©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 19-kDa Protein Belonging to a New Family Is Expressed in the Golgi Apparatus of Neural Cells (*)

(Received for publication, June 9, 1994; and in revised form, October 12, 1994)

Délara Sabéran-Djoneidi (1) Isabel Marey-Semper (1) Renée Picart (2) Jeanne-Marie Studler (1) Claude Tougard (2) Jacques Glowinski (1) Matthieu Lévi-Strauss (1)(§)

From the  (1)INSERM U.114, Chaire de Neuropharmacologie, Collège de France, 11, Place Marcelin-Berthelot, 75005 Paris, France and (2)Groupe de Biologie de la Cellule Neuroendocrine, Collège de France, URA CNRS 1115, 11, Place Marcelin-Berthelot, 75005 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mouse 8.5 mRNA encodes a 171-residue novel protein which displays a highly significant similarity with the product of the previously characterized neuronal p1A75 cDNA (Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E.(1983) Cell 33, 671-682). Northern blot and in situ hybridization experiments indicated that the 8.5 mRNA is specifically expressed in neural and neuroendocrine tissues. An affinity-purified antibody directed against the recombinant 8.5 protein demonstrated the existence of the 19-kDa natural protein in brain and evidenced its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural analysis of the same preparation revealed a specific labeling of all the Golgi saccules and of some vesicles in the Golgi zone. In transfected COS cells, the exogenous protein was also detected in the Golgi area, indicating, therefore, the presence of a Golgi targeting signal in its primary sequence.


INTRODUCTION

To identify mRNAs whose expression is enriched at embryonic day (E) (^1)17 or E20 in the embryonic striatum, we have previously performed a differential screening of a cDNA library with two radiolabeled probes corresponding to striatal mRNAs extracted at E17 and E20(1) . The 8.5 mRNA was selected since it was enriched at E20. The sequence of a partial cDNA clone showed a 60% similarity between the translation product of its main open reading frame and the protein encoded by the neuronal p1A75 cDNA whose organelle-like location within cells suggests an involvement in secretory, transport, or mitochondrial functions(2) . As a first step in the study of this new protein family, the structural and functional characterization of the 8.5 mRNA was undertaken. We describe here the complete nucleotide sequence of the 8.5 mRNA whose expression was detected only in neural and neuroendocrine tissues. Antibodies directed against the recombinant 8.5 protein demonstrated the existence of the natural protein, which was mainly localized in the Golgi apparatus. Furthermore, using transfected COS cells, the exogenous protein was also detected within the Golgi area, demonstrating therefore that its primary sequence contains the information for this specific subcellular compartmentalization.


MATERIALS AND METHODS

RNA Isolation and Northern Blotting

Total cellular RNA was extracted from fresh tissue or cells by the guanidium thiocyanate/phenol-chloroform extraction method(3) . Timed pregnant OFA rats (Iffa-Credo) provided a source of fetal and neonatal brains of precise gestational or post-natal ages. Other brain structures or non-neural tissues were dissected from adult Sprague-Dawley (Charles River) male rats. Dissociated neurons from cerebral hemispheres of E17 rat embryos were plated at high density (6 times 10^4/cm^2) according to Di Porzio et al. (4) and cultured for 2-4 days; astrocyte cultures were prepared from the same cerebral areas as described by Denis-Donini et al.(5) . Total cellular RNA (5 µg/lane) was fractionated on 1.2% agarose gel containing 3.7% (w/v) formaldehyde, using standard procedures(6) . Gels were blotted onto Hybond N (Amersham Corp.) nylon membranes. The 18 and 28 S ribosomal RNAs observed on the filter by UV light were used to check that equal amounts of RNA were loaded on each lane. Hybridization conditions were: 65 °C, 16 h in the presence of 2 times 10^6 cpm/ml of probe in the following solution: 5 times SSC, 5 times Denhardt's solution, 50 mM sodium phosphate, pH 6.5, and 0.4% SDS. Final washes were done in 0.2 times SSC and 0.1% SDS at 65 °C. The 8.5 full-length cDNA, labeled with [alpha-P]dCTP to a specific activity of 10^9 cpm/µg by random priming(7) , was used as a probe.

In Situ Hybridization

In situ hybridization experiments were performed as described previously(1) . Briefly, single-stranded RNA transcripts were labeled with [alpha-S]UTP (1000 Ci/mmol; Amersham) as the only source of this nucleotide. Whole embryos from Sprague-Dawley rats were sectioned on a cryostat and the sections (10-20 µm thick) were thaw-mounted on silanized glass slides, fixed with 4% paraformaldehyde, dehydrated, and stocked at -20 °C until use. Frozen sections were thawed, fixed with paraformaldehyde, permeabilized with proteinase K, and again fixed with paraformaldehyde. Prehybridization and hybridization were performed at 50 °C in a medium containing 50% formamide. Sections were then washed at 50 °C in a medium containing 50% formamide and subjected to RNase A digestion. Finally, sections were dehydrated and exposed to beta-Max films (Amersham) for 1-3 weeks. Autoradiograms were transformed into digitized images with an image analyzer (IMSTAR).

Characterization of the 8.5 cDNA

The cDNA library was constructed, as described previously(1) , from poly(A) RNA prepared from cerebral hemispheres of newborn BALB/c mice. The original, 1500-base pairs long, 8.5 cDNA was identified by differential screening of the library plated in the pBluescript (Stratagene) plasmid vector(1) . This cDNA was used as a probe to screen 2 times 10^5 cDNA clones from the same library plated in the gt10 phage vector. The insert of the longest hybridizing cDNA was excised by partial EcoRI restriction and was subcloned in both orientations in the pBluescript plasmid vector and in the M13mp19 phage vector. Both strands of the 8.5 cDNA were sequenced in M13 using the deletion method described by Dale et al.(8) coupled with the dideoxynucleotide chain termination technique (9) and the modified T7 polymerase(10) .

Recombinant 8.5 Protein and Antibody Production

The plasmid containing the 8.5 cDNA was amplified with the Taq DNA polymerase (11) using the two following primers: A, (5`-GCGGATCCATCGAAGGTAGAATGGTGAAGCTGAACAGCAA-3`); and B, (5`-ACCCACCCGGATCCCAGCTG-3`). Primer A was designed to add a BamHI restriction site and a factor Xa (IEGR) cleavage site fused to the initiating methionine of the 8.5 protein. Primer B was designed to create, by site-directed mutagenesis, a BamHI restriction site at the position 692 of the sequence shown in Fig. 1. The amplified DNA fragment was restricted using the BamHI endonuclease and cloned in the BamHI site of the pQE-8 (Qiagen) expression vector which produces a recombinant protein with a 6xHis tag on the N terminus. The construction was sequenced in order to verify the absence of mutations, and the synthesis of the recombinant protein was demonstrated by immunoblot using an antibody directed against a synthetic peptide (residues 138-155). The recombinant 8.5 protein was produced and purified using nickel-chelate affinity chromatography (12) in denaturing conditions according to the protocols provided by Qiagen. The very low solubility of the 8.5 recombinant protein in non-denaturing buffers precludes its cleavage in bulk quantities with the factor Xa and therefore the whole fusion protein was used to immunize rabbits. To this end, 0.5 mg of the recombinant protein solubilized in 8 M urea were loaded onto a 14% preparative SDS-PAGE. The gel was stained in 0.25 M KCl, and the band corresponding to the 8.5 protein was cut and washed in phosphate-buffered saline. New Zealand white rabbits were immunized three times subcutaneously with a piece of gel containing approximately 100 µg of protein. Human keratin (Sigma) coupled to a CH-Sepharose 4B (Pharmacia Biotech Inc.) column was used to get rid of a contaminating anti-keratin immunoreactivity of the serum(13) . Antibodies to the 8.5 protein were affinity-purified by passage through a column of CH-Sepharose 4B coupled in 8 M urea to the purified recombinant protein and subsequent elution in 0.1 M glycine buffer, pH 2.2.


Figure 1: A, nucleotide sequence of the murine 8.5 cDNA and translation of its longest open reading frame. B, alignment of the 79 C-terminal amino acids of the 8.5 protein (top) with the partial published (2) sequence of the product of the p1A75 cDNA (bottom). A double bar indicates an identity and a single bar indicates a replacement by an isofunctional amino acid.



Subcellular Fractionation and Western Blot Analysis

Subcellular fractions were prepared from rat post-natal day 1 brain according to Huttner et al.(14) . Briefly, neonatal rat brains were homogenized in buffered sucrose (320 mM sucrose, 4 mM Hepes, pH 7.4) using a glass-Teflon homogenizer. This homogenate (H) was centrifuged for 10 min at 800 times g; the pellet was discarded, and the supernatant was centrifuged for 15 min at 10,200 times g to yield a pellet (P2) and a supernatant (S2). The S2 fraction was then centrifuged for 1 h at 165,000 times g to yield a pellet (P3) fraction and a cytosolic fraction (S3). Equivalent fractions of each preparation, corresponding to 150 µg of homogenate, were run on a 14% SDS-PAGE and blotted onto poly(vinylidene fluroide) membranes (Bio-Rad)(15) . Carbonate extractability was tested on the P2 and P3 fractions as described previously(16) . Briefly, pellets were resuspended in a large volume of ice-cold 100 mM Na(2)CO(3) buffer, pH 11.5 (or phosphate-buffered saline for the control experiment), incubated for 1 h on ice, and centrifuged for 1 h at 200,000 times g. After saturation with 5% nonfat dry milk and 0.05% Tween-20, membranes were incubated with a 1/200 dilution of the affinity-purified antibody (corresponding to a 1/200 dilution of the rabbit serum) followed by a goat anti-rabbit IgG coupled to alkaline phosphatase. Enzymatic activity was detected with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate substrates.

Cell Cultures, Transfection, and Immunofluorescence

Dissociated neurons from cerebral hemispheres of E17 rat embryos were plated in 35-mm culture dishes at high density (6 times 10^4/cm^2) in serum-free medium according to Di Porzio et al.(4) and cultured for 2 days. For transient transfection experiments the 8.5 cDNA was amplified (11) using the primer B described above and primer C (5`-CCGCACAGTCTCGAGTAAAA-3`) which creates a XhoI site at position 141. The amplified DNA fragment was restricted using the BamHI and XhoI endonucleases and inserted in the pSVL (Pharmacia) expression vector. Transfections of COS-7 cells with this construction were performed in 35-mm culture dishes using the DEAE-dextran protocol and reagents provided in the Stratagene mammalian transfection kit. For immunofluorescence experiments, dissociated neurons or COS cells were seeded on glass coverslips and fixed with periodate-lysine-paraformaldehyde (17) for 2 h at room temperature, permeabilized with 0.005% saponin, and immunocytochemically stained using the rabbit affinity-purified specific antibody and goat immunoglobulins (IgG) against rabbit IgG labeled with tetramethylrhodamine (Biosys, Compiègne, France).

Immunoperoxidase Electron Microscopy

The immunoperoxidase procedure was performed on dissociated neurons using a preembedding approach, in situ in Petri dishes, as described previously (18) . Briefly, cells were fixed with periodate-lysine-paraformaldehyde (as described above) and permeabilized with 0.005% saponin before incubation with the rabbit affinity-purified specific antibody and then with sheep IgG against rabbit IgG labeled with peroxidase (Institut Pasteur, Paris). After postfixation in 1% glutaraldehyde, detection of peroxidase activity, postfixation in 1% osmium tetroxide, cells were embedded in situ in Epon according to Brinkley et al.(19) . After observation at the light microscopic level, selected areas of immunoreactive cells were sectioned and ultrathin sections were examined under the electron microscope without further staining.


RESULTS

Cloning and Sequence Analysis

We used a previously described screen (1) in order to identify mRNAs whose expression is enriched at E17 or E20 in the embryonic striatum. The original 8.5 plasmid was selected due to its strong hybridization with the E20 mRNA probe. Since its 1500-base pair long cDNA insert was smaller than the guessed size of the corresponding mRNA (approximately 2500 bases; see above), it was used to screen a mouse embryonic brain cDNA library constructed in the gt10 phage vector. This procedure led to the isolation of an apparently full-length cDNA whose size (2260 base pairs) corresponds to the estimated length of the mRNA if one takes into account the poly(A) tract. Its insert was subcloned in the M13mp19 phage vector and sequenced on both strands (Fig. 1A) using the deletion technique described by Dale et al.(8) . This cDNA terminates at its 3` end by a poly(A) tract preceded by a typical polyadenylation signal (AATAAA) at position 2223. The longest open reading frame encodes a polypeptide of 171 residues with a calculated molecular mass of 19 kDa. This open reading frame is initiated by the first ATG (position 160), surrounded by a favorable consensus sequence for the initiation of translation(20) , and preceded by a stop codon (TGA, position 106) in the same reading frame.

Study of hydropathy of this polypeptide by the method of Kyte and Doolittle (21) indicated that it is moderately hydrophilic with the exception of two adjoining 22-residue long stretches, being, respectively, strongly hydrophilic (residues 47-69) or hydrophobic (residues 70-92) (Fig. 2). The N terminus of the polypeptide does not fit the outline proposed for signal sequences (22) .


Figure 2: Hydropathy plot of the amino acid sequence of the murine 8.5 protein. Negative values indicate hydrophobic regions.



Comparison of this predicted polypeptide with protein data base (23) (release 37 of NBRF) revealed a highly significant similarity of its C-terminal part with the C-terminal, 83-residue long, partial sequence of the p1A75 cDNA product (2) (Fig. 1B). Indeed, 35 residues of the putative 8.5 protein were found to be identical to the partial sequence of the product of the p1A75 cDNA, and 12 were replaced by an amino acid belonging to the same biochemical group. One should note that the similarity of these two polypeptides does not include the 15 most C-terminal residues which are completely divergent.

The 8.5 protein contains also a cysteine-rich segment (residues 85-112) remotely related to the epidermal growth factor (EGF)-like regions(24) . This segment, even if it lacks cysteine 1, 2, and 4 of the type 2 sequence(24) , displays a similarity with six EGF-like regions belonging to types 2 and 3 described by Appella et al.(24) (Fig. 3).


Figure 3: Alignment of the atypical EGF-like region of the 8.5 protein (amino acids 85-112) with the partial sequence of the product of the p1A75 cDNA (2) and with the following sequences: Agrin (amino acids 1727-1747)(30) , Notch (amino acids 270-290)(31) , TGF-binding protein (TGF-bp) (amino acids 850-874)(32) , coagulation factor IX (F.IX) (amino acids 108-126)(33) ; low density lipoprotein receptor (LDL-R) (amino acids 330-353)(34) . Identical or isofunctional residues are boxed.



Expression of the 8.5 mRNA

The 8.5 mRNA was further characterized by Northern blot analysis as well as by in situ hybridization. The pattern of hybridization of the 8.5 cDNA probe to RNA from a variety of rat tissues (Fig. 4A) revealed an absence of expression in liver, spleen, testis, kidney, and heart and a strong but variable signal in all brain and spinal cord samples tested. The 8.5 mRNA was also strongly expressed in the pituitary and to a much lesser extent in the adrenal gland. The 8.5 probe recognized an apparently unique mRNA with a migration rate slightly slower than the 18 S ribosomal RNA. Comparison of the 8.5 hybridization signal in RNA samples extracted from cultured astrocytes or neurons suggested a neuronal origin for this mRNA whose faint expression in the astrocyte culture can be attributed to a neuronal contamination. As shown in Fig. 4B, the 8.5 mRNA was present as early as E14 in cerebral hemispheres, where, as expected, its expression increased during embryogenesis. The maximal abundance of this mRNA was found around E20, and its expression faded afterward to reach adult levels at P15.


Figure 4: A, neural expression of the 8.5 mRNA Northern blot hybridization of total RNA samples from various adult rat organs, brain structures, or cultured cells using the full-length 8.5 cDNA as a probe. At 4 days of exposure of the autoradiogram, the migration of the 18 and 28 S ribosomal RNAs is indicated. B, Northern blot hybridization of total RNA samples from rat cerebral hemispheres from various embryonic (E) or post-natal ages (P). Overnight exposure of the autoradiogram.



In situ hybridization to 8.5 mRNA, performed on an oblique section of the whole E17 head (Fig. 5A), showed an intense labeling in several brain areas, this mRNA being mostly expressed in the striatal rudiment, the cerebral cortical plate, and in germinal zones containing proliferating cells. The spinal cord and the nearby dorsal root ganglia were faintly labeled. This is in contrast with in situ hybridization experiments performed on a similar section with the neuron-specific SCG10 probe indicating a very intense staining of these structures(1) . A frontal section of the anterior part of the whole E20 head (Fig. 5B) revealed a strong labeling of the retinas and of the brain but not of the rest of the head.


Figure 5: In situ hybridization to 8.5 mRNA. A, oblique section of a rat E17 head; B, anterior frontal section of a rat E20 head.



Expression of the 8.5 Protein

Western blot analysis (Fig. 6A) of 8.5 expression using an affinity-purified antibody raised against the recombinant 8.5 protein showed the existence of one predominant band in post-natal day 1 rat brain. The apparent molecular mass of the corresponding protein (19 kDa) is very similar to that of the recombinant 8.5 protein after removal of its added polyhistidine tail by proteolytic cleavage with coagulation factor Xa (data not shown). The 8.5 protein was absent from the soluble cytosolic fraction (S3) and was found at comparable levels in the post nuclear pellet (P2) and the supernatant (S2) in which it was fully associated with the pellet (P3) obtained after ultracentrifugation. We also performed (Fig. 6B), on the P2 and P3 fractions, a carbonate treatment which allows to discriminate between integral and peripheral membrane proteins(16) . Fig. 6B shows that the 8.5 protein is not released from the P2 and P3 fractions after carbonate treatment. Control experiments, performed with an anti-synapsin antibody indicated that this peripheral protein is, as expected, completely extracted from the P2 and P3 pellets by the carbonate treatment (data not shown). Another protein of apparent molecular mass 4O kDa was also weakly recognized by the antibody. The presence of this protein in tissues in which the 8.5 mRNA was not expressed suggests that it does not correspond to a dimer of the 8.5 protein (data not shown). Moreover, the molecular mass of this protein, very different from that of the p1A75 cDNA product (28 kDa) (2) indicates that it must be different from this latter protein.


Figure 6: Immunoblot detection of the 8.5 protein in subcellular fractions of a post-natal day 1 rat brain homogenate. A, fractions equivalent to 150-µg homogenate were loaded on each lane. H, homogenate; S2, post-nuclear supernatant; P2, post-nuclear pellet; S3, cytosol; P3, ultracentrifugation pellet. The migration of the molecular mass markers is indicated. B, carbonate extractability of the 8.5 protein. P2 and P3 fractions equivalent to 100 µg of the S3 fraction were treated with sodium carbonate (+) or with phosphate-buffered saline as a control(-) as described under ``Materials and Methods.''



Cellular and Subcellular Localization of the 8.5 Protein

Immunocytochemical analysis of primary cultures from the embryonic striatum and cerebral cortex demonstrated the presence of a prominent juxtanuclear Golgi-like labeling in the majority of neurons (Fig. 7A). This signal can be exclusively attributed to the 8.5 protein immunoreactivity since Western blot experiments indicated an extremely low expression of the 40-kDa protein in these primary cultures (data not shown). In contrast, the few contaminating glial cells were devoid of immunolabeling (Fig. 7A). Analysis of the same preparation by immunoelectron microscopy showed that most of the specific labeling is on the membrane of all the Golgi saccules and on some vesicles in the Golgi zone (Fig. 8, A and B). Some dispersed small vacuolar structures and multivesicular bodies were also observed in the cell body or in neurites (Fig. 8C).


Figure 7: A, immunofluorescence of cultured rat neurons using affinity-purified anti 8.5 antibody. The juxtanuclear area of the majority of neurons is conspicuously immunolabeled. One can notice the immunostaining of a neurite (arrowhead) but the majority of the neurites and a contaminating glial cell (arrow) are devoid of labeling. The bar represents 10 µm. B, immunofluorescence, using affinity-purified anti 8.5-antibody, of COS cells transfected with the 8.5 cDNA inserted in the pSVL expression vector. Note the strong juxtanuclear immunolabeling. The bar represents 10 µm.




Figure 8: A and B, ultrastructural localization of the 8.5 protein in the Golgi apparatus (G) of cultured neurons. The stack of all Golgi saccules is labeled. Note the strong labeling of two Golgi vesicles (arrowheads). C, a labeled multivesicular body (arrowhead) located within a neurite. N, nucleus. The bar represents 0.5 µm.



Immunocytochemical analysis of transfected COS cells expressing the 8.5 cDNA revealed a conspicuous immunolabeling of a juxtanuclear zone (Fig. 7B). No specific immunoreactivity was detected in wild-type COS cells (data not shown).


DISCUSSION

In this study, we have characterized the second member of a new protein family. This new 19-kDa protein (p19) is specifically expressed in neural and neuroendocrine cells where it is localized mainly in the Golgi apparatus.

The similarity of the primary sequences of p19 and of the p1A75 cDNA product (2) indicates that these proteins belong to the same family. Moreover, these two proteins are specifically expressed in the cytoplasm of neural cells. In addition to the atypical epidermal growth factor-like motif, a search in the protein data bases for conserved regions revealed that these two proteins share a highly similar short segment with secretogranin III (Fig. 9)(25) . p19, whose calculated isoelectric point is basic (9.48), cannot belong to the granin family of acidic secretory proteins(26) , even if its precocious expression peak is reminiscent of this family(25) . Nevertheless, this motif, shared by p19 and secretogranin III, respectively, expressed in the Golgi apparatus and in intracellular vesicles of neural cells, could characterize proteins involved in the process of neurosecretion. The Golgi-like localization of transfected p19 in COS cells indicated that its primary sequence contains a Golgi targeting signal. Comparison of the p19 primary sequence with that of other proteins known to be expressed in this cellular compartment did not provide any significant similarity. However, immunofluorescence and immunoelectron microscopy analyses clearly indicated that the Golgi apparatus is indeed the main expression site of p19 in embryonic neurons (Fig. 8A) which express high amounts of the corresponding mRNA (Fig. 4).


Figure 9: Alignment of residues 142-153 of the 8.5 protein with residues 51-62 of the partial sequence of the product of the p1A75 cDNA (2) and with residues 137-146 of the product of the 1B1075 cDNA (secretogranin III)(25) . Identical or isofunctional amino acids are boxed.



Our data do not allow the determination of the topology of p19 with respect to the membrane of the Golgi apparatus. Nevertheless, the presence in p19 of an highly hydrophobic segment (residues 70-92, Fig. 2) and its carbonate-resistant membrane association (Fig. 6B) indicated that it is an integral membrane protein. The absence of a characteristic sequence for signal peptides, on the one hand, and the migration to the same apparent molecular mass of native and factor Xa-cleaved recombinant p19 (data not shown), on the other hand, suggests that p19 have no signal sequence and that it could therefore belong to the class of membrane protein with a C-terminal anchor(27) .

The recognition, by our affinity-purified antiserum, of a 40-kDa protein could be due to a reactivity against the non-8.5 added part (MRGSHHHHHHIEGR, see ``Materials and Methods'') of the recombinant protein. However, another antiserum directed against an unrelated recombinant protein which bears the same N-terminal tail did not recognize the 40-kDa protein. (^2)The 40-kDa band could therefore correspond to a cross-reacting protein, possibly belonging to the same family of that of p19. Southern blot analysis of rat and human DNA indicated the presence of a single 8.5 gene (data not shown). In addition, the chromosomal assignment, in 5q35, of the human 8.5 gene, that we determined in a complementary study (28) in which we sought for a possible colocalization with a disease gene, does not correspond to any known neurological hereditary disease.

Although the Golgi apparatus is the main expression site of p19 in cultured neurons, its presence in the P3 fraction of a neonatal rat brain homogenate (Fig. 6) suggests that this protein is also expressed in small organelles. Indeed, the labeling of some vacuolar structures and multivesicular bodies, which could be involved in neuronal endocytosis(29) , indicates that the function of p19 may be related to some intraneuronal vesicular trafficking. These data provide a working hypothesis that should be tested by functional studies.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17259[GenBank].

§
To whom correspondence should be addressed. Tel.: 33-1-44-27-12-54; 33-1-44-27-15-85; Fax: 33-1-44-27-12-60.

(^1)
The abbreviations used are: E, embryonic day; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor.

(^2)
J.-M. Studler, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Michèle Gelman for expert technical assistance with the primary cultures, to Claude Pennarun for the microscopy pictures, and to Flancia Guirouard for technical assistance.


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