In vivo embryonic expression of laminin and its involvement in cell shape change in the sea urchin Sphaerechinus granularis

During embryogenesis, the interaction of cells with the basement membrane results in changes in cell shape, cell growth and differentiation (Hay, 1981; Bernfield, Banerjee, Koda & Rapraeger, 1984, for reviews). Laminin has been shown to be an abundant protein component of the basement membrane in vertebrate embryos and adults which, in cultured cells, induces morphological changes of cell spreading, elongation and neurite outgrowth (Von der Mark & Kiihl, 1985, for review). As part of an extracellular matrix (ECM) substrate of cultured cells, laminin has also been implicated in the maintenance of epithelial cell shape, in the regulation of cell adhesion and differentiation of cultured epithelial cells (Gospodarowicz, Greenburg & Birdwell, 1978; Sugrue & Hay, 1981; Greenburg & Hay, 1986; see Watt, 1986, for review). During the development of the mouse embryo, laminin is one of the earliest of the ECM components to appear, preceding the establishment of an organized basement membrane. These results have suggested a coordinating role with other ECM components in the formation of basement membrane (Leivo, Vaheri, Timpl & Wartiovaara, 1980; Wu, Wan, Chung & Damjanov, 1983; Dziadek & Timpl, 1985). Its expression on the surface of early mouse blastomeres also indicates that it may play a role in the establishment of cell polarity, cell adhesion during compaction or in the mediation of cell interactions during preimplantation development (Dziadek & Timpl, 1985).

In the early sea urchin embryo, a thin fibrous layer or basal lamina has long been known to exist on the basal surfaces of cells beginning at the early blastula stage (Endo & Uno, 1960; Wolpert & Mercer, 1963; Okazaki & Niijima, 1964). The basal lamina has been thought to play a role in many morphogenetic events of early sea urchin development such as cell-cell adhesion and cell shape change (Gustafson & Wolpert, 1967), primary mesenchyme cell migration (Okazaki, Fukushi & Dan, 1962; Katow, Yamada & Solursh, 1982) and gastrulation (Spiegel, Burger & Spiegel, 1980, 1983). Some evidence points to extracellular matrix components as mediators of these events in the sea urchin embryo. In sulphate-deprived embryos, primary mesenchyme cell migration and gastrulation are blocked along with normal metabolism of sulphated glycosaminoglycans, glycoproteins and proteoglycans (Sugiyama, 1972; Karp & Solursh, 1974; Solursh & Katow, 1982). Exposure of embryos to /5-xylosides will inhibit proteoglycan synthesis, leading to a block of gastrulation and primary mesenchyme cell migration (Kinoshita & Saiga, 1979; Akasaka, Amemiya & Terayama, 1980). Tunicamycin, an inhibitor of protein and lipid glycosylation, will prevent gastrulation (Schneider, Nguyen & Lennarz, 1978; Akasaka et al. 1980) under conditions where glycosaminoglycan synthesis is normal (Heifetz & Lennarz, 1979). Little is known, however, of the mechanisms by which the extracellular matrix components influence these morphogenetic events.

Monoclonal antibody BL1 (Mab BL1) recognizes two proteins in the sea urchin embryo. Metabolic and embryo surface labelling studies have shown that one protein is restricted to the hyaline layer. The other protein is a sea urchin basal lamina component which is structurally related to the vertebrate basement membrane protein laminin (McCarthy, Beck & Burger, 1987). In the present study, we use the Mab BL1 to study the expression and involvement of laminin in epithelium formation in vivo. Microinjection of fluorescently labelled Mab BL1 antibody demonstrates that laminin is secreted on the basal cell surface at the early blastula stage. Expression of laminin is correlated in time with the change in cell shape of embryonic blastomeres. Injection of Mab BL1 results initially in a dramatic cell shape change and a rounding up of cells, leading, in some cases, to embryonic deformation. These results are consistent with the hypothesis that laminin functions in vivo as a cell adhesion protein of the basal lamina and that it may have a role in the regulation of cell polarity and cell shape during early embryogenesis.

Adult Sphaerechinus granularis were obtained from Laboratoire Arago, 66650 Banyuls-sur-Mer, France. Gametes were collected by electrical stimulation into Woods Hole formula artificial sea water containing Tris buffer instead of sodium carbonate (MBLSW; Cavanaugh, 1956). Eggs were fertilized as described for other sea urchin species (McCarthy & Spiegel, 1983) and embryos were raised at 16°C to the appropriate developmental stage.

Monoclonal antibody IgG was prepared from hybndoma supernatant as previously described (McCarthy et al. 1987) and labelled with fluorescein isothiocyanate (FITC). Briefly, 0·5–1 mg of purified Mab IgG was mixed with 6 mg ml^-1 cytochrome C in 2·0 ml of 0·05M-sodium carbonate buffer pH9·5, 0·15M-sodium chloride and dialysed against the same buffer. The dialysis tubing was placed into 20 ml sodium carbonate buffer pH 9·5, 0·15 M-sodium chloride containing 0·2 mg ml⁻¹ FITC and dialysed 4h in the dark at room temperature. After dialysis, free FITC was removed from the protein mixture by chromatography on a Sephadex G-25 column and the FITC-labelled Mab was reisolated by protein-A affinity chromatography. The FITC-Mab preparation was concentrated to 1 mg ml^-1 and dialysed extensively against PBS.

Embryos at specific stages were injected using the pressure injection method of Hiramoto (1962) with the modifications described by Kiehart (1981). Before loading into the injection chamber, the embryos were washed three times by hand centrifugation in MBLSW. The pellet of embryos was then suspended in four volumes of MBLSW containing 0·2 % agarose which had been melted and quickly brought to room temperature. The suspension of embryos was immediately loaded into the injection chamber. The cooled agarose formed a loose gel within the microinjection chamber which allowed the ciliated embryos to be observed over long periods. The amount of IgG injected was computed by calibrating the micropipette with vegetable oil injected into the sea water.

Embryos were observed and photographed with a Zeiss Axiomat or ICM microscope equipped with Nomarski differential interference contrast (DIC) and epifluorescence optics. Kodak Tri-X Pan film was used and processed at 1600 ASA using Ilford Microphen developer.

The blastula epithelium undergoes complex morphogenetic changes during the early development of Sphaerechinus granularis (Fig. 1). These events include polarization of the blastomeres and dynamic changes in cell-cell contacts and cell shape. After the fifth cleavage, the embryo consists of 32 rounded cells attached at their apical ends to the hyaline layer (Fig. 1A, arrow). A dramatic cell shape change occurs at approximately the ninth cleavage (Fig. 1B) during which the apparent volume of the blastocoelic cavity is reduced by approximately two thirds. The cells remain attached to the hyaline layer and take on a wine-glass shape and elongated appearance (Fig. 1C, large arrow). During the next few hours of development as the embryo enlarges (Fig. 1D), cells occasionally round up and lose their wine-glass shape and adhere closely to the hyaline layer (Fig. 1C, small arrow).

By 21 h after fertilization (Fig. 1E), a well-ordered epithelium begins to form and by 24 h the embryo consists of cells bounded on their apical surfaces by the hyaline layer and on their basal surfaces by the basal lamina. At this time, the primary mesenchyme cells enter the blastocoel and begin to migrate (Fig. 1F, arrows). Other morphogenetic changes occur in the epithelium after primary mesenchyme cell ingression. These are localized thickenings of the epithelium visible in the vegetal half of the 24-30 h embryo (Fig. 1G, arrows) in which cells are often present in a wine-glass shape with apparent spaces between adjacent cells as in the 35–48 h embryo in the region of the animal plate (Fig. 1H, arrows).

Monoclonal antibody BL1 recognizes two large molecular weight proteins: a protein restricted to the hyaline layer and a basal lamina protein, sea urchin laminin. Sea urchin laminin has been isolated from the sea urchin basal lamina and is analogous to the vertebrate basal lamina protein laminin (McCarthy et al. 1987), a major component of vertebrate basement membranes. In order to investigate the expression and distribution of sea urchin laminin in the early development of the sea urchin embryo, we directly coupled FITC to Mab BL1 (FITC-Mab BL1) and microinjected the fluorescently labelled antibody into the blastocoel of living embryos at various stages.

Binding of FITC-Mab BL1 is first detected as spotty fluorescence on the basal surfaces of the cells, indicating secretion and cell binding of laminin (Fig. 2B, embryo upper left). As cells begin to elongate at approximately 12 h after fertilization (Fig. 2C, white arrows, uninjected control and Fig. 1B), fluorescence is associated with the basal cell surfaces in the areas where blastula cells appear elongated (Fig. 2C,D, arrow, embryo upper right). Injection of a control FITC-Mab results in diffuse fluorescence (Fig. 2B,D, embryos lower left). FITC-Mab 6A4 is directed against a hyaline layer protein and will bind to the hyaline layer if administered to the external surface of the embryo (not shown). This antibody was used routinely to determine specific binding of FITC-Mab BL1.

At 17h after fertilization, most of the cells of the embryo are wine-glass shaped (Fig. 2E). FITC-Mab BL1 binds extensively to the basal cell surfaces, demonstrating the presence of laminin (Fig. 2F) and, as is the case in the mesenchyme blastula stage embryo (Fig. 2G,H), fluorescence is coincident with the position of the basal lamina. Particularly interesting is the result of injection into the midgastrula-stage embryo. Although present on most basal cell surfaces, the fluorescence is heterogeneous with intense labelling noted at the animal pole in a position corresponding to the animal plate (Fig. 2J, arrow). At the locations of increased fluorescence, the ectodermal cells are elongated (Fig. 2I, arrow). Additionally, fluorescence is limited to the basal cell surface of ectodermal cells and is absent from primary mesenchyme cells and the invaginating archenteron. At the gastrula stage, fluorescence is distributed over most of the basal surfaces of ectodermal cells (Fig. 2L) and is additionally present on the archenteron over the basal surface of the endodermal cells (arrow). Binding is also absent or reduced from primary mesenchyme cells.

The development of individual embryos that had been injected with FITC-Mab BL1 was observed in order to investigate possible functions of laminin during early sea urchin morphogenesis. Laminin is first detected over most of the basal cell surfaces at 16 h after fertilization (see Fig. 2F), The cells of the embryo are wine-glass shaped and the blastocoel is delimited by the basal cell surfaces (Fig. 3A). Within 1min after injection, the basal cell surfaces of these embryos become more distinct (Fig. 3C, arrows). The same embryo viewed with fluorescence optics after 3 min demonstrates fluorescent binding of the FITC-Mab BL1 antibody to the basal cell surfaces (compare Fig. 3C and D). At 7min after injection, the cells begin to elongate and the apparent blastocoel volume is reduced by 60–75 % (compare Fig. 3E and A). The cells remain elongated and by 30min the embryo has taken on the appearance of an earlier embryonic stage (compare Figs 1B and 3G). The small volume of the blastocoel and extensive cell elongation is similar to the 12 h control embryos at a time when laminin is first detected on the basal cell surface. The degree of cell elongation varies, however; in some areas cells are twice the length recorded before injection (compare Fig. 3A with F and G). After 1 h the cells of the injected embryo begin to round up and adhere to the hyaline layer (Fig. 3H, arrow).

The rounding up of cells after antibody injection is shown more clearly in Fig. 3I—L. Bright fluorescence is associated with the basal surfaces of cells after FITC-Mab BL1 is injected (Fig. 3I, upper embryo); however, only diffuse fluorescence is observed in the case of control FITC-Mab 6A4 (Fig. 3I, lower embryo). Nomarski DIC micrographs of an injected embryo show little effect of the injection of FITC-Mab 6A4 over a period of 2 h (Fig. 3J-L). In the case of Mab BL1, the blastocoel volume is reduced within 15min and the cells elongate (Fig. 3J,K). After 2h, some cells lose their wine-glass shape. These cells round up and remain closely associated with the hyaline layer (Fig. 3L, arrows). The cells return to a normal shape over a period of hours and the embryo develops normally.

Microinjection of FITC-Mab BL1 at 15-23 h after fertilization results in the elongation of cells in the blastula epithelium and subsequent rounding up of cells (Table 1). At this time, most cells are part of the blastula epithelium and it is not possible to determine any polarity of the cell elongation event with respect to the animal-vegetal axis of the embryo. FITC-Mab BL1 was therefore microinjected into mesenchyme blastula embryos where the primary mesenchyme cells leave the vegetal epithelium and designate the vegetal pole of the embryo (Fig. 4A, arrows). Immunofluorescence of FITC-Mab BL1 binding demonstrates that laminin is confined to the basal surfaces of the ectodermal cells and absent from inwardly migrating primary mesenchyme cells (Fig. 4B; Table 1). After 1h (Fig. 4C) and 3h (Fig. 4D), the cells of the ectoderm elongate. However, there is little effect on primary mesenchyme cells, which continue to migrate. Comparison of Fig. 4D and E demonstrates that the fluorescent Mab is still associated with the basal surfaces of the ectodermal cells and that the elongation is restricted to these cells. A heterogeneous distribution of laminin in the early embryo is supported by the result of microinjection of FITC-Mab BL1 into the midgastrula stage where it is also absent from primary mesenchyme cells and the archenteron (see Fig. 2I,J).

Normally, at the mesenchyme blastula stage, the cells of the blastula wall are of an equivalent length within the ectodermal epithelium (Fig. 4A,F; see also Fig. 1E,F). After injection of FITC-Mab BL1, the cells at the animal pole elongate to a greater extent than the cells lateral to the animal-vegetal axis (Fig. 4G). These injected embryos, with elongated animal pole cells, resemble older control embryos that are in the process of forming the animal plate (compare Fig. 1H to 4G,D). Comparison of Fig. 4I and J demonstrates that the fluorescently labelled antibody is distributed over the basal surfaces of cells remaining in the epithelium. Microinjection of a control FITC-Mab 6A4 (Fig. 4K-O) has little effect on either the shape of ectodermal epithelial cells or the normal progression of primary mesenchyme cells during the injection period (arrows).

FITC-Mab BL1 initiates epithelial elongation at specific stages when injected at amounts of approximately 300 pg (in 300 pl) of antibody (Table 1). Injections of larger amounts of Mab BL1 can cause additional deformation of the ectodermal epithelium (Table 1). Fig. 5 demonstrates the effect of microinjection of approximately 600 pg FITC-Mab BL1 into a mesenchyme blastula stage embryo. The binding of the FITC-Mab BL1 antibody to the basal cell surfaces is apparent after 4 min (Fig. 5A) and, at 10min, there is a slight elongation of cells (Fig. 5B). After 2h, however, major changes occur in the blastula epithelium (Fig. 5C). Cells begin to round up (Fig. 5C) and there is a general deformation of the epithelium. Cells initially elongated have flattened and other cells have aggregated into clumps (compare Fig. 5B,C), presumably over the surface of the basal lamina since the overall internal shape of the blastocoel is maintained. The embryo is able to regulate this effect and will go on to form a normal gastrula stage embryo (Fig. 5D). During the process of embryonic reorganization, an epithelium is reformed consisting of elongate cells. Fig. 5E-H demonstrates an additional embryo in which the binding of the antibody to the basal cell surface (Fig. 5E) results in deformation of the epithelium and loss of embryonic shape (Fig. 5F,G). After 3h, however, some cells begin to reform an epithelium and are once again arranged as elongated cells (Fig. 5H, arrow).

Table 1 is a compilation of many injections of FITC-Mab BL1 into embryos of various stages. Basal cell surface binding becomes evident in embryos injected at 11–12 h after fertilization. Since at this stage the blastula cells undergo shape changes and the blastocoel is quite small, no effect on cell elongation could be attributed to the antibody. Cell elongation in response to Mab BL1 injection is first noted at approximately 15 h after fertilization and can be detected until the mesenchyme blastula stage. In early gastrula stage embryos, FITC-Mab BL1 binding could be detected; however, no effects of the binding on cell shape were noted. Embryos from all embryonic stages were injected with an equivalent amount and volume of FITC-Mab 6A4 IgG. No effects could be attributed to these injections.

In this study, we have microinjected a fluorescently labelled monoclonal antibody, which recognizes sea urchin laminin, into living sea urchin embryos and demonstrate a correlation between the time of laminin expression on the basal surfaces of embryonic blastomeres and a change in the shape of the blastomeres. Laminin is expressed on the basal cell surface just prior to the organization of the blastomeres into an epithelium. During the formation of an epithelium the blastomeres elongate and a very thin basal lamina is observed by electron microscopy (Gibbins, Tilney & Porter, 1969). The appearance of laminin at early stages in mouse embryoic development (Leivo et al. 1980; Wu et al. 1983; Dziadek & Timpl, 1985) and the fact that it is a major component of adult basement membranes (Timpl et al. 1979) has led to the idea that laminin may be involved in the organization of the cytoskeleton and establishment of epithelia. Our experiments support the idea that the secretion and appearance of laminin on the basal cell surface is an important step in the organization of blastomeres into an epithelium during early development.

In the early sea urchin blastula, the cells are not yet organized into a differentiated cell epithelium. The basal surfaces are attached to a newly formed basal lamina. However, the cells do not yet have a columnar shape typical of an epithelium but instead have a wine-glass shape (Gibbins et al. 1969) as if under tension. In this regard, the cells may be considered to be in the very early stages of epithelium formation. Since it is at this time that laminin is detected on most basal cell surfaces, it is interesting to speculate that the organization and cell binding of the basal lamina components, including laminin, may be sufficient to cause temporally regulated changes in cell shape early in development which would not occur after a more highly structured basement membrane is present.

Microinjection of FITC-Mab BL1 results in an initial elongation, a rounding up of cells and loss of epithelial morphology. In the mesenchyme blastula, the elongation is asymmetric with longer cells associated with the animal pole ectoderm. These changes in cells and in the overall form of the embryo mimic those that occur in the normal development of the blastula and of an animal plate ectoderm.

The mechanism by which Mab BL1 accomplishes its effect is open to interpretation. If we assume that the effect of the antibody is due to its interaction with laminin, several interesting possibilities exist which pertain to basal lamina structure. As laminin is present over most of the basal surfaces of the blastula cells, microinjection of the bivalent antibody may crosslink the laminin, reducing the intermolecular distances of laminin molecules within the basal lamina. Cell elongation could then occur either directly if the cells are attached to laminin or indirectly since the cells are attached to the basal lamina. Cell rounding and epithelium disruption may occur if the cells release from the basal lamina. Alternatively, the antibody may affect the ability of laminin to provide a structural support for the newly forming basal lamina by interfering with the interaction of laminin with cells or with other extracellular matrix components. It is known that laminin interacts with other structural components of basement membranes (Martin & Timpl, 1987). Elimination of structural constraints in the basal lamina imposed by laminin may have the result of disrupting the basal lamina and affecting the molecular interactions of other components.

Traditionally, cell shape change has been thought to involve extracellular matrix interactions with the cytoskeleton (Gospodarowicz et al. 1978; Hay, 1983). It is possible that the antibody binding affects the cytoskeleton by the induction of microtubule polymerization (Porter, 1966; Burnside, 1971) or interference with microfilaments or cell junctions. It is interesting, in this regard, that concentrations of colchicine that disrupt microtubules have little effect on the shape of the blastula ectoderm and only treatments that disrupt cell-cell contacts result in epithelium disruption (Tilney & Gibbins, 1969).

We have recently been able to isolate small amounts of laminin from embryonic sea urchins and assess its structure at the electron microscopic level (McCarthy et al. 1987). We are initiating experiments in order to identify the binding site of the Mab BL1 on the laminin molecule and to study the effect of Mab binding on the interaction of laminin with other extracellular matrix components in vitro. Such studies should provide relevant information with which to evaluate the effects of the antibody in vivo.

Binding of FITC-Mab BL1 is limited to the ectodermal cells being absent or reduced on primary mesenchyme cells. This is also observed in indirect immunofluorescence using Mab BL1 on paraffin sections of embryos (McCarthy et al. 1987). Immunological assays are limited in their ability to determine the presence of an antigen and a lack of signal may represent a masking of the Mab BL1 antigenic site of laminin by another extracellular molecule. In this regard, a mesenchyme-specific molecule has been described which appears on the cell surface of mesenchyme cells upon the entry of these cells into the blastocoel (Wessel & McClay, 1985). The present results are, however, consistent with studies on the distribution of laminin in vertebrate embryos and adult tissues which show that laminin is most often associated with epithelial cells and absent from mesenchymal cells (Wartiovaara, Leivo & Vaheri, 1980; Foidart et al. 1980).

Electron microscopic evidence demonstrates that presumptive primary mesenchyme cells initially elongate and bind to the blastula basal lamina (Gibbins et al. 1969). After the primary mesenchyme cells have migrated into the blastocoel, their cell surface changes. The cells lose their ability to bind the hyaline layer protein, hyalin, and gain an affinity for fibronectin (Fink & McClay. 1985; Venkatasubramanian & Solursh, 1984) and for the lectin, wheat germ agglutinin (DeSimone & Spiegel, 1986). Katow et al. (1982) have also demonstrated that primary mesenchyme cells bind a polyclonal antibody directed against vertebrate fibronectin and increase their migration speed in vitro on surfaces coated with vertebrate fibronectin and dermatan sulphate but not on laminin or collagen IV (Katow, 1986). In vivo, primary mesenchyme cells change in their ability to bind the anti-laminin Mab BL1 upon entry into the blastocoel. The above results suggest a possible mechanism for primary mesenchyme ingression and migration. The cessation of laminin synthesis or masking of laminin by other extracellular molecules may interfere with the cells’ ability to bind to the basal lamina. A decreased affinity for hyalin and an increased affinity for fibronectin may allow the cells to leave the epithelium and to migrate.

It is not known whether primary mesenchyme cells actively degrade the basal lamina components upon entry into the blastocoel or whether a developmentally regulated switch in the synthesis or binding of extracellular matrix components, such as laminin, would result in local disruptions of the basal lamina. We are currently investigating the developmental expression of laminin by primary mesenchyme cells. Whatever the mechanism of primary mesenchyme ingression, our results demonstrate that primary mesenchyme cells change in their ability to bind the antilaminin Mab BL1 during the epithelial-mesenchymal transformation.

FITC-Mab BL1 binding suggests that laminin is present over the basal surfaces of cells upon secretion at the blastula stage. Differences in FITC-Mab BL1 binding within the basal lamina are noted at the midgastrula stage where more intense fluorescence is apparent on the basal surfaces of ectodermal cells which comprise the animal plate in both living embryos and tissue sections (McCarthy, unpublished data). Heterogeneity of sea urchin cell surface components has been previously noted using fluorescently labelled lectins as probes (Spiegel & Burger, 1982; Katow & Solursh, 1982; DeSimone & Spiegel, 1986). In the midgastrula embryo, specific binding of Concanavalin A is detected at the region of the animal plate, the site of attachment of the secondary mesenchyme cells (Spiegel & Burger, 1982). Microinjection of Concanavalin A, proteases or collagenase break adhesions of attached secondary mesenchyme cells and interfere with gastrulation (Spiegel & Burger, 1982). The molecular basis for this effect is not known; however, collagenase treatment strongly implies that collagen is involved. These results suggest that not only the individual components but also their distribution within the basal lamina may be important during morphogenesis.

The apparent heterogeneity of the basal lamina for Mab BL1 binding may be explained by either sequential assembly of extracellular matrix components, as in the mouse embryo (see Wartiovaara et al. 1980, for review), or differential expression of laminin by epithelial cells. Support for sequential assembly of extracellular matrix components in the sea urchin embryo has been obtained using indirect immunofluorescence (Wessel, Marchase & McClay, 1984). In the above study, however, cross-reactive antigens were not identified as bona fide sea urchin extracellular matrix proteins. Nevertheless, the differences in laminin distribution at the midgastrula stage may represent the addition of extracellular matrix components which interfere with the FITC-Mab BL1 binding in specific regions of the basal lamina. Alternatively, the observed fluorescent binding pattern may reflect differences in synthesis or binding of laminin by subsets of epithelial cells. The present results do demonstrate, however, that heterogeneities arc present in the distribution of sea urchin basal lamina components on epithelial and primary mesenchyme cells and are correlated with differences in cell shape.

Clearly, many components are involved in the complex interactions of cells with the extracellular matrix during early embryogenesis. The present data demonstrate a method by which extracellular matrix components may be characterized and their function assayed in vivo. This represents a new approach that offers major advantages over traditional localization techniques involving tissue sections. First, the microinjection experiments permit spatial and temporal localization of the laminin molecule in the living embryo and allow one to correlate the expression of laminin with important developmental events. Second, because the antibody interferes with normal development, but only when injected into specific stages, it suggests a specific role for laminin in embryonic epithelium formation and cell shape change.

We thank Katrina Saladin for excellent technical assistance and Drs D. Smith, K. Beck, D. DeSimone, J. Engel, J. Fessler and L. Fessler for stimulating discussions during the course of this work. We also thank Dr H. J. Marthy for his kindness and support in providing us with animals from Laboratoire Arago. The work was supported by Swiss National Science Foundation Grant No. 3.269–0.82. and 3.169–0.85.