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Previous Article | Next Article
The Journal of Neuroscience, March 1, 1998, 18(5):1795-1805
Functional Cooperation of
1-Integrins and Members of the Ig Superfamily in Neurite Outgrowth Induction
Ullrich Treubert and Thomas Brümmendorf Max-Planck-Institut für Entwicklungsbiologie, D-72076 Tübingen, Germany
ABSTRACT
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Abstract
Introduction
Neurite outgrowth is a central aspect of the ontogenetic formation of neural networks and is regulated by distinct groups of cell surface molecules. One protein involved in neurite elongation and fasciculation is the neural Ig superfamily member F11/contactin. We have shown previously that F11 promotes neurite extension of chick tectal neurons by interaction with the tectal receptor NrCAM, a member of the L1 subgroup of the Ig superfamily. By contrast, it does not induce outgrowth of retinal neurons despite the fact that these cells also express NrCAM, suggesting that in retinal cells the F11-NrCAM interaction alone is not sufficient to induce neurite extension. In this report we present a novel image analysis procedure to quantify neurite outgrowth and use it to demonstrate that F11 enhances the fibronectin-induced outgrowth response of embryonic retinal neurons. We reveal that NrCAM is the neuronal receptor mediating the enhanced outgrowth of retinal neurons, whereas the related F11-binding molecule NgCAM is not involved. Furthermore, we provide evidence that a
1-integrin may represent the fibronectin-dependent receptor that cooperates indirectly with the F11-NrCAM pathway. Our results support the concept of a combinatorial labeling of cells in nervous system histogenesis by different classes of cell surface proteins, in particular by integrins and molecules of the Ig superfamily.
Key words: retina; neurite growth; neural development; Ig superfamily; integrins; F11; NrCAM
INTRODUCTION
Outgrowth of neurites in nervous system ontogenesis can be divided into at least two aspects, pathfinding of elongating axons on their way to their target regions and formation of the dendritic tree that is typical for a particular neuron. In the last decade several classes of ligands and receptors have been described to regulate neurite outgrowth and axon guidance (Dodd and Schuchardt, 1995
; Keynes and Cook, 1995
Retinal neurons of embryonic day 6 (E6) are well suited to studying the interplay between integrin- and IgSF-mediated neurite outgrowth for two reasons. First, these neurons simultaneously express molecules of both protein classes on their surface during the period of neurite outgrowth. For instance, identified integrin subunits in embryonic chick retina include
1,
The F11 molecule, also referred to as F3 (Buttiglione et al., 1996
MATERIALS AND METHODS
Antibodies. The preparation and specificity of antibodies directed to F11, NCAM, NgCAM, and NrCAM have been described (Rathjen et al., 1987
Recombinant soluble F11. Because we intended to evaluate hexahistidine tagging as a means of isolating native, soluble recombinant protein from tissue culture supernatants, we inserted a DNA sequence encoding this tag after the leader sequence of F11. To this end, the DNA sequence corresponding to mature F11 (Thr22-Ala988) was amplified by PCR on the plasmid pSG5/F11 (Brümmendorf et al., 1993
The plasmid was used to transfect COS cells transiently by the DEAE-dextran method as outlined previously (Brümmendorf et al., 1996
Neurite outgrowth assay. To prepare fibronectin substrates, a solution containing 20 µg/ml fibronectin (Dianova, Hamburg, Germany) in PBS was spotted as 3 µl spots on Petriperm dishes (20 cm2; Heraeus, Osterode, Germany) and incubated for 3 hr in a humidified atmosphere at 37°C. To generate a fibronectin-F11 substrate, fibronectin (20 µg/ml) and F11 (4 µg/ml, recombinant or isolated from adult chick brain as described; Rathjen et al., 1987
To isolate retinal cells, retinas were dissected from E6 chick embryos in HBSS without Ca2+ and Mg2+ and incubated for 15 min at 37°C with 0.05% trypsin in PBS and 0.02% EDTA (Boehringer Mannheim, Mannheim, Germany). After aspiration of the trypsin solution, cells were dissociated by trituration in PBS containing 1 mg/ml soybean trypsin inhibitor (Sigma), 1 mg/ml DNase (Worthington, Freehold, NJ) and 3 mg/ml BSA (Sigma). The cells were pelleted and carefully resuspended in serum-free N2 culture medium with 0.5-1.5 × 105 cells/ml, followed by seeding 0.5-4.5 × 105 cells in a protein-coated Petriperm dish. The dishes were incubated at 37°C in an atmosphere of 5% CO2 for 20 hr. In contrast to tectal cells, which extend neurites if grown on a pure F11 substrate in serum-free N2 medium or DMEM with 10% FCS, retinal cells do not in either medium (our unpublished observations).
For antibody perturbation or peptide inhibition experiments, spots were prepared, and cells were seeded as described. After attachment of the cells (2 hr at 37°C), medium was aspirated, and the region between spots was dried with wicks. The medium was replaced with 100 µl of fresh medium containing antibodies or the GRGDSP peptide (Nova-Biochem, Bad Soden, Germany). Polyclonal rabbit antibodies directed to F11, NgCAM, or NrCAM or their Fab fragments were used at 200 µg/ml. JG22 hybridoma culture supernatant was dialyzed against PBS and diluted 1:10 in N2 culture medium. This resulted in a functionally saturating (with respect to retinal ganglion cell binding to FN) concentration of the monoclonal antibody (mAb), as determined by serial dilution experiments (data not shown). The peptide GRGDSP was used with 200 µg/ml, a functionally saturating concentration if retinal cell binding to FN is examined.
Immunofluorescence analysis of retinal ganglion cell cultures. Cultures were fixed in PBS containing Ca2+ and Mg2+ (PBSCM) and 4% formaldehyde for 1 hr at room temperature. After three washes with PBSCM containing 0.02% BSA, cultures were processed for immunofluorescence analysis. Cell bodies and neurites were detected by incubating for at least 1 hr with NCAM-specific antibodies, followed by Cy3-conjugated secondary antibodies from goat (Dianova). To reveal cellular nuclei, the DNA-staining reagent bisbenzimide (H33258, Boehringer Mannheim) was added to the secondary antibodies at a final concentration of 50 ng/ml. Alternatively, to ensure that effects measured on neurite growth were attributable to increased growth of axons originating from retinal ganglion cells, polyclonal antibodies specific for NgCAM, a marker predominantly expressed on retinal ganglion cell axons at stage E6 (de la Rosa et al., 1990
Quantification of neurite growth by digital image analysis. To determine parameters reflecting neurite outgrowth, images of neurons and their nuclei were captured at the appropriate wavelength by an intensified CCD camera (Proxitronic, Bensheim, Germany) attached to a fluorescence microscope (Axiophot; Zeiss, Oberkochen, Germany). Spectral overlap of the images was not observed. Images with a resolution of 512 × 512 pixels were processed with an image analysis card [Machine Vision System IV120(C); AIT Göhner, Stuttgart, Germany] on a standard personal computer. With the help of the JL Genias image analysis software (AIT Göhner) an algorithm was developed that was suited to evaluating automatically the number of attached cells, the number of putative neurites, and their individual length (see Results and Fig. 1). Raw data generated by JL Genias were processed with a spreadsheet program (Excel; Microsoft, Unterschleißheim, Germany) and evaluated statistically using the Mann-Whitney U test implemented in the SPSS (Chicago, IL) package. The final layout of figures was created with graphics programs (Freehand; Altsys Inc., Richardson, TX; and Photoshop; Adobe Systems, Mountain View, CA).
RESULTS
Quantification of neurite outgrowth in vitro by an automatic image analysis procedure
The study of potential synergistic or cooperative effects between different adhesion receptor
ligand pairs with respect to neurite outgrowth requires the quantification of process outgrowth of a large number of neurons. To this end we developed an automatic digital image analysis algorithm, which is suited to evaluation of several hundreds of neurons for each experimental condition by a standardized procedure. This enabled us to measure parameters reflecting neurite outgrowth without any manual processing of the micrographs to be compared with each other. Two different parameters have been chosen to quantify neurite outgrowth in this study, namely, the number of neurites observed and their average length.
Retinal cells were grown in a tissue culture system in vitro on different substrates composed of the proteins to be analyzed. After incubation for 20 hr, the cultures were fixed, and the neurons were detected by immunofluorescence analysis with NCAM-specific antibodies (Fig. 1a). The nuclei of the cells were simultaneously stained with bisbenzimide, a reagent intercalating in DNA (Fig. 1d). Two digitized images representing the neurons and their nuclei, respectively, were then captured separately by a CCD camera. The images were processed automatically to estimate the number of attached cells, the number of neurites, and their average length (Fig. 1).
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Figure 1. Digital image analysis procedure to evaluate neurite outgrowth in vitro. Retinal cells were seeded on a substratum composed of a mixture of FN and F11. After cultivation for 20 hr, cell bodies and processes were detected by immunofluorescence analysis using antibodies directed to NCAM (a), and cellular nuclei were identified by bisbenzimide staining of DNA (d). To discriminate neurites from cell bodies, both fields were processed as follows. The field showing cells and neurites (a) was processed by applying a staining intensity threshold and size exclusion criteria to distinguish the significant structures representing neurons from background and from small artifactual signals of subcellular size (b, blue objects). These structures were then eroded to a skeleton of a single-pixel width (c). The field comprising cellular nuclei (d) was also processed by applying a staining intensity threshold and size exclusion criteria to label cellular nuclei (e, green objects). These were used to estimate the number of attached cells by dividing the total area representing cell nuclei by the average area covered by a single nucleus. Because we are interested in quantifying primarily the long neurites with a length of more than two cell diameters, all structures within this distance of every cell nucleus had to be excluded from the analysis. To this end, every object that represents a cell nucleus was enlarged correspondingly (f). A comparison of the resulting image with the image representing putative neurites (c) allows the identification of long neurites and fragments thereof, because they do not overlap with the enlarged nuclei (g). To enhance the stringency of the procedure further, objects below a defined length threshold were discarded. The remaining objects (h) were counted, and their individual lengths were measured automatically. The values obtained were normalized with respect to the cell number to correct for intra-assay variations in the number of attached cells. In this procedure we did not attempt to measure the real number of neurites in the observed field or their real length, but we were merely interested in parameters that reflect the outgrowth and can be used to quantify the response of a large number of neurons. Scale bar, 50 µm.
To examine the performance of this automatic procedure, we compared it with a manual method that we had used earlier to measure neurite lengths of tectal neurons. In our previous studies, we had shown that immobilized F11 or NgCAM promotes neurite outgrowth of tectal neurons and that NgCAM is a more potent substratum than F11 (Morales et al., 1993
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Figure 2. Comparison of methods to evaluate neurite outgrowth. Neurite extension of tectal neurons on immobilized F11 was compared with that on immobilized NgCAM. A manual evaluation, which was performed as described (Brümmendorf et al., 1993
F11 enhances FN-induced neurite outgrowth of retinal cells
Because retinal cells do not extend neurites on F11 (data not shown) and only poorly on FN, these neurons are well suited to study synergistic promotion of outgrowth elicited by a mixture of both proteins. Retinal cells of embryonic day 6 were isolated and plated on FN alone or on an FN-F11 mixture. On the FN substrate, a significant neurite outgrowth response is elicited (Fig. 3a). If retinal cells are plated on a mixed substrate of FN and F11, the number of attached cells is almost indistinguishable from that on FN alone (Fig. 3c,g). However, the neurite outgrowth response on the FN-F11 mixture is enhanced if compared with FN alone (Fig. 3a,e). To quantify this enhancement, the image analysis procedure that was outlined in detail above (Fig. 1) was applied. Cell bodies and neurites that had been detected by immunofluorescence analysis (Fig. 3a,e) were identified by application of a staining intensity threshold and size exclusion criteria (Fig. 3b,f). In parallel, cell bodies were identified analogously by staining of their nuclei (Fig. 3c,g). Both images were then processed to identify the neurites (Fig. 3d,h) and to estimate their number and length by applying the stringent criteria outlined in Figure 1.
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Figure 3. Enhancement of FN-based retinal neurite outgrowth by F11. Retinal cells of embryonic day 6 were cultured for 20 hr on FN alone (left column) or on a mixture of FN and F11 (right column). Cells and neurites were identified by immunofluorescence analysis using NCAM-specific antibodies, and cellular nuclei were identified by bisbenzimide staining. The first row shows immunofluorescence micrographs, and the second through fourth rows show objects that have been processed by the image analysis procedure outlined in detail in Figure 1. In the second row cells and neurites are depicted; in the third row, the cellular nuclei; and in the fourth row, the identified neurites. Scale bar, 100 µm.
To compare the neurite outgrowth-promoting effect of FN and the FN-F11 mixture, 10 different images were evaluated by the image analysis procedure for each experimental condition. For each image the number of attached cells, the number of observed neurites, and the sum of the lengths of these neurites were determined to estimate the number of neurites per cell and the average length of neurites per cell. In a typical experiment, the average number of cells attached to the FN substrate is not significantly different from that on an FN-F11 mixture (Fig. 4a, open bar, filled bar). However, the neurite outgrowth response is enhanced in the presence of F11 (Fig. 4b,c). Mixing of FN with an equal amount of the biochemically similar control protein IgY did not modulate the outgrowth-promoting effect of FN (Fig. 4, open bars, hatched bars).
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Figure 4. F11 enhances FN-dependent neurite outgrowth of retinal ganglion cells. Retinal cells were cultured on FN alone, on an FN-IgY mixture, or on an FN-F11 mixture. Cultures were evaluated, and data are presented as outlined in the legend of Figure 2. The average number of attached cells is indistinguishable on the three substrates (a). If F11 is added to FN before coating, the average number of neurites per cell (b) and the average length of neurites per cell (c) are larger (filled bars) on this mixture than on FN alone (open bars). By contrast, substitution of F11 by an equal amount of IgY (hatched bars) did not enhance the outgrowth response of retinal neurons. Data from one representative experiment of at least four independent experiments are depicted. **Statistical significance of the difference (Mann-Whitney U test, p < 0.001).
The F11 protein used in the previous experiment was isolated from embryonic chick brain by immunoaffinity chromatography as outlined previously (Rathjen et al., 1987
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Figure 5. F11-induced enhancement of neurite outgrowth can be inhibited by F11-specific antibodies and can also be observed if recombinant F11 is analyzed. Retinal cells were cultured on an FN-IgY mixture and on an FN-F11 mixture in the presence or absence of polyclonal F11-specific antibodies. Cultures were evaluated, and data are presented as outlined in the legend of Figure 2. The number of substrate-bound cells was indistinguishable on both substrates and was not influenced by the application of the antibodies (a). On the FN-IgY substratum the antibodies did not interfere with the average number of neurites per cell (b) and the average length of neurites per cell (c). However, the antibody neutralized the outgrowth-promoting effect mediated by F11 contained in the FN-F11 substratum (b, c). In another set of experiments, the outgrowth-promoting effect of soluble recombinant F11, expressed in eukaryotic cells, was evaluated. Two different preparations of recombinant F11 were found to enhance neurite outgrowth to a similar extent as F11 isolated from embryonic chick brain (d-f). Data from one representative experiment of at least four independent experiments are depicted. **Statistical significance of the difference (Mann-Whitney U test, p < 0.001).
As a further specificity control, recombinant F11, which was heterologously expressed in eukaryotic cells, was also tested. To express soluble recombinant F11 in COS cells, the C-terminal hydrophobic stretch involved in the posttranslational attachment of the GPI anchor was deleted on the cDNA level in a eukaryotic F11 expression plasmid. Recombinant F11 was isolated from tissue culture supernatants by immunoaffinity chromatography using a monoclonal antibody (Rathjen et al., 1987
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Figure 6. Characterization of heterologously expressed soluble F11. Soluble F11 that was isolated from culture supernatants of transfected COS cells by immunoaffinity chromatography was characterized by SDS-PAGE followed by silver staining (a-c) or Western blot analysis with an F11-specific monoclonal antibody (d-f). F11 isolated from chick brain (a, 600 ng; d, 120 ng) is compared with recombinant soluble F11 (b, 1.6 µg; e, 320 ng). The latter contains a 53 kDa contaminant (b), which is unrelated to F11 (e) and which is also found in preparations of other recombinant proteins isolated from tissue culture supernatants, for instance, the 23 kDa DiFc protein (c, 3.2 µg; f, 640 ng), which consists of two IgG Fc domains (our unpublished data). Because preparations of the DiFc protein did not enhance neurite outgrowth of retinal neurons on FN (data not shown), suggesting that the 53 kDa protein does not interfere with aspects of this study, we did not attempt to separate the contaminant from recombinant F11.
Taken together, we showed that the addition of F11 to an FN substrate did not affect retinal cell attachment to FN but enhanced retinal neuron outgrowth on this substrate.
The enhancement of retinal neurite outgrowth is mediated by neuronal NrCAM
The enhancement of retinal neuron outgrowth, which is triggered by F11 in the FN-F11 substrate, is likely to be mediated by a receptor on the retinal neurons. The two similar IgSF cell surface molecules NgCAM and NrCAM are expressed on retinal neurons (Morales et al., 1996
Fab fragments of NgCAM-specific antibodies were not found to interfere with attachment of retinal cells to FN-IgY or FN-F11 substrates (Fig. 7a), indicating that they have no nonspecific toxic effect. Furthermore, addition of the Fab fragments to the culture medium of cells grown on an FN-F11 substrate did not neutralize the F11-mediated enhancement of neurite outgrowth (Fig. 7b,c). We therefore conclude that NgCAM is unlikely to be involved in the enhancement effect.
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Figure 7. Perturbation of F11-induced enhancement of neurite outgrowth by different reagents. Retinal cells were cultured on an FN-IgY or an FN-F11 substrate in the presence or absence of polyclonal NgCAM-specific Fab fragments (a-c), NrCAM-specific Fab fragments (d-f), a
To test whether NrCAM is the F11-binding molecule on the E6 retinal neurons, Fab fragments of NrCAM-specific antibodies were tested and were not found to interfere with the cell attachment to FN-IgY or FN-F11 substrate (Fig. 7d), which demonstrates that they are not toxic for the neurons. Interestingly and in contrast to the finding concerning NgCAM, the antibodies directed to NrCAM neutralized the F11-mediated enhancement of neurite outgrowth and reduced it to the level observed with FN alone (Fig. 7e,f).
Taken together, our data suggest that F11-triggered enhancement of retinal neuron outgrowth is mediated by the neuronal F11 receptor NrCAM but not by NgCAM, a related F11-binding molecule that is also expressed by these neurons.
Involvement of
It has been shown previously that FN is an outgrowth-promoting substratum for E6 retinal cells (Akers et al., 1981
Addition of functionally saturating amounts of mAb JG22, which interferes with
To get a first clue to which
One possible interpretation of these findings might be that a
DISCUSSION
Evaluation of neurite outgrowth by an automatic image analysis procedure
Analysis of multiple variables affecting neurite outgrowth and detection of subtle modulatory effects requires the quantification of outgrowth from a large number of neurons. In the present study, we introduce an automatic procedure to analyze hundreds of neurons in vitro for each experimental condition to determine parameters reflecting the number of analyzed cells, the number of neurites, and their length. Neurite outgrowth was quantified by two criteria, the average number of neurites per cell and the average length of neurites per cell. Both criteria were found to be correlated and to be equally suited to describe neurite outgrowth in the present study under all experimental conditions. Comparison of this novel procedure with a manual method that we have used previously to quantify tectal neurite outgrowth (Morales et al., 1993
In an attempt to analyze as many neurons as possible, we used a higher cell density of retinal neurons in the present study than we did previously with tectal neurons. Therefore, in our automatic procedure, we are more conservative and underestimate neurite outgrowth for two reasons. First, fasciculated neurites cannot be distinguished from single isolated neurites. This is attributable to the process of eroding to a single pixel width (Fig. 1), which is done before the neurites are measured and leads to an underestimation of the number of neurites. Second, growing neurites are more likely to encounter other neurons than in the low-density cultures used previously. Therefore the very long neurites are undetectable, which leads to an underestimation of neurite length. The cultures of retinal cells analyzed in this study contained a variable number of single cells and cell aggregates, which differed between distinct preparations of retinal cells and which were also dependent on the cell density at the time of plating. To avoid an influence of the total cell numbers and the size distribution of the aggregates to the outcome of the analyses, only data obtained in one single experiment were compared with each other, and data of consecutive experiments have not been pooled.
The main advantage of our approach is that any bias that might be introduced by the investigator (if neurites have to be chosen by eye and labeled manually for processing) can be excluded. Because a large number of neurons can be evaluated, subtle modulatory effects on neurite outgrowth can be revealed with this method, which therefore might facilitate future studies to elucidate signal transduction mechanisms leading to neurite extension.
NrCAM and NgCAM as receptors involved in neurite outgrowth
In the present study we provided strong evidence that NrCAM on retinal neurons mediates F11-triggered enhancement of FN-induced neurite outgrowth, either as a receptor or as component of a receptor complex. Therefore, the NrCAM molecule plays the role of a receptor in F11-induced neurite outgrowth of two distinct types of neurons, namely, retinal neurons (this study) and tectal neurons, as shown previously (Morales et al., 1993
Our approach does not allow us to distinguish between cis and trans interactions of F11 and NrCAM, because the F11 molecule is most likely bound to the substratum in random orientations. Therefore, concerning the retina, our model may reflect an in vivo interaction of NrCAM with F11 in the same cell membrane, with released F11 in the ECM or with F11 on the opposing cell membrane. A related situation has been analyzed recently in another cellular context, namely, outgrowth of dorsal root ganglion neurons. These cells extend neurites on immobilized NgCAM (Kuhn et al., 1991
on glia (Peles et al., 1995
Binding to F11 is not the only heterophilic molecular interaction that has been characterized for the NrCAM protein, which also binds homophilically (Mauro et al., 1992
In contrast to the functional role of the F11-NrCAM interaction in neurite outgrowth, the significance of the F11-NgCAM interaction is currently unknown. We therefore examined in this study whether NgCAM could play a role in F11-enhanced retinal neuron outgrowth, but we could not provide evidence for involvement of this molecule (Fig. 7a-c). One possible reason for this may be that binding of NgCAM on the neuronal membrane to substrate-bound F11 does not elicit a signal or may be undetectable. Binding of F11 to NgCAM has been shown using NgCAM-coated covaspheres, on which the molecule is most likely immobilized in random orientations (Brümmendorf et al., 1993
Cooperativity between different outgrowth-mediating receptor systems
Because the size of the vertebrate genome is to small to encode all types of cellular interactions in neurohistogenesis by distinct receptor-ligand pairs, any combinatorial principle of cell surface labeling is of great conceptual interest. In the present experiments we provided evidence of cooperativity between two different receptor systems on retinal neurons, namely, integrins and IgSF molecules, with respect to neurite outgrowth. Our data suggest that the retinal neuron outgrowth response is based on stimulation of neuronal integrins by FN and enhanced by triggering of neuronal NrCAM by F11. Similar cooperative effects have been reported recently for other molecules involved in neurite outgrowth. Concerning retinal neurons, it has been demonstrated that purified NgCAM strongly promotes outgrowth, but purified NrCAM does not. However, if both are mixed, a synergistic growth-promoting effect is observed (Morales et al., 1996
Enhancement of
We have shown that F11-triggered enhancement of FN-dependent retinal neurite outgrowth can be blocked by an mAb that interferes with
In our model, which is shown in Figure 8, a functional cooperation of the
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Figure 8. Model of NrCAM-mediated enhancement of
Currently, we cannot formally exclude a second explanation for our data. We observed that even if a functionally saturating concentration of the
The molecular mechanisms of cooperation between the NrCAM and the integrin pathway remain uncharacterized. However, it may be important in this context that NrCAM and other members of the L1 subgroup that are involved in neurite outgrowth (for review, see Brümmendorf and Rathjen, 1995
Because we used preparations of freshly dissociated whole E6 retinas, cells examined in our study are composed of at least two cell populations, undifferentiated neuroepithelial precursor cells and differentiated retinal ganglion cells. We therefore refer to the cells that extend neurites in our assays as early embryonic retinal cells, but we assume that most of them represent retinal ganglion cells (RGCs) for four reasons. First, RGCs are the first differentiating neurons in the retina at E6 (for review, see Mey and Thanos, 1992
Our model of a functional cooperation of integrin- and IgSF-mediated neurite outgrowth is related to a similar model proposed previously for dorsal root ganglion (DRG) neurons. These neurons are known to extend neurites in response to axonin-1/TAG-1 (Kuhn et al., 1991
FOOTNOTES Received Aug. 18, 1997; revised Oct. 31, 1997; accepted Dec. 19, 1997.
This study was partly supported by European Union Biotechnology Programme Project BIO4-CT96-0450, The Role of the Neural Cell Adhesion Molecule L1 and its Ligands in Normal Brain Development and Hereditary Brain Diseases. We thank Dr. A. Gierer for generous support and encouragement and Dr. F. G. Rathjen for stimulating discussions. The kind gift of antibodies by Drs. G. Morales, H. Volkmer, and G. E. Pollerberg is gratefully acknowledged. We thank Drs. H. Volkmer, F. G. Rathjen, and T. Lufkin for helpful comments on this manuscript, A. Schöffski for technical assistance, and C. Hug for secretarial help.
Correspondence should be addressed to Dr. Thomas Brümmendorf, Max-Delbrueck-Centrum für Molekulare Medizin, Robert-Rösslestrasse 10, D-13122 Berlin, Germany.
Dr. Treubert's present address: Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029.
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