Basement membrane composition in the early mouse embryo day 7

Authors


Abstract

Basement membranes (BM) are specialized structures of the extracellular matrix known to be involved in various early developmental processes. Despite numerous investigations on the localization of BM components, it remains unknown which molecules are expressed in early developmental stages and by which germ layers these proteins are produced. Therefore, we tested for all known laminin chains, nidogens, collagen type IV, and perlecan by means of light microscopic immunostaining and performed in situ reverse transcriptase-polymerase chain reaction to detect the mRNAs specific for laminin α1, laminin β1, the α1 chain of collagen type IV, nidogen-2, and perlecan in the early mouse embryo, day 7, in vivo. Only the laminin chains α1, β1, and γ1 were detected immunohistochemically throughout the entire endodermal and ectodermal BM zones of the embryo proper. The mRNA of laminin α1, laminin β1, collagen type IV, nidogen-2 and perlecan were expressed in the ectoderm-derived mesoderm, in the endoderm as well as in the ectoderm. In contrast, Reichert's membrane was positive for all laminin chains except for the α4, α5, β3, and γ3 chains. Moreover, maternal epithelial as well as mesenchymal cells expressed laminins, nidogen-1 and nidogen-2, collagen type IV, and perlecan. In conclusion, laminin-1 might be the only laminin isoform in the early mouse embryo that, together with the other main BM components, nidogens, collagen type IV, and perlecan, is synthesized by all three germ layers. Developmental Dynamics 233:1140–1148, 2005. © 2005 Wiley-Liss, Inc.

INTRODUCTION

Basement membranes (BMs) are special structures of the extracellular matrix with multiple functions such as division of tissues into compartments, provision of structural support, as well as regulation of cell behavior (Timpl, 1996; Timpl and Brown, 1996). The formation of BMs is an important morphogenetic factor in embryonic development (Miner and Yurchenco, 2004), and the precise organization and stabilization of the major components into a stable supramolecular structure is probably a prerequisite for the appropriate biological activity of such membranes (Leivo et al., 1980; Dziadek and Timpl, 1985; Miosge et al., 1993; Timpl and Brown, 1996). Moreover, changes in the distribution of BM components seem to constitute a modulating factor in epithelial–mesenchymal interactions (Willem et al., 2002). Laminin-1 (Timpl et al., 1979), nidogen-1 (Carlin et al., 1981), nidogen-2 (Kohfeldt et al., 1998), collagen type IV (Kühn et al., 1995), and perlecan (Noonan and Hassell, 1993) have been identified as major components of BMs.

The laminins are heterotrimeric glycoproteins, each composed of one α, one β, and one γ chain. Currently, five α chains, three β chains, and three γ chains have been identified, which can form up to 15 different laminin isoforms (Engvall and Wewer, 1996; Koch et al., 1999; Libby et al., 2000; Tunggal et al., 2000). Laminins fulfill a large number of biological functions, e.g., tissue regeneration, cell migration, cell differentiation, cell proliferation, and cell adhesion (Ryan and Christiano, 1996; Timpl, 1996; Miner and Yurchenco, 2004).

Nidogen-1, also referred to as entactin, has an elongated shape and is composed of three globular domains, G1 to G3, connected by a flexible link and a rod (Carlin et al., 1981; Timpl et al., 1983; Fox et al., 1991; Mayer et al., 1995). Nidogen-1 shows a high in vitro binding activity to a single laminin-type epidermal growth factor-like module (γ1III4) on the laminin γ1 chain (Mayer et al., 1993). Nidogen-1 also contains a binding site for collagen type IV (Mayer and Timpl, 1994). It was shown that nidogen-1 mediates the formation of ternary complexes with laminins, perlecan, and collagen type IV (Brown et al., 1994; Mayer et al., 1995), thus stabilizing the BM and integrating other proteins. Nidogen-2 is a recently identified member of the nidogen family (Kohfeldt et al., 1998). The molecule shows 46% sequence identity with nidogen-1 and has a similar structure.

Collagen type IV is the most abundant constituent of the BM and belongs to the network-forming collagens (Kühn, 1995). With the help of cDNA sequencing, four additional α chains (α3, α4, α5, and α6) were isolated besides the α1 and α2 chains. Further isoforms with the composition 2α3[IV]1α4[IV] and 2α5[IV]1α6[IV] thus were identified (Hudson et al., 1993; Ninomiya et al., 1995).

Perlecan is a heparan sulfate proteoglycan that is expressed in almost all BMs, in cartilage, and in several other mesenchymal tissues during development (French et al., 1999; Hassell et al., 2002). Its core protein is 400 kDa in size and consists of five distinct structural domains (Noonan et al., 1991). Several in vitro studies suggest multiple functions for perlecan in cell growth and differentiation processes and in tissue organization. It also has been shown to bind BM proteins such as laminin isoforms, collagen type IV variants, nidogen-1, and nidogen-2 (Iozzo, 1994). The presence of perlecan in BMs and its abilities suggest that it may be involved in cell–matrix interactions and BM assembly (Rheinhardt et al., 1993; Hopf et al., 1999; Miosge et al., 2003).

The early mouse embryo, day 7, is an excellent model for the investigation of BM formation. Previous studies have demonstrated that laminin-1 and collagen type IV are found between the germ layers before a ultrastructural visible BM is seen. In contrast, nidogen-1 is only present in fully developed BMs (Miosge et al., 2000a, b).

Despite the wealth of information on BM components, a systematic localization of these molecules in the early mouse embryo is still not available. Therefore, we tested for all known laminin chains, nidogen-1, nidogen-2, collagen type IV, and perlecan. Furthermore, we detected the mRNA for laminin chains α1 and β1, nidogen-2, collagen type IV, and perlecan with the help of in situ reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate which cells produce these BM components in the mouse embryo on day 7.

RESULTS

Light Microscopic Immunohistochemistry for Each of the Laminin Chains, Nidogen-1, Nidogen-2, Collagen Type IV, and Perlecan in the Embryonic Tissue of the Mouse Embryo on Day 7

On day 7, consistent with Theiler stages 12,13 (1989), the mouse embryo has reached the stage of mesoderm formation and BM development has started. The mesodermal layer has started to develop between the ectoderm and the endoderm in the caudal (primitive streak) and the lateral regions of the transition zones between the extraembryonic and the embryonic tissues.

The endodermal BM zone was positive for laminin-1 (α1, β1, and γ1 chains; Fig. 1B–D). Also nidogen-1, nidogen-2 (Fig. 1A), collagen type IV, and perlecan were localized within the endodermal BM zone (Table 1). The ectodermal BM zone was stained for laminin-1 (α1, β1, and γ1 chains) as well as nidogen-1, nidogen-2, collagen type IV, and perlecan. The laminin chains α2, α3, α4, and α5, β2, and β3, γ2 (Fig. 1E), and γ3 were not found in the ectodermal and the endodermal BM zone. The cytoplasm of the cells of the endoderm, the mesoderm and the ectoderm were not stained for the laminin α chains (α1, α2, α3, α4, α5), the laminin β chains (β1, β2, β3), the laminin γ chains (γ1, γ2, γ3), nidogen-1, nidogen-2, collagen type IV, and perlecan.

Figure 1.

A–G: Light microscopic immunostaining for nidogen-2 (A), the laminin α1 chain (B), the laminin β1 chain (C), the laminin γ1 chain (D), the laminin γ2 chain (E) in a day 7 mouse embryo, the laminin β2 chain (F) in the decidua, and the laminin α4 chain in the maternal tissues (G). A: Staining for nidogen-2 in the basement membrane (BM) zones of the decidua capillaries (de), Reichert's membrane (open arrow), the extraembryonic BM zones, and within the embryo proper, between the ectoderm (open star) and endoderm (black arrow) and the mesoderm (asterisk). B–F: Higher magnifications of the staining for the laminin α1 chain (B), the laminin β1 chain (C), the laminin γ1 chain (D), the laminin γ2 chain (E), and the laminin β2 chain (F). Note the linear staining for Reichert's membrane (open arrows) and the BM zones between the ectoderm (open star) and the mesoderm (asterisk). No staining is seen for the laminin γ2 chain (E) in the BM zones between the ectoderm (open star) and the mesoderm (asterisk) or between the mesoderm (asterisk) and the endoderm (black arrow). G: Staining for the laminin α4 chain in the cytoplasm of the cells lining the decidua capillaries (asterisk). Scale bars = 140 μm in A; 30 μm in B–G.

Table 1. Light Microscopic Immunostaining for the Laminin Chains α1, α2, α3, α4, and α5; β1, β2, and β3; γ1, γ2, and γ3; Nidogen-1; Nidogen-2; the α1 Chain of Collagen Type IV; and Perlecan in the Embryonic Part and the Extraembryonic Part of the Mouse Embryo on Day 7 and in Maternal Tissuesa
Mouse embryo day 7 Laminin chainsnid-1nid-2α1 of col. IVPerlecan
α1α2α3α4α5β1β2β3γ1γ2γ3
  • a

    nid-1, nidogen-1; nid-2, nidogen-2; α1 of col. IV, the α1 chain of collagen type IV.

EmbryonicEndoderm
 Endodermal basement membrane+++++++
 Ectoderm
 Ectodermal basement membrane+++++++
 Mesoderm
ExtraembryonicEndoderm
 tissuesEndodermal basement membrane+++++++
 Ectoderm
 Ectodermal basement membrane+++++++
 mesoderm
Ectoplacental conus 
Decidua cells +
Basement membrane zones of the decidua capillaries++++++++++++
Reichert's membrane+++++++++++

Light Microscopic Immunohistochemistry for Each of the Laminin Chains, Nidogen-1, Nidogen-2, Collagen Type IV, and Perlecan in the Extraembryonic Tissue of the Mouse Embryo on Day 7

In the extraembryonic tissues, endodermal and ectodermal BMs had developed between the extraembryonic ectoderm and yolk sac cells. Laminin chains (α1, β1, and γ1) were localized over the entire extraembryonic endodermal BM zone (Fig. 1B–D). Nidogen-1, nidogen-2 (Fig. 1A), as well as collagen type IV and perlecan were also found within the extraembryonic endodermal BM zone (Table 1). The extraembryonic ectodermal BM zone was stained for α1, β1 and γ1 chains of laminin, but not for any of the other chains. Also nidogen-1 and nidogen-2, as well as collagen type IV and perlecan, were localized in the extraembryonic ectodermal BM zone. No staining for nidogen-1 or nidogen-2, or any other laminin chains was seen in the cytoplasm of the cells in the three germ layers. Furthermore, collagen type IV and perlecan were not detected in the cells of endoderm, mesoderm, and ectoderm at this developmental stage.

Light Microscopic Immunohistochemistry for Each of the Laminin Chains, Nidogen-1, Nidogen-2, Collagen Type IV, and Perlecan in the Maternal Tissues

In the present study, we also examined the maternal tissues that surround the embryo. The entire ectoplacental conus was neither stained for any of the laminin chains nor for nidogen-1, nidogen-2, collagen type IV, or perlecan (Table 1). With the exception of the laminin β2 chain (Fig. 1F), the decidua cells also did not show any staining for any of the laminin chains. Also nidogen-1 and nidogen-2, collagen type IV, and perlecan were not localized in the decidua cells. In contrast, the BM zones of the decidua capillaries were stained for the laminin chains α1, α2, α3, α4, and α5, β1, as well as γ1 and γ2. In addition, nidogen-1, nidogen-2, collagen type IV, and perlecan were localized within the BM zones of the decidua capillaries.

The laminin chains α1 (Fig. 1B), α2, α3, β1 (Fig. 1C), and β2 (Fig. 1F), γ1 (Fig. 1D), and γ2 (Fig. 1E), nidogen-1, nidogen-2 (Fig. 1A), as well as collagen type IV and perlecan were localized in Reichert's membrane. Reichert's membrane remained unstained for the laminin α4 and α5 chains, as well as for the laminin β3 chain and the γ3 chain. The BM zones of the capillaries of the decidua were positive for the laminin chains α1, α2, α3, α4 (Fig. 1G), and α5, β1, γ1 and γ2, nidogen-1, nidogen-2, collagen type IV, and perlecan.

In Situ RT-PCR for the mRNA of the Laminin α1 Chain, the Laminin β1 Chain, Nidogen-2, Collagen Type IV, and Perlecan in the Embryonic Part of the Mouse Embryo on Day 7

In the embryonic part, the endoderm was stained for the mRNA of the laminin α1 chain (Fig. 2B), the laminin β1 chain (Fig. 2C), nidogen-2 (Fig. 2A), as well as perlecan (Fig. 2D) and collagen type IV (Table 2). In addition, the ectoderm as well as the mesoderm were positive for the mRNA of the laminin α1 chain and the laminin β1 chain, nidogen-2, collagen type IV, and perlecan.

Figure 2.

A–G: In situ reverse transcriptase-polymerase chain reaction (RT-PCR) for the mRNA of nidogen-2 (A), of the laminin α1 chain (B), of the laminin β1 chain (C), of perlecan (D) in a mouse embryo on day 7, negative control (E), and in the maternal tissues (F,G). A: Staining for the mRNA of nidogen-2 in the parietal endoderm of Reichert's membrane (open arrow), the extraembryonic tissue, and within the embryo proper, between the ectoderm (open star) and endoderm (black arrow) and the mesoderm (asterisk). B: Staining for the mRNA of laminin α1 in the ectoderm (open star), mesoderm (asterisk), endoderm (black arrow). C: Localization of the mRNA of the laminin β1 chain in the embryonic tissues, ectoderm (open star), mesoderm (asterisk), endoderm (black arrow). D: Staining for the mRNA of perlecan in the extraembryonic tissues. E: Negative control, ectoderm (open star), mesoderm (asterisk), endoderm (black arrow), and the Reichert's membrane (open arrow). F,G: Staining for the mRNA of the α1 chain of collagen type IV (F), and for the α1 chain of laminin in the cytoplasm of the cells lining the decidua capillaries (G, asterisk). Scale bars = 140 μm in A, 30 μm in B–G.

Table 2. mRNA Detection of Laminin α1, Laminin β1, Nidogen-2, Collagen Type IV and Perlecan with the Help of In Situ RT-PCRa
Mouse embryo day 7 Laminin chainnid-2col. IVPerlecan
α1β1
  • a

    nid-2, nidogen-2; col. IV, collagen type IV; RT-PCR, reverse transcriptase- polymerase chain reaction.

Embryonic tissuesEndoderm+++++
 Ectoderm+++++
 Mesoderm+++++
 Endoderm+++++
Extraembryonic tissuesEctoderm+++++
 Mesoderm+++++
Ectoplacental conus +++++
Decidua cells 
Cells lining the decidua capillaries +++++
Parietal endodermal cells +++++

In Situ RT-PCR for the mRNA of the Laminin α1 Chain, the Laminin β1 Chain, Nidogen-2, Collagen Type IV, and Perlecan in the Extraembryonic Part of the Mouse Embryo on Day 7

The mRNA of the laminin chains α1 and β1, nidogen-2 (Fig. 2A), collagen type IV, and perlecan were localized in the extraembryonic part of the endoderm (Table 2). In addition, the laminin chains α1 and β1, nidogen-2, collagen type IV, and perlecan were expressed in the extraembryonic part of the ectoderm and the mesoderm.

In Situ RT-PCR for the mRNA of the Laminin α1 Chain, the Laminin β1 Chain, Nidogen-2, Collagen Type IV, and Perlecan in the Maternal Tissues

In the surrounding maternal tissues, we localized the mRNA of the laminin α1 chain, the laminin β1 chain, nidogen-2, collagen type IV, and perlecan in the ectoplacental conus (Table 2). The decidua cells were not stained for the mRNA of laminin α1, laminin β1, nidogen-2, collagen type IV, or perlecan. The mRNA of the laminin α1 chain (Fig. 2G), the laminin β1 chain, nidogen-2 (Fig. 2A), collagen type IV (Fig. 2F), and perlecan were expressed in the cells lining the decidua capillaries. The parietal endodermal cells were stained for the mRNA of laminin α1, laminin β1, nidogen-2, collagen type IV, and perlecan.

DISCUSSION

In this study, we applied well-characterized and frequently used polyclonal antibodies to perform a routine peroxidase–antiperoxidase staining (Miosge et al., 2000a) to localize all known laminin chains, nidogen-1 and nidogen-2, as well as collagen type IV and perlecan in the mouse embryo, day 7, and in the maternal tissues at the light microscopic level. In addition, we applied in situ RT-PCR to detect even the smallest amounts of mRNA of laminin α1, laminin β1, nidogen-2, the α1 chain of collagen type IV, and perlecan in tissue sections. The laminin α1 chain first appears in the mouse 16-cell stage (Cooper and MacQueen, 1983), and, in the course of embryonic development, the mRNA of the α1 chain can be localized still in fairly low amounts (Klein et al., 1990). In adult mice, laminin α1 can only be seen in a few epithelial tissues (Falk et al., 1999). The laminin β1 chain is the first chain appearing in the embryonic development of the mouse. On the mRNA level, the β1 chain can already be localized in the two-cell stage (Shim et al., 1996), as is the protein itself (Dziadek and Timpl, 1985). The laminin β1 chain is expressed throughout organogenesis. For example, in early human glomerular development, it is a prerequisite and only in the late developmental stages is the β1 chain replaced by the laminin β2 chain (Virtanen et al., 1995). The existence of the mRNA of laminin γ1 was already described in the two- to four-cell stage (Dziadek and Timpl, 1985). Laminin γ1 knockout mice are not able to form BMs (Smyth et al., 1999), and its absence causes early embryonic death. This finding means that, in early murine embryonic development, laminin γ2 and laminin γ3 cannot compensate for the absence of laminin γ1 and, indeed, we did not detect these γ chains in the mouse embryo, day 7. In contrast, in the maternal tissues, for example the BM zones of the decidua capillaries, we localized all of the laminin chains except β2, β3, and γ3. In vitro–cultivated parietal endodermal cells synthesize laminin chains (Fowler et al., 1990). Here, we demonstrate that the mRNAs for the laminin α1 and β1 chains are found in the cytoplasm of the parietal endodermal cells in vivo and that the laminin chains α1–α3 are present in the Reichert's membrane, indicating that, for example, the laminin isoforms -1, -2, or -10 might be found in this structure.

We detected the α2 chain of laminin in the extraembryonic tissues of the mouse embryo and the γ2 chain in Reichert's membrane. This discrepancy to the results of Klaffky et al. (2001) could be due to the differing antibodies applied or the different processing of the tissues, as we have embedded them in paraffin. Also of concern are the differences in the localization of the α5 chain of laminin. We did not detect this chain in the embryo proper, whereas an elegant study (Miner et al., 2004) found this chain in the embryonic BM zones, proposing that laminin-10 might be a component of early BM. We embedded the embryos in paraffin and exposed them to formaldehyde for a long time, whereas Miner et al. (2004) used briefly fixed frozen tissues. It might be possible, that our tissue processing altered the structure of the α5 chain, so that one and the same antibody is no longer able to detect it. Furthermore, we detected some of the laminin chains in the blood vessels of the decidua. It should be noted that, during early formation of blood vessels in the deciduas, vessels are ensheathed by epithelium (trophoblast cells), which may express laminin α1, α2, and α3 chains. At the time of implantation, the epithelial trophectoderm cells transform into an invasive population, the trophoblast giant cells. These are the cells that mediate penetration of the embryo into the uterus and formation of connections with the maternal blood supply.

Collagen type IV appears on day 5 in embryonic murine development (Adamson and Ayers, 1979). On day 7, collagen type IV can be localized at the ultrastructural level (Herken and Barrach, 1985) in BMs and in Reichert's membrane. In this study, the mRNA of the α1 chain of collagen type IV can be localized in all three germ layers as well as in the parietal endodermal cells in the mouse embryo on day 7. Nevertheless, the collagen type IV isoform [α1(IV)]2α2(IV) is not a precondition for early embryonic BM formation, because α1 and α2 knockout mouse embryos die around day 9.5. Further development is prevented by deficits in the structure and integrity of the BMs and Reichert's membrane (Pöschl et al., 2004).

With the help of immunofluorescence, nidogen-1 was localized at the 8- to 16-cell stage (Dziadek and Timpl, 1985). In investigations at later embryonic stages (mouse embryos on day 11 and day 12.5), the mRNA of nidogen-1 was only localized in cells of mesenchymal origin (Thomas and Dziadek, 1993; Ekblom et al., 1994). In contrast, in early murine embryogenesis on day 7, the mRNA of nidogen-1 was localized in all three germ layers by in situ hybridization (Miosge et al., 2000a). In this study, the day 7 embryo shows a similar nidogen-2 distribution at the mRNA level in all three germ layers and in the parietal endodermal cells synthesizing Reichert's membrane. We assume that in early stages of embryogenesis both nidogens participate in BM formation, even though nidogen-2 exhibits a variable expression pattern in adult mice (Kohfeldt et al., 1998; Murshed et al., 2000). However, in adult murine kidney, both nidogens are colocalized with laminin-1 in the glomerular, tubular, and capillary BMs (Miosge et al., 2000b). Mice with a deletion of the nidogen-1 gene show no overt abnormalities and are fertile, and their BM structures appear normal at the ultrastructural level (Murshed et al., 2000), where nidogen-2 compensates for the absence of nidogen-1 (Miosge et al., 2002b). The same is true for nidogen-2 knockout mice (Schymeinsky et al., 2002), whereas nidogen-1/nidogen-2 double-knockout mice do exhibit some BM defects at the ultrastructural level later during fetal development (unpublished observations).

Perlecan can be localized already at the two-cell stage of murine development (Smith et al., 1997). It has been assumed that the early expression of perlecan is connected with the implantation of the embryo into the uterine wall (Carson et al., 1993). The mouse embryo on day 10.5 shows a high mRNA concentration of perlecan, especially in regions of the vessel formation, for example, in heart, pericardium and in the major blood vessels (Handler et al., 1997). During the implantation of the embryo, it seems that perlecan plays no limiting role, because perlecan knockout mice develop at least until day 9.5 (Costell et al., 1999). In this study, we localized perlecan in the embryonic and extraembryonic BM zones of endoderm and ectoderm in the mouse embryo on day 7. The perlecan mRNA was detected in all three germ layers as well as in parietal endodermal cells.

In summary, our immunohistochemical data show that the laminin chains α1, β1, and γ1 might be the main laminin chains seen in the mouse embryo on day 7; therefore, laminin-1 might be the only isoform present in the early mouse embryo. In addition, nidogen-1 and nidogen-2 are localized in the embryonic and extraembryonic BM zones in the early mouse embryo, as well as collagen type IV and perlecan. Furthermore, in situ RT-PCR demonstrates that these main BM components are synthesized by all three germ layers in the early mouse embryo on day 7.

EXPERIMENTAL PROCEDURES

Animals

Female NMRI mice were kept on a normal day/night cycle and received Altromin commercial food and water ad libitum. Day 0 of gestation was defined as starting at 11 hr on the day on which a vaginal plug was detected after a mating period of 3 hr.

Tissue Processing

On day 7 of gestation, the pregnant mice were anesthetized with ether and killed by cervical dislocation. After dissection of the uterine horns, the embryos were removed. Ten specimens were fixed in 4% paraformaldehyde in phosphate buffer pH 7.2 at 4°C overnight. They were then dehydrated in an ascending series of ethanol from 30% to 100% and embedded in paraffin. Serial paraffin sections of 5 μm were cut with a Reichert's microtome. All embryos were staged in comparison to the appropriate Theiler stages (Theiler, 1989).

Sources of Antibodies

The following primary antibodies were used for immunohistochemistry, and except for anti–collagen type IV and anti-laminin α3, they were all obtained from Prof. Dr. R. Timpl. Monoclonal rat anti-mouse antibody was prepared against the E1 and E8 fragments of murine laminin α1 (Miosge et al., 1995). For the α2 chain, a rabbit antibody against the mouse laminin fragments α2LG1 and α2LG4 was used (Talts et al., 2000). An anti-α3 antibody against domain I of the α3 chain (Champliaud et al., 1996) was kindly provided by Prof. Dr. R. E. Burgeson. An affinity purified rabbit anti-mouse antiserum against the two fragments of the laminin α4 chain, α4LG1-3 and α4LG4-5, was used for the laminin α4 chain (Talts et al., 2000) and an affinity purified antibody was used for the laminin α5 chain (Miner et al., 1995). An affinity purified antibody specific for laminin β1IV and laminin β2IV was used to detect the laminin β1 and β2 chains, respectively (Sasaki et al., 2002). The laminin β3 antibody was a kind gift of R. Timpl. A polyclonal rabbit anti-mouse antibody specific for the γ1 chain (Mayer et al., 1993) and a polyclonal antibody against the murine laminin γ2 chain were applied (Sasaki et al., 2001). The antibody against the murine laminin γ3 chain was a kind gift of R. Timpl. Antibody JF4 against murine recombinant nidogen-1 was used (Fox et al., 1991) as was an affinity-purified rabbit antibody against recombinant nidogen-2, which showed no cross-reactivity with nidogen-1 (Kohfeldt et al., 1998). Polyclonal antibody against murine collagen type IV (Andujar et al., 1985) was purchased from Quartett, Berlin, Germany. An affinity purified polyclonal antibody against the perlecan fragments IV-1 and IV-2 was applied (Hopf et al., 1999). The secondary antibody was an anti-rabbit or anti-rat swine IgG (Dakopats, Hamburg, Germany).

Light Microscopic Immunohistochemistry

Sections were deparaffinized, rehydrated, and rinsed for 10 min in phosphate-buffered saline (PBS) pH 7.2. Endogenous peroxidase was blocked by incubation in 3% H2O2 in methanol for 45 min in the dark. Each of the reaction steps was followed by rinsing for 10 min in PBS. Sections were pretreated for 5 min with 10 μg/ml protease XXIV (Sigma, Deisenhofen, Germany). The antibodies against the laminin chains α2 to α5, β1 to β3, and γ1 to γ3, nidogen-1, nidogen-2, collagen type IV, and perlecan were used at a dilution of 1:100 and the antibody against the laminin α1 chain at a dilution of 1:20 in PBS for 1 hr at room temperature. Before the incubation of the γ3 and α5 antibodies, sections were pretreated with Protex I (Quartett, Berlin, Germany), a urea-containing reagent. The secondary antibody (anti-rabbit or anti-rat swine IgG; Dakopats, Hamburg, Germany) was diluted 1:50 in PBS and applied, followed by the PAP complex (diluted 1:150 in PBS), each for 30 min at room temperature. Finally, the sections were treated with 3,3′-diaminobenzidine and counterstained with hematoxylin.

Controls

As negative controls, normal rabbit or rat IgG was used instead of the primary antibodies, at similar concentrations. No immunostaining was observed after this replacement.

Primers

The laminin α1 primers produce a 521-bp PCR product (Flores-Delgado et al., 1998). The primers for the laminin β1 chain (sense, 5′-GATAACTGTCAGCACAACACC-3′ and antisense, 5′-GTGAAGTAGTAACCGGACTCC-3′) produce a 563-bp fragment; for nidogen-2 (sense, 5′-GAATCTGATACCTACGCCCTCTTTCTCT-3′ and antisense, 5′-GTGGTCCTCC AGTCCTATCACATACTTC-3′) an amplification of 909 bp between the base 612 in exon 3 and the base 1521 in exon 4 with a genomic contamination of 4312 bp; for the α1 chain of collagen type IV (sense, 5′-CCAGCCTGGAGCTAAGGGAGA-3′ and antisense, 5′-TCCAGGTGTACCGGGAATGC-3′) an amplification of 700 bp between the base 1696 in exon 20 and 2396 in exon 25; and for perlecan (sense, 5′-ACCTTCGCTGGCTCAAGGAG-3′ and antisense, 5′-CTCGGGGCGAACTGATGGTA-3′) an amplification of 614 bp between the base 8829 in exon 61 and the base 9443 in exon 65. They were all designed by primer 3 shareware.

In Situ RT-PCR

To test the primers, we performed RT-PCR with the RNA derived from day 7 mouse embryos. For the in situ RT-PCR, tissue sections were deparaffinized, rehydrated, and treated for 5 min with proteinase K (10 μg/ml) and rinsed in distilled water and 100% ethanol under DNAse-free conditions. Treatment with RNAse-free DNAse (desoxyribonuclease I, Roche Diagnostics, Mannheim, Germany) in DNAse buffer and rinsing followed. The sections were covered with 50 μl of the RT-PCR reaction mix (5 units rTth DNA polymerase [Perkin Elmer, Weiterstadt, Germany], 2.5 mM Mn(OAc)2, 1× EZ buffer, dNTPs, and primer as stated above) sealed under Ampli-Cover-Discs and –Clips, and placed in the Thermocycler 1000 (Perkin Elmer, Weiterstadt, Germany). The cycling began with a 60-min hold at 62°C for the transcription and a 7 min hold at 93°C, followed by 40 cycles of 90 sec at 93°C, 90 sec at 63°C, 120 sec at 68°C, and a final hold for 7 min at 68°C. Thereafter, the discs and clips were removed and the sections were rinsed in TBS. A second reaction mix was prepared as follows: 5 units of IS Taq polymerase (Perkin Elmer, Weiterstadt, Germany), 0.3 μl of digoxigenin-11-UTP (Roche, Mannheim, Germany), 1× PCR buffer II, and 3 mM MgCl2. The dNTPs and the primers were identical to those listed above. This PCR reaction mix was pipetted onto the tissue sections. The slides were then cycled for 3 min at 93°C, 2 min at 55°C, and a final hold for 2 min at 58°C. The detection of the labeled cDNA was performed as described before (Miosge et al., 2002a) and leads to a blue stain of the amplified DNA. Counterstaining was carried out with nuclear fast red (Merck, Germany).

Controls

Negative controls were performed for each reaction by either omitting primers, enzymes, or digoxygenin-11-dUTP. All controls showed no results.

Acknowledgements

We thank the late Rupert Timpl for the antibodies, Christina Zelent for technical assistance, and Cyrilla Maelicke, B.Sc., for editing the manuscript. Parts of our work were taken from the doctoral theses of Stephan Hübner and Ralf Poschadel.

Ancillary