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Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs is already available, a systematic localization of the laminin β1 and β2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and β2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin β1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin β2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.
The formation and maintenance of normal epithelial organization requires a basement membrane (Ekblom 1989; Virtanen et al. 1995a, b). Basement membranes are thin, specialized structures surrounding and separating layers of cells from different lineages. Basement membranes first appear during the blastocyst stage, between the primitive endoderm and the inner cell mass (Timpl and Brown 1996). They are the earliest extracellular matrices (ECM) produced during embryogenesis (Graham and Lehtonen 1979). At this stage, all main basement membrane components are synthesized, including laminin, nidogen, perlecan and collagen type IV (Leivo et al. 1980). Although basement membranes at various body sites are similar on the ultrastructural level (Merker 1994), there is evidence for their organ-specific heterogeneity (Ljubimov et al. 1995; Hallmann et al. 2005; Roediger et al. 2009). Though the molecular basis for such heterogeneity is not yet fully defined, many studies have shown organ-specific expression of the main basement membrane components, such as laminins (Glukhova et al. 1993; Virtanen et al. 1995a, b; Otonkoski et al. 2008).
The laminin family of glycoproteins is a major constituent of the basement membrane (Timpl 1996). During metazoan embryogenesis, laminins are required for basement membrane assembly and cell polarization, and have subsequent effects on cell survival and tissue organization (Li et al. 2003). In addition to their role in the assembly and architectural integrity of the basement membrane, laminins interact with cells to influence proliferation, differentiation, adhesion, and migration processes. In mammals, there are at least 15 laminins, each an αxβyγz glycoprotein heterotrimer derived from five α, three β, and three γ subunits (Sasaki and Timpl 1999; Colognato and Yurchenco 2000; Libby et al. 2000). In 2005, a new heterotrimer identification system was introduced, using three Arabic numerals based on the α, β, and γ chain numbers (Aumailley et al. 2005). The earliest-expressed laminins during mammalian embryogenesis are laminin-111 and laminin-511, which show widespread expression and are crucial during mammalian embryonic development (Li et al. 2003). These two laminins mostly overlap in developmental expression and function, but only laminin-111 appears to be essential during the periimplantation period (Smyth et al. 1999).
We have previously shown that perlecan is present during embryonic and fetal human development, particularly at those stages of human development when epithelial–mesenchymal interactions occur (Roediger et al. 2009). In this work, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and laminin β2 chains in various developing embryonic and fetal human organs between gestational weeks (gw) 8 and 12. Up to now, distribution of the laminin β1 chain during human embryogenesis has generally been examined during late stages of fetal development, i.e., from gw 12 onwards. During this later stage, there is evidence for expression in the kidney (Virtanen et al. 1995a), several parts of the nervous system (Jaakola et al. 1993), pancreas, heart and skin (Iivanainen et al. 1995). However, laminin β1 chain expression in skeletal muscle (Wewer et al. 1997) and smooth muscle cells in the aorta and intestine (Glukhova et al. 1993) could only be found during gw 10. Laminin β2 chain expression was detected in the choroid plexus, skin, heart and pancreas during gw 17–19 (Iivanainen et al. 1995) and was shown not to be expressed before gw 9 in the lens capsule of the eye (Byström et al. 2006).
Aborted human embryos and fetuses were obtained according to the regulations of the Ethics Committee of the Medical Faculty of the University of Goettingen, Germany. The cut-off point for the differentiation between embryos and fetuses has been defined as ranging from gw 8 to 9 (Moore 1982). We investigated three embryos of gestational week gw 8, three fetuses of gw 10, two fetuses of gw 11 and two fetuses of gw 12. The ages were determined according to Carnegie stages (O’Rahilly and Müller 1987). No malformations or abnormalities were observed in these specimens. The abortion procedure leads to the destruction of much of the embryonic tissue; our results are, therefore, limited to those tissues we were able to follow to completion.
The abortion material was transported to the laboratory in a histidine-tryptophane-ketoglutarate solution at 4°C to ensure good preservation of the tissues (Bretschneider 1980; Koelling et al. 2006) and were fixed in 3.7% paraformaldehyde in phosphate buffer, pH 7.2, at 4°C for 24 h. The tissues were then serially dehydrated in ethanol from 30 to 100% and embedded in paraffin according to standard protocols (Gersdorff et al. 2005). Serial sections of 5 μm thickness were cut with a Reichert’s microtome. Every fifth section was stained with hematoxylin and eosin (HE) for topological orientation within the anatomical regions examined.
Rat monoclonal antibodies against the laminin β1 chain were purchased from Chemicon International (Temecula, USA; catalogue number: MAB1928). An affinity purified rabbit anti-mouse antiserum specific for laminin β2IV was used to detect the laminin β2 chain and has been shown not to cross-react with the β1 chain (Sasaki et al. 2002). The secondary antibodies used for the light microscopic immunostaining were purchased from Dakopats (Hamburg, Germany).
Immunoperoxidase staining was performed on paraffin-embedded tissue sections as follows: Tissues were deparaffinized, rehydrated and rinsed for 10 min in phosphate buffered saline (PBS), pH 7.2. Endogenous peroxidase was inhibited with a solution of 3% H2O2 in methanol for 45 min in the dark (Koelling et al. 2006). The sections were pretreated for 5 min with 10 μg/ml protease XXIV (Sigma Deisenhofen, Germany). The antibodies against the laminin β1 and β2 chains were used at a 1:100 dilution in PBS/BSA 1% and incubated for 1 h at room temperature. The tissues were rinsed in PBS for 10 min following each of the steps. The secondary antibody was applied at a 1:50 dilution in PBS/BSA. A standard peroxidase-antiperoxidase procedure followed, applying a peroxidase-coupled goat-anti-rabbit antibody (Dako, Hamburg, Germany) at a dilution of 1:150 in PBS for 30 min at room temperature. The sections were counterstained with hematoxylin. Negative controls omitted the primary antibody.
A list of our results regarding the tissue distribution of the laminin β1 chain is given in Table 1 and Fig. Fig.1.1. Comparative findings regarding the tissue distribution of the laminin β2 chain are shown in Table 2 and Fig. Fig.2.2. Results for the developing human eye are mentioned in text below and shown in Fig. 3.
The choroid plexus consists of many capillaries and is the part of the ventricular system where cerebrospinal fluid is produced. The invagination of the choroid plexus in the developing telencephalon starts at gw 5 (Sadler 2005). We found the laminin β1 and laminin β2 chains in basement membrane zones (BMZs) and mesenchymal cells of choroid plexus at gw 8 and 9. BMZs of the neuroectoderm and BMZs of capillaries stained positive for both laminin chains from gw 8, whereas no staining was detected for either laminin chain in neuroectodermal cells.
The laminin β1 and β2 chain expression was found in the endoneurium of peripheral ganglia and BMZs of capillaries from gw 8 onwards. Neuronal cells were negative for the laminin β1 and laminin β2 chains at all stages examined.
The differentiation of angioblasts into endothelial cells begins in the visceral mesoderm at gw 3 (O’Rahilly and Müller 1996). Endothelial BMZs of arteries and veins, as well as BMZs of capillaries, between gw 8 and 12 stained positive for the laminin β1 chain.
The development of the liver is initiated by the budding of entodermal cells from the distal end of the foregut (O’Rahilly and Müller 1996). The laminin β1 chain was detected in BMZs from sinusoids from gw 8 to 12. Hepatocytes did not express the laminin β1 chain before gw 10. The presence of the laminin β1 chain was detected at gw 12.
During kidney development, three renal systems evolve: the pronephros, the mesonephros and the metanephros (Saxen and Sariola 1987). The Bowman’s capsule and BMZs of the glomeruli and tubules stained positive for the laminin β1 chain at all developmental stages examined, whereas mesenchymal cells exhibited no expression of the laminin β1 chain.
Skin development starts from a separation of the ectodermal and mesodermal compartments. The epidermis derives from the ectoderm, whereas the dermis derives from the mesoderm (Smith and Holbrook 1986). In BMZs of dermal-epidermal junctions and capillaries, the laminin β1 chain was expressed from gw 8 onwards, and positive staining for the laminin β2 chain could be shown during gw 11. Cells of the loose mesenchyme were negative for the laminin β1 and β2 chain staining at all developmental stages examined.
BMZs of the developing exocrine gland were positive for the laminin β1 chain from gw 8 onwards. Furthermore, BMZs of glandular capillaries were positive for the laminin β1 chain staining, whereas epithelial cells exhibited no staining for the laminin β1 chain.
The early foregut forms by gw 4. The musculature and connective tissues of the gastro-intestinal tract derive from the surrounding mesenchyme (O’Rahilly and Müller 1996). In the developing intestine, the laminin β1 chain was detected in BMZs of capillaries and in the mesenchyme from gw 8 onwards. The laminin β1 chain was absent in epithelial cells of the intestine at all stages investigated.
The early heart anlage consists of two compartments separated from each other by the cell-free, homogeneous cardiac jelly (CJ): the endocardial tube and the developing myocardial layer, both already present at gw 4. The CJ also surrounds individual myocytes (Wenink 1976). The laminin β1 chain was first detected at gw 8 in BMZs of the endo- and the pericardium, as well as BMZs of capillaries, and was present until gw 12. Developing cardiomyocytes were negative for the laminin β1 chain staining, but the ECM of the cardiomyocytes revealed positive staining.
Additionally, the expression of the laminin β2 chain during gw 8 and 9 had a similar pattern as the distribution of the laminin β1 chain during the same period.
The laminin β1 chain is present in developing bone. In cartilage, we found the laminin β1 chain from gw 10 onwards, whereas the detection of the laminin β2 chain was limited to gw 8 and 9, another difference between the embryonic distribution of these two laminin chains. BMZs of the skeletal muscle cells were positive for staining of the laminin β1 chain from gw 10 to 12, whereas the laminin β2 chain was not detected.
The laminin β1 chain was detected in the lens capsule of the developing eye from gw 10 to 12. The laminin β2 chain was only detected in the lens capsule during gw 10.
Laminin is the first extracellular matrix protein to appear during embryonic development, first detectable in the 16-cell (morula) embryo (Leivo et al. 1980). From the blastocyst stage onwards, laminin or its subunits are required for development, maintenance of cellular polarity and compaction of the cells (Timpl et al. 1983; Smyth et al. 1999).
An overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs (gw 17–19) has been provided by Iivanainen et al. (1995). In organs like the eye, choroid plexus, kidney, pancreas and skin, both chains showed similar expression patterns. We identified the laminin β1 and β2 chain proteins in the endocardium of the heart and in the choroid plexus. Additionally, laminin β1 chain mRNA was detected in the dermis of the skin and endothelial cells of blood vessels (Iivanainen et al. 1995). We found that BMZs of these tissues contain the laminin β1 chain protein. These findings support the idea that the structural components of the subepithelial basement membrane are produced both by the epithelial cells and by stromal fibroblasts of the upper dermis (Iivanainen et al. 1995).
Localization of laminin β1 chain mRNA to the glomeruli of the kidney could not be demonstrated, but we found positive staining for the protein in the relevant BMZs. Miner (1998) highlighted the important role of the laminin chains β1 and β2 during the development of the kidney. In the maturing glomeruli of wild type mice, the laminin β2 chain is gradually replaced by the laminin β1 (Noakes et al. 1995). This is in agreement with our results, which show the presence of the laminin β1 chain in BMZs of glomeruli and tubuli in the developing kidney from gw 8 to 12 during human development.
We found the laminin β1 chain in the lens capsule of the eye between gw 10 and 12, and the laminin β2 chain at gw 10, as did Byström et al. (2006) up to gw 20. Furthermore, another study demonstrated that laminin β1 is essential for BM integrity in the zebrafish eye (Lee and Gross 2007).
We have previously shown that perlecan is present during embryonic and fetal human development in BMZs of blood vessels and capillaries in various organs (Roediger et al. 2009). Now, we present evidence for a similar localization of the laminin β1 chain from embryonic gw 8 onwards in BMZs of endothelial cells of blood vessels. The laminin β1 chain has been described in human smooth muscle cells of fetal developing vasculature and intestine up to gw 22. Based on these results, an important role for laminins in the maintenance of differentiated smooth muscle cells has been postulated (Glukhova et al. 1993). Additionally, we found positive staining for the laminin β1 chain in BMZs of capillaries and in the mesenchyme from gw 8 to 12, demonstrating the presence of this protein in the developing embryonic and fetal gut. Another study showed the omnipresent localization of the laminin β1 chain in all BMZs of human adult gastric mucosa (Virtanen et al. 1995b).
In skeletal muscle cells, we found the laminin β1 chain in BMZs between gw 10 and 12, whereas the presence of the laminin β2 chain could not be shown before gw 15 (Wewer et al. 1997). This indicates that the laminin β2 chain is only present in later stages of skeletal muscle development.
Previous studies demonstrated the involvement of laminin fragments in the development of cartilage. In human fetal cartilage (gw 17 and 24), a strong pericellular immunohistochemical reaction for laminin-111 was shown (Dürr et al. 1996). In embryo chick sternum and mouse limb bud, laminin β1 and β2 chains are present in the cytoplasm of chondrocytes (Lee et al. 1997). We found laminin β1 present in cartilage from gw 10 onwards, but not during gw 8 and 9. This suggests that the laminin β1 chain does not play a role in human cartilage development until the fetal stage. According to the results of our study, the laminin β2 chain could only be detected during gw 8 and 9, indicating a laminin β switch in which laminin β2 is eventually replaced by laminin β1 during cartilage development, as has been demonstrated in the glomeruli of the kidney (Noakes et al. 1995).
In our current work, laminin β1 and β2 chains were detected in various neuronal tissues. The first laminin subunits detectable within the human nervous system are β1 and β2 chains, present in BMZs of Schwann cells at gw 11, and in the perineurium at gw 17 (Jaakola et al. 1993). Based on a study of the development of the rat central nervous system, it has been postulated that laminin β1 has an influence on rat nervous system development (Hunter et al. 1992).
Taken together, our data demonstrate the ubiquitous presence of the laminin β1 chain during human embryonic and fetal development from gw 8 to 12. The laminin β2 chain was not as widely distributed during these developmental stages. In some organs, such as kidney or cartilage, a chain switch from the laminin β2 chain to the β1 chain seems to be a common developmental pattern.
We would like to thank Dr. Takako Sasaki (Shriners Hospital in Portland, Oregon) for the laminin β2 chain antibody.
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