of the cells of the three germ layers and revealed the strongest labeling in the cytoplasm of the mesoderm (Figure 3D). As for all hybridizations performed, the corresponding sense probe revealed only sparse labeling, regarded as nonspecific background (Figure 3E). In addition, the cytoplasm of the ecto- and endoderm (Figure 3F) was positive for nidogen-1 mRNA. Again, the corresponding hybridization with the sense probe revealed a low labeling intensity, regarded as nonspecific background (Figure 3G).
Because of the very high labeling intensity seen for nidogen-1 mRNA in all three germ layers and the fact that nidogen-1 mRNA has previously been detected mainly in mesenchymal cells, we performed several control experiments. Hybridization for nidogen-1 was carried out on EHS tissue (an epithelial tumor), the previous primary source of nidogen-1 (
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Discussion |
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Nidogen-1 is produced by mesenchymal cells during fetal mouse development (
We found strong expression of nidogen-1 mRNA in the developing mesodermal cells, and the cells of the endo- and ectoderm also produced nidogen-1 mRNA. This supports the emerging concept that although nidogen-1 is predominantly a product of mesenchymal origin (
One of the major functions of nidogen-1 is its strong interaction with laminins, giving rise to further interaction sites for basement membrane assembly (1-chain of laminin. These mice develop no basement membranes at all and die early during embryogenesis (
Only a few authors have investigated basement membrane components in the early mouse embryo (
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Acknowledgments |
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Supported by a grant from the German Research Council (DFG) to N. Miosge (Mi 573/1-2).
We wish to thank Dr Rupert Timpl, MPI for Biochemistry, Martinsried, for the kind gifts of cDNA, and Cyrilla Maelicke, BSc, for editorial correction of the manuscript. Parts of the work were taken from the doctoral thesis of Tobias Fahl.
Received for publication June 9, 1999; accepted October 7, 1999.
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