©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Novel Alternatively Spliced Agrin mRNA That Is Preferentially Expressed in Non-neuronal Cells (*)

Guoshan Tsen (1), Audrey Napier (1), Willi Halfter (2), Gregory J. Cole (1)(§)

From the (1)Neurobiotechnology Center and Department of Cell Biology, Neurobiology and Anatomy, The Ohio State University, Columbus, Ohio 43210 and the (2)Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel agrin isoform was identified based on the isolation of an agrin cDNA from E9 chick brain that lacked 21 base pairs (bp) in the NH-terminal encoding region of the agrin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) of E9 chick brain mRNA confirmed the existence of this agrin isoform in brain, although the novel splice variant represents a minor fraction of agrin mRNA in brain. However, upon analysis of chick brain astrocyte mRNA, smooth muscle mRNA, and cardiac muscle mRNA by RT-PCR, we show that this novel agrin isoform is the predominant agrin isoform in these non-neuronal cell populations. We extended our analyses to examine the expression of this agrin mRNA isoform during chick development and show that the agrin mRNA lacking this 21-bp exon is up-regulated with brain development, consistent with the increase in glial number during brain development, while the agrin isoform that does not undergo splicing and thus contains the 21-bp exon is down-regulated in brain development. Because the 21-bp exon is inserted in the region of chick agrin which encodes the putative signal sequence of agrin, with the signal peptidase site immediately preceding the putative first amino acid of the mature protein being deleted as a result of splicing, these data raise the interesting possibility that the presence or absence of this alternatively spliced exon may differentially regulate processing of the agrin protein in neuronal and non-neuronal cells, respectively.


INTRODUCTION

Critical to the proper development of the nervous system is a highly regulated pattern of gene expression that defines processes necessary for the interaction of cells with their environment and neighboring cells. Numerous studies have documented the importance of extracellular signals in neural development, with these signals regulating processes as diverse as cell-matrix interactions, cell motility, and cell differentiation. A well-characterized model system for the study of the role of extracellular matrix proteins in nervous system development is agrin and its association with neuromuscular synaptogenesis (for reviews see Refs. 1 and 2). Agrin was initially identified as a neuronal basal lamina protein capable of inducing the aggregation of acetylcholine receptors (AChR)()in the developing neuromuscular junction(3, 4) , with its ability to also induce the aggregation of other synapse-associated proteins suggesting a key role for agrin in synaptogenesis(5, 6) . Although it was believed that agrin could not be isolated in its intact form from basal lamina(3) , it was recognized that an agrin proteolytic fragment of about 100 kDa was capable of mediating the AChR clustering effects of agrin(3, 7) . However, recent studies have demonstrated that the agrin mRNA encodes a 400-kDa heparan sulfate proteoglycan, which contains an approximately 250-kDa core protein(8) . In light of the wealth of data implicating a role for heparin and/or heparan sulfate proteoglycans in agrin function(9, 10, 11) , it remains to be determined if heparan sulfate proteoglycans other than agrin are involved in AChR aggregation during neuromuscular synaptogenesis.

Recent studies have focused on a molecular characterization of agrin mechanisms and have shown that agrin has alternatively spliced mRNA isoforms(12, 13, 14, 15) . The demonstration that alternative splice sites, designated A and B, are restricted to the region of the mRNA that codes for the functional COOH-terminal domain of agrin has been of particular interest(12, 14) , since these data indicate that the function of agrin can be regulated in a cell- or tissue-specific manner. For example, it is clear that the agrin isoform, which lacks the alternative exons, is not functional in AChR clustering assays(12, 14) . In addition, single-cell RT-PCR experiments have shown convincingly that the agrin isoform is the predominant isoform expressed by non-neuronal cells, with combinations of alternative splicing of exons occurring in neurons(16) . Thus, these data imply that functional forms of agrin, defined in terms of their ability to aggregate AChR, are restricted to neurons. However, the role of alternative splicing in agrin functions becomes clouded, based on recent studies describing the role of -dystroglycan as a putative receptor for agrin(17, 18, 19, 20) . The binding of agrin to -dystroglycan occurs in a heparin-dependent manner(17) , consistent with earlier analyses of agrin function, but the agrin-dystroglycan interaction is not isoform-dependent(20) . Likewise, the ability of agrin-dystroglycan interactions to aggregate AChR in muscle is not inhibited by antibodies that perturb agrin-dystroglycan binding (20). Thus, it remains to be determined by what mechanism(s) alternative splicing of the agrin mRNA regulates the expression of functional forms of the protein.

Our recent studies demonstrating that agrin is a HSPG are noteworthy, since we have shown previously that this HSPG is capable of binding to and modulating NCAM function(21) . In view of the demonstration that the majority of agrin expression occurs in brain(8, 22) , but agrin's function in brain has remained elusive, our studies raise the interesting possibility that brain agrin has a role in cell adhesion processes. Accordingly, our studies suggest that the heparan sulfate chains of agrin, which appear to be localized to the NH-terminal half of the molecule, are critical to cell adhesion to NCAM(8, 21) , but are not likely involved in the AChR clustering activity of agrin. In the course of our molecular cloning of agrin, we isolated a cDNA lacking 21 bp in the corresponding 5` region of the mRNA. In the present study, we show by RT-PCR that splicing of this novel exon occurs in a cell- and tissue-dependent fashion, with splicing occurring predominantly in non-neuronal cells. Thus, in accord with the studies of Smith and O'Dowd(16) , it appears that the inclusion of alternatively spliced exons in agrin mRNA is preferentially restricted to neurons. We have named this new agrin isoform in non-neuronal cells agrin-related protein-3 (ARP-3), and the alternative splice site C, in keeping with the previous nomenclature for agrin isoforms in chicken(14) .


EXPERIMENTAL PROCEDURES

Isolation of Agrin cDNA

A cDNA encoding agrin, and lacking 21 bp when compared to the previously published chick agrin cDNA(14) , was isolated by screening a random-primed E9 chick brain cDNA library with a restriction fragment from a previously isolated agrin cDNA(8) . Briefly, a 711-bp SstI restriction fragment from the 5` region of agrin clone pPG3 (8) was used to screen 600,000 recombinants from the E9 chick brain cDNA library according to published protocols (8). One resulting agrin clone, named 801, was found to lack 21 bp when compared to the published chick agrin nucleotide sequence(14) .

RT-PCR Analysis of ARP-3 mRNA Expression

Total RNA from various ages of embryonic chick brain and heart, or from cultured chick brain astrocytes, was isolated according to Chomczynski and Sacchi (24). Poly(A) RNA was then isolated using the Straight A's mRNA isolation system (Novagen). First strand cDNA was then synthesized from 1 µg of mRNA using the reverse transcriptase SuperScript (Life Technologies, Inc.). RNA was then hydrolyzed with NaOH at 65 °C for 30 min, and, following neutralization with acetic acid, the cDNA was purified using Geneclean (Bio 101). Following isolation of the cDNA by precipitation with glycogen and ethanol, 10% of the isolated cDNA was used for PCR amplification.

For PCR analysis at different developmental ages, the use of equivalent amounts of cDNA was confirmed by RT-PCR of cDNA using chicken -actin PCR primers. For PCR amplification, 1 µl of the cDNA synthesis mixture described above was used. Primer pairs used for RT-PCR of agrin mRNA were a forward 19-mer: 5`-TGGAGCACTGCGTGGAAGA-3` and a backward 20 mer: 5`-TGCAGACACAGGAGGCTTGG-3`. cDNA was amplified for 28 cycles at an annealing temperature of 52 °C, and PCR products were then analyzed by electrophoresis on 3% agarose gels, with DNA visualized by ethidium bromide staining. To confirm the authenticity of the PCR products, they were subcloned into the pBluescript vector pCR-Script SK(+) (Stratagene) and subsequently sequenced by the dideoxy chain termination method(23) . The lower M PCR product was shown to contain 146 bp and lack the 21 bp insertion, while the larger PCR product was 167 bp in length and contained the 21-bp insertion.


RESULTS

Identification of Novel Agrin mRNA Splice Variant

During the course of our molecular cloning of an NCAM-binding HSPG, which was subsequently shown to be identical to agrin(8) , we isolated an agrin cDNA that exhibited sequence differences to the published chick agrin cDNA(22) . This partial cDNA, named clone 801, was isolated by rescreening of a random-primed E9 chick brain cDNA library with a restriction fragment from agrin clone pPG3, which encoded nucleotides 560-1271 of agrin(8) . Upon sequence analysis, it was determined that clone 801 was identical with the published chick agrin cDNA at its 5` and 3` ends, but a 21-bp region from nucleotides 169-189 was absent from clone 801 (Fig. 1). Since these data raised the possibility that this cDNA defines a new splice variant of agrin, we named this cDNA agrin-related protein-3 (ARP-3), and the alternative exon encoding this domain was named C, in accord with the previous nomenclature for alternative splice sites in chicken agrin(14) .


Figure 1: Comparison of nucleotide and deduced amino acid sequences for agrin and ARP-3 cDNAs. The nucleotide position is indicated at top left, with the corresponding amino acid sequence shown at the bottom. The underlined nucleotide sequence denotes the forward primer sequence used for RT-PCR studies. The dashed line in the ARP-3 sequence indicates the absence of the 21-bp insertion observed in the previously published chick agrin cDNA. The double-underlined amino acid sequences represent consensus signal peptidase sites in the NH-terminal region of agrin, showing that the first signal peptidase site would be eliminated by alternative splicing.



In order to confirm whether the ARP-3 cDNA was present in chick brain as an mRNA, we designed PCR primers flanking this region. The forward primer sequence is indicated in Fig. 1, and the reverse primer was from nucleotides 319-300 of chick agrin. As shown in Fig. 2, by PCR of the clone 801 plasmid, a 146-bp product is obtained as predicted by the cDNA sequence. Upon PCR amplification of E9 chick brain mRNA that had been reverse-transcribed to yield single strand cDNA, the predominant product obtained was a 167-bp fragment, with the 146-bp product representing a minor mRNA species in E9 chick brain (Fig. 2). The authenticity of these PCR products was confirmed by subcloning of the products into PCR pBluescript, followed by dideoxy sequencing. The nucleotide sequence of each PCR product confirmed that the 167-bp fragment is identical with the published chick agrin cDNA, and that the 146-bp fragment is identical with the ARP-3 cDNA.()Thus, these data show that a novel agrin mRNA species is expressed in chick brain, albeit at extremely low levels.


Figure 2: RT-PCR analysis of E9 chick brain agrin mRNA expression. mRNA was isolated from E9 chick brain, reverse-transcribed with SuperScript reverse transcriptase and amplified by PCR using primers flanking the 21-bp region not inserted into ARP-3. PCR products were then separated on 3% agarose gels, with DNA visualized by ethidium bromide staining. Lane 1, positive control using clone 801 plasmid, showing that a 146-bp PCR product is obtained; lane 2, PCR amplification of E9 chick brain cDNA, showing the presence of a major 167-bp product, as well as the 146-bp product corresponding to the ARP-3 cDNA; lane 3, negative control using E9 chick brain mRNA not treated with reverse transcriptase, showing absence of any PCR products; lane 4, negative control showing absence of PCR products when E9 brain cDNA is omitted from the PCR reaction mixture.



Tissue Distribution of ARP-3 mRNA

Since previous analyses of agrin mRNA expression have suggested that non-neuronal cell agrin mRNA is subjected to alternative splicing, usually resulting in splicing out of all identified alternatively spliced exons, with only various combinations of splicing occurring in neurons(16) , we were interested in ascertaining whether the alternatively spliced ARP-3 mRNA is also restricted to non-neuronal cells. To address this question, mRNA was isolated from chick brain astrocyte cultures, E10 gut (smooth muscle), and E10 heart (cardiac muscle). Following reverse transcription, these cDNAs were amplified using the PCR primers that flank the 21-bp region absent from ARP-3. These data show that the major PCR product in glia is 146 bp, indicating that the primary transcript in chick glia is the ARP-3 mRNA, lacking the 21-bp insertion (Fig. 3). In addition, both muscle tissues show an enrichment for the ARP-3 mRNA, as exhibited by the abundant signal for the 146-bp PCR product (Fig. 3). Although it is also evident that the 167-bp PCR product is obtained using muscle and glia mRNA, it remains to be determined what cellular source is responsible for expression of the unspliced form of agrin (i.e. containing the 21-bp insertion).


Figure 3: RT-PCR analysis of agrin isoform mRNA expression in embryonic chick brain, glia, and muscle. mRNA was isolated from E9 chick brain, cultured chick brain astrocytes, E10 smooth muscle (gut), and E10 cardiac muscle, and subjected to RT-PCR. brain, E9 chick brain, showing that the predominant agrin isoform contains the 21-bp insertion; glia, chick astrocytes, showing that the major agrin isoform lacks the 21-bp sequence and corresponds to ARP-3; gut, E10 smooth muscle cDNA after PCR, with this tissue exhibiting significant amounts of both isoforms, although the ARP-3 isoform predominates; heart, E10 chick heart, showing the presence of both agrin isoforms.



Developmental Expression of Agrin Isoforms

To extend our understanding of the expression pattern of the ARP-3 agrin mRNA, we analyzed by RT-PCR the expression of this mRNA during chick brain and heart development. Based on our demonstration that glia exhibit an enrichment for the ARP-3 mRNA, we predicted that its expression should become augmented during brain development, consistent with gliogenesis during late development. As shown in Fig. 4A, during chick brain development the ARP-3 mRNA does display an increase in abundance, while the major agrin mRNA species, containing the 21-bp insertion, is diminished. In parallel analyses, we employed -actin primers for RT-PCR, and these data indicate that different cDNA levels did not account for the observed alteration in agrin expression by RT-PCR, as similar levels of actin could be detected at each developmental age (Fig. 4).


Figure 4: Analysis of developmental expression of agrin isoforms by RT-PCR. A, E9 and E18 chick brain mRNA was analyzed by RT-PCR, followed by separation of PCR products on 3% agarose gels. It can be seen that while the major brain agrin isoform is diminished with development, the level of the ARP-3 agrin isoform is augmented. B, E7-E18 chick heart mRNA was analyzed by RT-PCR, followed by agarose gel electrophoresis. These data show a significant diminution of both agrin isoforms occurring subsequent to E10 of chick heart development. In both brain and heart cDNAs, -actin levels were determined by RT- PCR, and these data show that the 340-bp -actin PCR product is expressed at similar levels during development.



We also examined by RT-PCR the expression of agrin mRNA isoforms during chick heart development, and these data indicate that both agrin isoforms are reduced during heart development (Fig. 4B). The pattern of expression of these isoforms is similar in chick heart, with both species displaying a decrease in abundance subsequent to E10 of heart development. Thus, it appears that distinct non-neuronal cell populations display differences in expression of the ARP-3 agrin isoform, with glial expression in brain resulting in a developmental up-regulation, while in heart this isoform is down-regulated during development.


DISCUSSION

We demonstrate in the present study that an additional spliced isoform of agrin is expressed in brain and non-neuronal tissues. However, unlike the previous identification of alternatively spliced isoforms of agrin (12, 14) where the splice sites were restricted to the region of the agrin mRNA encoding the COOH-terminal domain of the protein, our studies indicate a novel splice site in the 5`-region of the agrin mRNA (Fig. 5). It is therefore interesting to note that in previous studies on the agrin gene in rat the 5`-region of the gene was not mapped, and thus the presence of this alternatively spliced exon had gone undetected(26) . Our data strongly suggest that in brain this 21 bp insertion into chick agrin appears to be localized to neuronal cells, since analysis of chick astrocyte mRNA by RT-PCR indicates the presence of agrin mRNA lacking the 21-bp domain. Upon analysis of non-neuronal chick tissues, in particular smooth and cardiac muscle, we have also observed an abundance of the agrin isoform lacking the alternatively spliced 21-bp insert. It is presently unclear why the agrin isoform containing the 21-bp insert is detected in chick glia and muscle, although it is likely that slight neuronal contamination of the astrocyte cultures can account for the minor levels of agrin mRNA containing the 21-bp exon in these cultures(25) . It remains to be determined whether non-neuronal cell populations in gut and heart are expressing the spliced form of agrin, or if neuronal components such as cardiac neural crest account for this expression. However, it has been shown previously that the low levels of the mRNA encoding the agrin isoform can be expressed in muscle and non-neuronal tissues(14, 16, 27, 28) , indicating that not all alternative exons are spliced out in non-neuronal agrin. Thus, it remains possible that the splicing at site C is regulated in non-neuronal cells. Although this remains to be confirmed, our data do suggest that in non-neuronal cells the majority of agrin isoforms lack the C exon, and thus it appears that the prevalent agrin isoform in non-neuronal cells is agrin.


Figure 5: Schematic model of agrin, showing structural domains and alternatively spliced exons. The alternative splice sites are indicated with their corresponding amino acid sequences and show that the site C splice site is contained within the putative signal sequence peptide of agrin. -, putative signal sequence; , Kazal type protease inhibitor; &cjs2090;, laminin homology domain; , epidermal growth factor-like repeat; °°°&cjs0822;, potential GAG attachment site; &cjs0822;, potential N-linked glycosylation site; /&cjs0822;&cjs0822;, alternative splicing site.



Based on our observation that in chick brain the predominant agrin form in glia lacks the 21-bp insert at splice site C, we reasoned that with development we should see an augmentation in expression of this isoform, concomitant with gliogenesis. Since previous studies have shown a pronounced diminution in agrin during chick brain development (8, 22), this would raise the possibility that glial agrin could be mediating distinct functions as neuronal agrin is down-regulated. Our present studies demonstrate a significant increase in the expression of ARP-3 mRNA, coincident with a marked decrease in the unspliced form of agrin. Thus, these data indicate distinctive patterns of regulation of agrin during brain development in neurons and glia. In light of our recent demonstration that agrin is capable of modulating NCAM function (21), one can speculate that up-regulated glial expression of agrin during brain development may selectively modulate glial and/or glial-neuronal cell interactions, by contributing to localized increases in agrin expression during development.

It is presently unclear what function the alternative splicing of the 21-bp exon may impart on agrin, especially in light of previous studies which have shown that the splicing out of alternative exons in non-neuronal agrin results in an inability to induce AChR aggregation (12, 14, 28). It is of interest that the alternatively spliced exon we have identified is localized to a region of agrin which has been suggested to contain the signal sequence for this protein. Thus, splicing out of the 21-bp exon at splice site C would affect the putative signal sequence, conceivably regulating the processing of this protein in non-neuronal and neuronal cells. For example, the splicing out of the 21-bp exon removes 7 amino acids from the putative signal sequence of agrin, including the consensus signal peptidase site immediately preceding the putative first amino acid of the mature protein (Fig. 1). Thus, with this signal peptidase site deleted, two possibilities exist in terms of processing of this splice variant of agrin: 1) the next available signal peptidase site would be utilized, resulting in non-neuronal cells lacking 24 amino acids at their NH terminus (see Fig. 1); 2) the character of the signal sequence would be altered, possibly precluding its removal and thus resulting in an agrin protein that would remain associated with the plasma membrane, rather than being secreted directly. However, this latter possibility would seem unlikely since agrin has been shown to be a secreted protein in both neurons (29, 30) and glia(21) , although our analyses in glia used long-term metabolic labeling of cultures which may have masked cleavage of a membrane-associated protein, with its subsequent release into the conditioned medium(21) . Although we have not yet been able to elucidate the functional significance of the splicing at site C, an alternative possibility that must be considered is that the putative first amino acid in the mature agrin protein (Asp) is in fact not the NH terminus of the mature protein, and thus the putative signal sequence upstream of this amino acid is not agrin's signal peptide. This possibility warrants consideration, since the absence of splicing would then result in the insertion of 7 amino acids in the mature protein which could possibly modulate the structure of the protein. For example, the 7-amino acid insertion contains a cluster of basic amino acids, which could regulate the ability of agrin to interact with anionic molecules. Likewise, the 7-amino-acid insert could introduce a putative protease cleavage site that could then alter the structure of the protein containing the insert. Moreover, in view of recent studies which have shown that agrin encoded by the full-length agrin cDNA is not secreted, and that the signal peptide of hemagglutinin had to be employed to facilitate secretion of the full-length agrin protein(28) , it remains possible that the signal sequence of agrin has not been identified.

Since in previous studies it was also thought that agrin could not be isolated in its intact form(3) , the NH-terminal amino acid of the mature protein has never been conclusively identified. We therefore undertook protein sequencing experiments using immunopurified agrin from chick brain, which we have shown can be isolated intact as a HSPG(8, 21) . The heparan sulfate chains were removed from this agrin using heparitinase, and the protein was then separated by gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Unfortunately, the NH-terminal amino acid sequence could not be obtained from this preparation, or from soluble purified agrin, suggesting that the NH terminus is blocked. It therefore remains to be determined if splicing of this 21-bp exon alters the properties of the mature protein versus the signal peptide. Thus, our goal in future studies will be to elucidate the possible function of this splicing event in agrin, with a focus on whether the processing of agrin is affected by splicing at site C and whether this splicing may modulate the ability of agrin to interact with adhesion proteins, such as NCAM, or its ability to regulate AChR aggregation in muscle.


FOOTNOTES

*
This work was supported by a grant from the National Institutes of Health and a National Institutes of Health Minority Supplement grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Neurobiotechnology Center, The Ohio State University, 184 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-292-1205; Fax: 614-292-5379.

The abbreviations used are: AChR, acetylcholine receptor(s); RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); HSPG, heparan sulfate proteoglycan; NCAM, neural cell adhesion molecule; ARP-3, agrin-related protein-3.

G. Tsen and G. J. Cole, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Jeff Masters, John Oberdick, and Pappachan Kolattukudy for their helpful discussion during the course of this work.


REFERENCES
  1. McMahan, U. J. (1990) Cold Spring Harbor Symp. Quant. Biol.55, 407-418 [Medline] [Order article via Infotrieve]
  2. Hoch, W., Campanelli, J. T., and Scheller, R. H. (1994) J. Cell Biol.126, 1-4 [Medline] [Order article via Infotrieve]
  3. Nitkin, R. M., Smith, M. A., Magill, C., Fallon, J. R., Yao, Y. M., Wallace, B. G., and McMahan, U. J. (1987) J. Cell Biol.105, 2471-2478 [Abstract]
  4. Reist, N. E., Magill, C., and McMahan, U. J. (1987) J. Cell Biol.105, 2457-2469 [Abstract]
  5. Nitkin, R. M., and Rotschild, T. C. (1990) J. Cell Biol.111, 1161-1170 [Abstract]
  6. Wallace, B. G. (1989) J. Neurosci.9, 1294-1302 [Abstract]
  7. Godfrey, E. (1991) Exp. Cell Res.195, 99-109 [CrossRef][Medline] [Order article via Infotrieve]
  8. Tsen, G., Halfter, W., Kröger, S., and Cole, G. J. (1995) J. Biol. Chem.270, 3392-3399 [Abstract/Free Full Text]
  9. Wallace, B. G. (1990) J. Neurosci.10, 3576-3582 [Abstract]
  10. Gordon, H., Lupa, M., Bowen, D., and Hall, Z. (1993) J. Neurosci.13, 586-595 [Abstract]
  11. Ferns, M. J., Campanelli, J. T., Hoch, W., Scheller, R. H., and Hall, Z. (1993) Neuron11, 491-502 [Medline] [Order article via Infotrieve]
  12. Ferns, M., Hoch, W., Campanelli, J. T., Rupp, F., Hall, Z. W., and Scheller, R. H. (1992) Neuron8, 1079-1086 [Medline] [Order article via Infotrieve]
  13. Rupp, F., Payan, D. G., Magill-Solc, C., Cowan, D. M., and Scheller, R. H. (1991) Neuron6, 811-823 [Medline] [Order article via Infotrieve]
  14. Ruegg, M. A., Tsim, K. W. K., Horton, S. E., Kröger, S., Escher, G., Gensch, E. M., and McMahan, U. J. (1992) Neuron8, 691-699 [Medline] [Order article via Infotrieve]
  15. Thomas, W. S., O'Dowd, D. K., and Smith, M. A. (1993) Dev. Biol.158, 523-535 [CrossRef][Medline] [Order article via Infotrieve]
  16. Smith, M. A., and O'Dowd, D. K. (1994) Neuron12, 795-804 [Medline] [Order article via Infotrieve]
  17. Bowe, M. A., Deyst, K. A., Leszyk, J. D., and Fallon, J. R. (1994) Neuron12, 1173-1180 [Medline] [Order article via Infotrieve]
  18. Campanelli, J. T., Roberds, S. L., Campbell, K. P., and Scheller, R. H. (1994) Cell77, 663-674 [Medline] [Order article via Infotrieve]
  19. Gee, S. H., Montanaro, F., Lindenbaum, M. H., and Carbonetto, S. (1994) Cell77, 675-686 [Medline] [Order article via Infotrieve]
  20. Sugiyama, J., Bowen, D. C., and Hall, Z. W. (1994) Neuron13, 103-115 [Medline] [Order article via Infotrieve]
  21. Burg, M. A., Halfter, W., and Cole, G. J. (1995) J. Neurosci. Res.41, 49-64 [Medline] [Order article via Infotrieve]
  22. Tsim, K. W. K., Ruegg, M. A., Escher, G., Kröger, S., and McMahan, U. J. (1992) Neuron8, 677-689 [Medline] [Order article via Infotrieve]
  23. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A.74, 5463-5467 [Abstract]
  24. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem.162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  25. Kelly, M. M., Phanhthourath, C., Brees, D. K., McCabe, C. F., and Cole, G. J. (1995) Dev. Brain Res.85, 31-47 [CrossRef][Medline] [Order article via Infotrieve]
  26. Rupp, F., Ozcelik, T., Linial, M., Peterson, K., Francke, U., and Scheller, R. (1992) J. Neurosci.12, 3535-3544 [Abstract]
  27. Ma, E., Morgan, R., and Godfrey, E. W. (1994) J. Neurosci.14, 2943-2952 [Abstract]
  28. Gesemann, M., Denzer, A. J., and Ruegg, M. A. (1995) J. Cell Biol.128, 625-636 [Abstract]
  29. Magill-Solc, C., and McMahan, U. J. (1988) J. Cell Biol.107, 1825-1833 [Abstract]
  30. Reist, N. E., Werle, M. J., and McMahan, U. J. (1992) Neuron8, 865-868 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.