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Vol. 18, Issue 4, 1143-1152, April 2007

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Cornichon-like Protein Facilitates Secretion of HB-EGF and Regulates Proper Development of Cranial Nerves

Hideharu Hoshino^*, Tsukasa Uchida^*, Toshiaki Otsuki^*, Shoko Kawamoto, Kousaku Okubo, Masatoshi Takeichi, and Osamu Chisaka^*

*Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; Research Information Research Division, National Institute of Informatics, Tokyo 101-8430, Japan; Laboratory for Gene Expression Analysis, Center for Information Biology, National Institute of Genetics, Shizuoka 411-8540, Japan; and Laboratory for Cell Adhesion and Tissue Patterning, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan

Submitted August 24, 2006; Revised December 13, 2006; Accepted January 8, 2007
Monitoring Editor: Marianne Bronner-Fraser

	ABSTRACT

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During their migration to the periphery, cranial neural crest cells (NCCs) are repulsed by an ErbB4-dependent cue(s) in the mesenchyme adjoining rhombomeres (r) 3 and 5, which are segmented hindbrain neuromeres. ErbB4 has many ligands, but which ligand functions in the above system has not yet been clearly determined. Here we found that a cornichon-like protein/cornichon homolog 2 (CNIL/CNIH2) gene was expressed in the developing chick r3 and r5. In a cell culture system, its product facilitated the secretion of heparin-binding epidermal growth factor-like growth factor (HB-EGF), one of the ligands of ErbB4. When CNIL function was perturbed in chick embryos by forced expression of a truncated form of CNIL, the distribution of NCCs was affected, which resulted in abnormal nerve fiber connections among the cranial sensory ganglia. Also, knockdown of CNIL or HB-EGF with siRNAs yielded a similar phenotype. This phenotype closely resembled that of ErbB4 knockout mouse embryos. Because HB-EGF was uniformly expressed in the embryonic hindbrain, CNIL seems to confine the site of HB-EGF action to r3 and r5 in concert with ErbB4.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rhombomeres (r) are segmented structures that transiently appear in the embryonic hindbrain of vertebrates (Lumsden and Krumlauf, 1996). How the neural tube is regionalized and compartmentalized into these cell movement-restricted (Fraser et al., 1990) rhombomeres has gathered many researchers' attention, but the mechanisms involved have still not yet been fully elucidated. Because each rhombomere expresses not only different sets of transcription factors such as Hox genes, but also different sets of secreted signaling molecules, many processes of craniofacial development are dependent on the proper patterning of the hindbrain. One such example is the migration pattern of cranial neural crest cells (NCCs). NCCs are multilineage-potential cells that emigrate from the dorsal edge of the developing neural tube (reviewed by Le Douarin and Kalcheim, 1999). Cranial neural crest cells differentiate into many important cell types such as glia and sensory neurons of the cranial nerves, smooth muscle, connective tissue of the thymus, endothelial cells of the carotid arteries, etc. In the hindbrain region, NCCs do not enter the mesenchyme adjoining odd-numbered rhombomeres, i.e., r3 and r5; and, accordingly, the migration pattern becomes segmented. In avian embryos, this phenomenon is in part dependent on the apoptosis of NCCs in the odd-numbered rhombomeres by BMP4 and MSX2 function under the interactive control of adjacent even-numbered rhombomeres (Graham et al., 1993, 1994). In addition, it has been suggested that the mesenchyme residing around odd-numbered rhombomeres has the property of repulsing the migrating NCCs. Some signal(s) from the neural tube is thought to be the reason for this property. In chicken embryos, the SemaphorinIII/D (formerly collapsin-1)-neuropilin-1 (Nrp1) chemorepellent system is reported to be responsible for the migration-path selection of the trunk and hindbrain NCCs, as was found by use of the stripe-assay and by ectopic expression of Sema3A (Eickholt et al., 1999; Osborne et al., 2005). Another candidate gene that may govern selection of the migration pathway of NCCs is ErbB4, a member of the EGF receptor family. ErbB4 is expressed in r3 and r5, and its knockout (KO) mouse exhibits abnormal NCC migration and axon path finding (Golding et al., 2000). NCCs from r4 of the ErbB4 KO mouse embryos are able to sense the repulsion signal resident in the mesenchyme adjacent to r3 when grafted into wild-type embryos. On the contrary, wild-type NCCs from r4 migrate into the ErbB4(–/–) mesenchyme located beside r3. These findings indicate that ErbB4 signaling in the r3 instructs the mesenchymal cells surrounding r3 to repulse NCCs. Also, the surface ectoderm around r3 seems to be important for maintaining this repulsion signal (Golding et al., 2004a). ErbB4 has been shown to be necessary for proper barrier function against cranial NCCs and nerves in the mesenchyme surrounding r3 and r5, although the mechanism establishing the NCC barrier operating in r3 and r5 seems to be slightly different in these areas (e.g., a graft of ErbB4-expressing r5 neuroepithelium is not sufficient to induce an NCC-free zone surrounding the r3 area of a recipient) (Golding et al., 2004a). Also in mouse embryos, the NCC-free zone adjacent to r3 is maintained by combinatorial interactions between the r3 neuroepithelium and the adjacent mesenchyme/surface ectoderm (Trainor et al., 2002).

These signaling molecules mentioned above are repeatedly used in many aspects of embryonic morphogenesis; however, the outputs are quite different depending on when and where the signaling takes place. As the activities of these molecules must be tightly regulated, such molecules need to go through many regulatory steps to be secreted. In addition to regulation at transcriptional and translational levels, recent findings have revealed that the activities of some of these molecules are regulated at the transportation and secretion levels. For example, in Drosophila development, the activity of Spitz, one of the TGF-alpha homologues, is confined by the activity of the transporter Star and a Golgi apparatus-resident protease known as Rhomboid (Lee et al., 2001; Urban et al., 2002). In this present article we report another example of the regulation of a growth factor at the secretion level.

While searching for genes specifically expressed in the embryonic hindbrain/pharyngeal area, we found the CNIL gene to be transiently expressed in a restricted manner, i.e., only in r3 and r5. CNIL is one of the homologues of the Drosophila gene cornichon (cni), which is necessary for the transportation of Gurken (one of the TGF-alpha homologues) to a transitional endoplasmic reticulum (tER) site (Herpers and Rabouille, 2004) and promotes the incorporation of Gurken into coat protein complex II (COPII) vesicles (Boekel et al., 2006). Another important piece of information has come from the study of Erv14p, the yeast homolog of CNIL. Erv14p of budding yeast interacts with COPII components and is localized in the ER and Golgi membranes. Mutation of the COPII-binding site (carboxyl-terminus domain) of Erv14p causes the accumulation of the transmembrane protein Axl2p in the ER and thus a lack of Axl2p protein localization to the nascent bud tips or to the mother bud site (Powers and Barlowe, 2002). Likewise, we found that when CNIL function was suppressed by introducing a plasmid designed to express the carboxyl-terminus–deleted form (C) of CNIL, the distribution of cranial NCCs was perturbed, and resulted in a cranial nerve-misrouted phenotype quite resembling that of the ErbB4 KO mouse embryo. ErbB4 is known to bind many EGF motif–containing molecules such as HB-EGF, betacellulin (BTC), epiregulin (EREG), and neuregulins (Nrg1, 2, 3, 4; reviewed in Olayioye et al., 2000; Carpenter, 2003). Because Drosophila Cornichon (Roth et al., 1995) is known to be a transporter of Gurken (TGF-alpha homolog), in this study we searched for EGF motif–containing protein transported by CNIL. Among known ErbB4 ligands, we found that HB-EGF bound CNIL and was efficiently secreted into the culture medium when both molecules were coexpressed in cultured cells. Because HB-EGF expression is ubiquitous in the developing hindbrain, CNIL seems to restrict HB-EGF/ErbB4 signaling to r3 and r5 by its specific expression in these rhombomeres. Because signaling transmitted by ErbB receptors is involved in many aspects of embryonic development, these findings may better explain the precise spatial and temporal pattern of ErbB receptor activation during embryonic development.

	MATERIALS AND METHODS

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Animals
Fertilized hen eggs from Yamagishi Farm (Kyoto, Japan) were incubated at 38.5°C in a humidified environment to the stages required (Hamburger and Hamilton, 1951).

Antibodies
Rabbit anti-CNIL antibody was raised against a peptide (GNPARARERLKNIERIC) that is a part of chicken CNIL. For production of the rat anti-chicken HB-EGF antibody, the protein directed by the N-terminally 6xhistidine-tagged whole ORF was synthesized in Escherichia coli by using the pColdI vector (Takara, Tokyo, Japan).

Cloning of Chicken CNIL cDNA
A 186-base pair fragment of CNIL cDNA was amplified from a st-17/18 chicken embryo cDNA library by degenerate PCR using the primers recognizing sites where mouse cni and cnil amino acid sequences were conserved. The sequences of the primers were 5'-TTYGCNGCNTTYTGYTAYATG-3' and 5'-CCAYTCNGCNGCRCANARRAACAT-3'. The cDNA library was then screened with the fragment thus generated as a probe, after which 5'-RACE was performed to obtain the entire ORF.

Homology alignment was carried out with the ClustalW (European Molecular Bioinformatics Institute, http://www.ebi.ac.uk/clustalw/) computer program. The putative transmembrane regions were deduced by the use of SMART software (http://smart.embl-heidelberg.de/).

In situ Hybridization
Whole-mount in situ hybridization was performed as described earlier (Watari et al., 2001). Digoxigenin-labeled RNA probes were used at 0.5 µg/ml for the SOX10 probe and at 0.1 µg/ml for the chicken CNIL probe (0.8-kb cDNA fragment covering nearly the entire ORF). For transcription of the chicken SOX10 antisense riboprobe, a cDNA fragment amplified by RT-PCR with the following primers was used: 5'-AATGGCACTTGCCTGAGCACCTC-3' and 5'-CTCCGTGGCTGGTACTTGTAGTC-3'.

Construction of Vectors
Expression vectors were constructed by using either a modified pCApA vector (Niwa et al., 1991) or pcDNA3.1/Myc-His(+)A (Invitrogen, Carlsbad, CA). For some vectors, a C-terminal FLAG epitope (Hopp et al., 1988) or enhanced green fluorescent protein (eGFP) was added as a tag. A C-terminus deletion of CNIL was introduced by PCR. ARIA (NRG1), was cloned by RT-PCR with 5'-GATATCATGTGGGCCACCTCTGAAGG-3' and 5'-GTCGACTTATACAGCAATAGGGTCTT-3' as primers, and then the HA epitope was added as described earlier (Wang et al., 2001). The cDNA of Rat NTAK (NRG2) cDNA was kindly donated by S. Higashiyama. To make an expression vector of the secreted form of HB-EGF, sHB-EGF, we inserted a stop codon after the 152nd codon. To make sARIA-HA and sNTAK-3*FLAG, we amplified each cDNA fragment by using primer set (5'-GAATTCAATGTGGGCCACCTCTGAAGG-3'/5'-TCTAGATTAGAAGCTGGCCATTACG-TAGT-3') and (5'-GAATTCAATGCGGCAGGTTTGCTGCTC-3'/5'-CTCGAGCTTCTGGTACAGCTCCTCAG-3'), respectively. Then these fragments were subcloned into the p3XFLAG CMV13 (Sigma, St. Louis, MO). An ErbB4-eGFP expression plasmid was constructed by inserting human ErbB4 cDNA (a gift from N. E. Hynes) into pCApA vector together with an eGFP cassette for a C-terminal tag.

In Ovo Electroporation
In ovo electroporation of st-9 embryos was performed as described previously (Kubo et al., 2003). DNA solution (pCA-CNIL or pCA-C-CNIL: 7.5 mg/ml, pCAGAP-eGFP:1.6 mg/ml, and 0.05% W/V fast green) was injected into the lumen of the hindbrain. pCAGAP-eGFP drives the expression of eGFP (Ichii, unpublished results). The anode was placed just beside the hindbrain, and a cathode-tungsten microelectrode was inserted into the lumen of the hindbrain. Electric pulses were applied for 25 ms, three times, each at 7 V. The embryos were allowed to develop at 38.5°C up to the desired stage and processed for further experiments. For rescue experiments, pCA-sHB-EGF, p3XFLAG CMV13-sARIA-HA or p3XFLAG-CMV13-sNTAK was also added (sHB-EGF and sNTAK-3XFLAG at 8.4 mg/ml and sARIA-HA at 7.5 mg/ml).

CNIL Knockdown with Small Interfering RNA
For RNA interference (RNAi) of the CNIL gene in the chick developing hindbrain, 25 mer double-stranded RNAs (60 µM, Stealth RNAi; Invitrogen) were electroporated into the hindbrain of st-9 chick embryos. The target sequences of the small interfering RNAs (siRNAs) were as follows: CNIL 715, 5' CGCCATCCCTATGCGGCTCAATAAA 3'; CNIL 87, 5' CATCTGGCATATCATCGCTTTCGAT 3'; CNIL 236, 5' TCCATGGGCTCTTCTGTCTGATGTT 3'; HB-EGF 447, 5' CATCCTGCATATGCCAGCCAGGATA 3'; HB-EGF 461, 5' CAGCCAGGATATCATGGAGAGAGAT 3'; and HB-EGF 567, 5' TGTCCTCTCTGTGCCTTGTCATCAT 3'. The numbers indicate the start nucleotide positions in the genes (CNIL; GenBank Accession number AB232677, HB-EGF; AF131224). Medium GC-content control RNA (Invitrogen) was used for the negative control. The effectiveness of siRNAs was confirmed by transfecting HEK-293T cells with siRNA, and the protein produced by CNIL or HB-EGF expression plasmids was detected by Western blotting with anti-CNIL or anti-HB-EGF antibody. The concentration of the siRNAs was 60 µM in distilled water. The electroporation conditions were the same as for the other expression plasmid DNAs.

Whole-Mount Immunostaining of Chicken Embryos
Whole-mount immunostaining of st-19/20 embryos was performed as described previously (Davis et al., 1991) with mAb specific for chicken neurofilaments (Hatta et al., 1987). HRP-conjugated anti-mouse IgG (1:200; GE Healthcare, Chalfont St. Giles, United Kingdom) was used for the secondary antibody; and diaminobenzidine (1 mg/ml in PBST, Sigma), as the colorimetric substrate. Some embryos were dehydrated with methanol and cleared in benzylalcohol-benzylbenzoate (1:2).

Immunohistochemistry
St-12-13 embryos were sectioned at 20 µm and then treated with rat anti-cHB-EGF overnight at 4°C. After having been washed with PBS, the sections were treated with Alexa Fluor 488 goat anti-rat IgG (1:200; Molecular Probes, Eugene, OR) and Alexa Fluor 568 phalloidin (Molecular Probes). The samples were mounted by using FluorSave Reagent (Calbiochem, La Jolla, CA).

Cell Culture
Human embryonic kidney derived HEK-293 and HEK-293T cells were cultured in a 1:1 mixture of DMEM and Ham's F12 medium supplemented with 10% fetal bovine serum.

To establish ErbB4-eGFP stable transfectants (HEK-293::ErbB4-eGFP), we transfected HEK-293 cells with pCAErbB4-eGFP vector and pPGK-Puro by using Effectine Transfection Reagent (QIAGEN, Santa Clarita, CA) according to the manufacturer's protocol and then subsequently selected with puromycin.

Activation of ErbB4 by sHB-EGF, sARIA (sNRG1), or sNTAK (sNrg2)
HEK-293T cells were transfected with sHB-EGF, sARIA, or sNTAK expression vectors. After 24 h, the medium was changed to that without serum, and the cells were incubated for another 24 h. Each conditioned medium was centrifuged at 6000 x g relative centrifugal force to remove floating cells. HEK-293::ErbB4-eGFP cells starved overnight in medium without serum were incubated in these conditioned media for 5 min and then immediately lysed with Laemmli's sample buffer (Laemmli, 1970).

Coimmunoprecipitation
HEK-293T cells cultured in 10-cm dishes were transfected with 2 µg plasmid DNA of each construct. After 36 h of incubation, the cells were pelleted and lysed with 0.1% Triton X-100 in TBS. FLAG or eGFP fusion proteins were immunoprecipitated with the respective anti-FLAG M2 (Sigma) or rabbit anti-GFP antibody (Molecular Probes). The immunoprecipitates were dissolved in Laemmli's sample buffer and boiled for 5 min.

Cell-Surface Biotinylation
Biotinylation of the cell surface was performed as described previously (Gechtman et al., 1999). HEK293T cells were transfected with pcDNA3.1 cHB-EGF-eGFP and pcDNA3.1 CNIL, pcDNA3.1C-CNIL or pcDNA3.1/Myc-His(+)A vectors at a ratio of 1:3. After 36 h, the cells were washed with ice-chilled 1 mM Ca²⁺, 1 mM Mg²⁺/HCMF (HBSS), and subsequently biotinylated by using 0.1 mg/ml EZ-Link Sulfo-NHS-Biotin (Pierce, Rockford, IL) in HBSS. The reaction was quenched by 1 mM Ca²⁺, 1 mM Mg²⁺/TBS. The biotinylation procedure was performed on ice. Cells were lysed by 0.5% Triton X-100, 0.5% NP-40/TBS. cHB-EGF-EGFP was immunoprecipitated by use of mouse anti-GFP (Roche) and protein G Sepharose (GE Healthcare). The immunoprecipitates were dissolved in Laemmli's sample buffer.

Brefeldin A Treatment
HEK-293T cells were transfected with equal amounts of pcDNA3.1cHB-EGF-eGFP and pcDNA3.1CNIL, C-CNIL, or vector without insert. After 10 h of transfection, brefeldin A or the same volume of vehicle (ethanol) was added (20 mg/l) in order to block secretion. The cells were incubated overnight and harvested.

Concentration of sHB-EGF from Conditioned Medium
Equal amounts of pcDNA3.1cHB-EGF and pcDNA3.1CNIL, C-CNIL, or vector without insert were used to transfect HEK-293T cells. After 24 h, the medium was changed to medium without serum, and then the cells were cultured further for 24 h. The conditioned medium was centrifuged at 16,100 rcf for 10 min to remove floating cells, and sHB-EGF was concentrated with a Microcon YM-3 (Millipore, Bedford, MA).

Western Blotting
Antibodies used for Western blotting were the following: rabbit anti-GFP (1:400; Molecular Probes), anti-FLAG M2 (1:200; Sigma), rat anti-cHB-EGF (1:200; this study), rabbit anti-actin (1:800; Sigma), anti-activated MAP kinase (1:1000; Sigma), HRP-conjugated anti-rabbit IgG (1:5000; Jackson ImmunoResearch, West Grove, PA), biotin-conjugated anti-rat IgG (1:1000; GE Healthcare), HRP-conjugated anti-mouse IgG (1:2500; GE Healthcare), HRP-conjugated avidin (1:10,000; Zymed, South San Francisco, CA), and HRP-conjugated anti-rat IgG (1:2000; Jackson ImmunoResearch). Blots were detected with ECL Plus (GE Healthcare).

	RESULTS

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Cloning and Sequence Analysis of Chicken CNIL cDNA
We have been collecting cDNAs specifically expressed in the mouse embryonic hindbrain. Through this attempt, we found that the cnil gene (cDNA sequence in GenBank; Accession number AB006191, also BC059922 under the name of cornichon-homolog 2) was expressed in r3 and r5 of 8.5–9.25 embryonic day (E) mouse embryos (Hoshino and Chisaka, unpublished results). Mouse cnil is also expressed at the primitive node (E6.5). For analysis of cnil function in hindbrain/cranial nerve development, a conditional knockout system using a hindbrain-specific DNA recombinase-expressing transgenic mouse strain would be very useful. However, such a strain (Cre knock-in mouse at Krox20 loci; Voiculescu et al., 2000) might produce the recombinase at a stage not earlier than the desired stage for the cnil gene elimination (Krox20 transcription starts at nearly the same stage as does that of cnil; our unpublished result). Because chicken embryos are easier than mouse ones to manipulate at the embryonic stages of interest, we decided to isolate chicken CNIL cDNA for further analysis of this gene function at the stage of hindbrain segmentation. First, we amplified a portion of CNIL cDNA from a stage (st) 17–18 (Hamburger and Hamilton, 1951) chick embryo cDNA library with a pair of primers against the conserved region between mouse cnil and cni. The major PCR product was cloned into a plasmid vector and sequenced. The nucleotide sequence of the amplified cDNA fragment showed high homology, 82%, with mouse cnil and had 48-base pair span of nucleotide sequence that was specific to cnil. Therefore, we named the cDNA fragment CNIL. The entire coding sequence of CNIL was obtained by cDNA library screening with the amplified fragment and 5'-RACE. We deposited the CNIL cDNA sequence in the GenBank database as Accession number AB232677. The amino acid sequence of CNIL was compared with those sequences of cni and cnil proteins from human and mouse, Drosophila cni, and budding yeast homolog Erv14p (Figure 1A). It should be noted that cnil has a stretch of sequences (16 amino acid residues, Nos. 47–62) that is not present in cni. As these cnil-specific sequences are highly conserved among chicken, mouse, and human, this domain may have some important role(s) in the function of these molecules. The cni and cnil proteins have three putative membrane-spanning domains (Figure 1A, indicated in the CNIL sequences with underlines). There are no other marked domains or motifs found in cni or cnil sequences. The carboxy terminus–deleted form (C; truncated at amino acid residue No. 95 at the end of the second transmembrane domain) was constructed for perturbing the CNIL function in the present study. This truncation deletes the suspected COPII-interaction domain (corresponding to amino acid residues 97-101 of the Erv14p, underlined in Figure 1; Powers and Barlowe, 2002), and the resulting product is expected to act as a dominant-negative molecule. Schematic drawing of the protein topology is also shown (Figure 1B).

View larger version (41K): [in this window] [in a new window]   Figure 1. (A) Predicted protein-sequence alignment of chicken CNIL (c-cnil), mouse cnil (m-cnil), human CNIL (h-CNIL), mouse Cornichon (m-cni), human CORNICHON (h-cni), Drosophila cornichon (d-cni), and budding yeast Erv14p. For maximum matching, gaps were introduced among the sequences. The residues are shaded when they are identical to those conserved among cnil sequences. The residues in red are conserved among all the cnil family member proteins including Erv14p. The underlined sequences of chicken CNIL indicate the putative transmembrane domains. Also, the underlined part of the Erv14p sequence indicates the COPII interaction domain. Note that cnil sequences have a specific insert of 16 residues at residues 47–62. The truncation point for creating the carboxy-terminus–deleted form (C-CNIL) is between the 95th and 96th residue of chicken CNIL. (B) Schematic drawing of the topology of wild-type CNIL and C-CNIL.

View larger version (110K): [in this window] [in a new window]   Figure 2. Expression pattern of chicken CNIL during rhombomere formation. (A–H) In situ hybridization of chicken embryos for CNIL mRNA at st-9 to st-16. Dorsal views with rostral side at the top except for F (lateral view) are presented. (B) At st-10, CNIL expression starts in primordial rhombomere (r) 3. (D) At st-12, CNIL expression is observed in both r3 and r5 cells. (E and F) The CNIL expression level is high at the dorsal edge of the neural tube and at the alar plate at st-13. Arrowhead in F indicates the expression in the NCCs arising from r5. (G and H) At st-14-16, gradually CNIL expression decreases and fades out. (I) At st-13, an embryo was longitudinally cut, and each fragment was hybridized with either CNIL or Hoxb1 probe. Numbers beside the hindbrain area indicate the respective rhombomeres. (J) This cross section of a st-13 embryo at r5 level shows that CNIL expression is high at the alar plate. ov, otic vesicle.

View larger version (95K): [in this window] [in a new window]   Figure 3. Projection of the cranial nerves and distribution of the NCCs are affected by C-CNIL. (A and B) Whole-mount immunostaining with anti-neurofilament mAb of control (A, eGFP expression plasmid-electroporated) and C-CNIL expression vector-electroporated (B) embryos at st-9. V, trigeminal nerve; VII, facial nerve; IX, glossopharyngeal nerve; X, vagus nerve; ov, otic vesicle. (C and D) Whole-mount in situ hybridization for SOX10 (glial lineage NCC marker) of st-17 control (C) and C-CNIL expression vector-electroporated (D) embryos. Black arrow in A indicates spinal tracts in the neural tube. White arrows in B and D indicate abnormal axon bundles (not in the neural tube) and NCC distribution between the Vth and VIIth ganglia, respectively. Note that these abnormal axon bundles are located more lateral to the entry/exit points of the Vth and VIIth nerves. (E) Dorsal view of a representative specimen with green eGFP signals (pseudocolored) shows the area where expression vectors were introduced by in ovo electroporation. Arabic numerals indicate corresponding rhombomeres.

Forced Activation of ErbB4 by a Secreted Form of HB-EGF Can Prevent Cranial Nerve Defects Caused by C-CNIL Transfection

To examine if forced activation of the ErbB4 signaling pathway could prevent the cranial nerve defects that resulted from the expression of C-CNIL in the chicken embryonic hindbrain, we performed coelectroporation of st-9 chicken embryonic neural tubes with the C-CNIL expression vector together with one of the expression vectors of the secreted (s) form of known ErbB4 ligands ([HB-EGF, neuregulin-1 [NRG1, ARIA in chicken], and neuregulin-2 [NRG2, NTAK in rat]). The agonistic activities of these proteins were confirmed by downstream MAPK phosphorylation in ErbB4-expressing cultured cells (Figure 4A). All the ligands showed similar agonistic activities in this experiment. Because BTC and EREG were not detected in st12-13 chicken embryos, and NRG3 and NRG4 were not detected in E9.0 mouse embryos by RT-PCR, these ligands were not used for this experiment. Embryos after electroporation were collected at st-20, and their cranial nerves were immunostained with an anti-neurofilament antibody. Representative specimens for each combination of the expression vectors are shown in Figure 4, B–D. The frequency of the cranial nerve-defect phenotype was 3/21, 15/19, and 8/20 for C-CNIL + sHB-EGF, C-CNIL + sNRG1, and C-CNIL + sNRG2, respectively. These frequencies were statistically compared with the frequency when only C-CNIL was introduced (17/36, previous section) by using the Mann-Whitney U test. The results indicated that only the secreted form of HB-EGF (sHB-EGF) could prevent the cranial nerve defect caused by C-CNIL (Figure 4B; the p value between the C-CNIL vs. C-CNIL + sHB-EGF was 0.00127 <0.05). The introduction of sNRG1 caused a slightly higher frequency of the defect than that of C-CNIL (Figure 4C; p = 0.0246 < 0.05). The introduction of sNRG2 apparently did not rescue the embryos from the above phenotype (Figure 4D; p = 0.6057 > 0.05). When sHB-EGF, sNRG1 or sNRG2 alone was ectopically expressed, sHB-EGF alone did not cause any abnormality in the cranial nerve development at a statistically high frequency (4/19), but sNRG1 and sNRG2 elicited the abnormal cranial nerve phenotype (15/19 and 8/20, respectively) at frequencies comparable to those with C-CNIL. Because sNRG1 and sNRG2 can bind and stimulate not only ErbB4 but also ErbB2/ErbB3 (required for proper NCCs migration and proliferation, Britsch et al., 1998), overexpression of these ligands would seem to have caused the above cranial nerve phenotype. Also, the phosphorylation pattern of ErbB4 differs after stimulation by NRG1 or by NRG2 (Sweeney et al., 2000). This difference might be one of the reasons why the cranial nerve defect-frequencies differed between sNRG1- and sNRG2-expressing embryos. Altogether, only sHB-EGF could rescue the embryos from the cranial nerve defect caused by C-CNIL.

View larger version (41K): [in this window] [in a new window]   Figure 4. Secreted-form HB-EGF suppresses C-CNIL caused cranial nerve abnormality. (A) The production of biologically active secreted (s) growth factors was confirmed by the detection of activated MAPK after treating HEK-293::HER4(ErbB4) cells with conditioned medium from cultures of sHB-EGF, sNRG1, and sNRG2 expression plasmid-transfected HEK-293T cells. (B–D) C-CNIL and sHB-EGF, C-CNIL and sNRG1, and C-CNIL and sNRG2 expression plasmids, respectively, were electroporated into st-9 chick hindbrains, and nerve fibers were immuno-stained at st-20. Note that only in C and D do abnormal nerve connections (arrows) exist between the Vth (V) and VIIth (VII) ganglia. r3, rhombomere3.

View larger version (32K): [in this window] [in a new window]   Figure 5. HB-EGF expression and interaction with CNIL protein. (A and B) Immunostaining of coronal (A) and transverse (B) sections of st-12 chick embryos with anti-HB-EGF antibody (green). Actin was also stained (purple) for tissue outlining. HB-EGF is weakly but ubiquitously expressed. r3-6, rhombomeres 3-6; ov, otic vesicle. Pre-immune serum did not give specific signals (data not shown). (C) Immunoblot analysis of st-12/13 chick head extract (H) with anti-HB-EGF antibody (-HB-EGF). 293T is the control cell extract of HB-EGF expression plasmid-transfected HEK-293T cells. Right panel (–) shows the signals without anti-HB-EGF antibody. (D and E) Reciprocal immunoprecipitation experiments using extracts of HEK-293T cells transfected with HB-EGF-eGFP and the wild-type (WT) or delta-C form (C) of CNIL-FLAG expression plasmids reveal the interaction between HB-EGF and CNIL protein. GFP and FLAG peptide were used as tags. Antibodies used for immunoprecipitation (IP) and detection on the Western blot membranes (WB) are indicated under each panel. Left half of panels ("soluble") shows the amount of CNIL protein (D) or HB-EGF-eGFP protein (E) in the soluble cell extracts. Note that the C-CNIL can interact with HB-EGF more strongly than does the wild type. Also, HB-EGF-eGFP proteins appear as a doublet band due to posttranslational modification.

Extracellular Secretion of HB-EGF Is Facilitated by CNIL
To analyze the influence of CNIL on the secretion of HB-EGF, we first examined whether the amount of cell-surface HB-EGF was dependent on CNIL. HEK-293T cells were transfected with HB-EGF-eGFP and CNIL expression plasmid, and then the cell-surface proteins were labeled with biotin. After immunoprecipitation with anti-GFP antibody, cell-surface HB-EGF was detected with HRP-conjugated avidin and a chemiluminescence system. As seen in Figure 6A, the total amount of HB-EGF in the whole cell lysate was reduced by coexpression of CNIL. The ratio of the amount of cell-surface HB-EGF against the total amount of HB-EGF did not differ so much among the samples with or without CNIL or C. This reduction in HB-EGF in the lysate of CNIL-expressing cells might have been caused by promoted secretion of HB-EGF into the culture medium. Actually, this reduction was suppressed by inhibition of vesicle transport from the ER-to-Golgi apparatus by treating cells with brefeldin A (Figure 6B). We next examined whether CNIL facilitated the secretion of HB-EGF into the culture medium. HEK-293T cells were cotransfected with the HB-EGF expression plasmid and the wild-type or C-CNIL expression plasmid. After 24 h of incubation, the secreted HB-EGF (sHB-EGF) proteins in the culture media were concentrated with a centrifugal filter device. The specimens were separated on an SDS-PAGE gel, and the HB-EGF was detected with anti-HB-EGF antibody. The results indicated that the wild-type CNIL dramatically enhanced the secretion of the sHB-EGF into the culture medium (Figure 6C). This CNIL-enhanced secretion of HB-EGF well matches the facilitation of budding yeast Axl2p packaging into COPII vesicles in the reconstituted budding reaction by the cnil homolog Erv14p (Powers and Barlowe, 2002). Moreover, the results of the coexpression of both the wild-type CNIL and C-CNIL along with the HB-EGF indicated that C-CNIL acted as a dominant-negative form. C-CNIL suppressed the reduction in HB-EGF from cell lysates caused by wild-type CNIL (Figure 6D), as well as the enhancement of HB-EGF secretion into the culture medium (Figure 6E).

View larger version (27K): [in this window] [in a new window]   Figure 6. HB-EGF secretion is facilitated by CNIL. (A) Cell-surface HB-EGF was detected by biotinylation. HB-EGF-eGFP and wild-type or C-CNIL expression plasmids were used to cotransfect HEK-293T cells. The biotinylated-HB-EGF-eGFP was detected with HRP-conjugated avidin after immunoprecipitation and SDS-PAGE by using anti-GFP antibody. Total HB-EGF-eGFP in the whole lysates and immunoprecipitates was also detected with anti-HB-EGF antibody. (B) Inhibition of protein transport from the ER to the Golgi apparatus by brefeldin A caused accumulation of HB-EGF-eGFP in wild-type CNIL coexpressing cells. (C) Wild-type CNIL promotes secretion of HB-EGF into the culture medium. HEK-293T cells were transfected with HB-EGF and wild-type CNIL or C-CNIL expression plasmids. Then, secreted HB-EGF from the culture medium was concentrated and detected by immunoblotting. (D) HB-EGF accumulated in C-CNIL, or wild-type CNIL- and C-CNIL–expressing cells. Whole cell lysates were subjected to immunoblotting with anti-HB-EGF antibody. (E) C-CNIL acts as a dominant-negative molecule. HEK-293T cells were transfected with cHB-EGF and wild-type CNIL expression vectors with or without an additional C-CNIL expression or mock plasmid. HB-EGF was quantified as in C. Note that secretion of HB-EGF is reduced in the C-CNIL–expressing cells.

View larger version (49K): [in this window] [in a new window]   Figure 7. RNAi of CNIL or HB-EGF causes cranial nerve defects. (A) siRNAs suppress CNIL expression from a plasmid vector when cotransfected into HEK293T cells. (B) siRNAs suppress HB-EGF expression. (C–E) Control siRNA- (C), CNIL siRNA- (D), and (E) HB-EGF siRNA-treated chick embryos were stained with anti-neurofilament antibody. The siRNAs were electroporated into st-9 chick hindbrains; and nerve fibers were immunostained at st-20. Note that only in D and E do abnormal nerve connections (arrows) exist between the Vth (V) and VIIth (VII) ganglia. Numbers after siRNAs indicate their nucleotide start positions in the gene.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (29K): [in this window] [in a new window]   Figure 8. Summary of the CNIL function focusing on r3 of the chick hindbrain. In rhombomeres not expressing CNIL, HB-EGF is not secreted from the cell surface. In normal r3, HB-EGF is efficiently secreted from the cell surface with the aid of CNIL. Subsequently, the mesenchyme adjacent to r3 and r5 inhibits NCC migration into these areas. On the contrary, in C-CNIL–expressing r3, the secretion of HB-EGF is blocked; and NCCs migrate into the mesenchyme beside r3.

In the developing vertebrate hindbrain, so far NRG1 has been the only suspected ligand of ErbB4, but our present finding indicates that HB-EGF is another ligand for Erb4 in this area. Knockout mice of nrg1 also showed cranial nerve defects, but the phenotype is different from that of ErbB4 KO mice and that of C-CNIL–expressing chick embryos. The proximal ganglia of cranial nerves appeared to be missing in nrg1 KO mouse embryos, and NCC generation or survival seemed to be dependent on NRG1 protein (Meyer and Birchmeier, 1995). In the mouse hindbrain, the protein encoded by cnil, which is expressed in r3 and r5, does not seem to carry Nrg1, because nrg1 is strongly expressed in r2, 4, and 6, as reported previously (Dixon and Lumsden, 1999). Also, we could not detect any physical interaction between CNIL and NRG1 in coexpressing cells (data not shown). The expression patterns of ErbB4 and NRG1 in the developing hindbrain slightly differ between the chick and mouse. As mentioned above, strong expression domains of ErbB4 and Nrg1 do not overlap in the mouse hindbrain. In the chick hindbrain, the expression domains of ErbB4 and NRG1 partially overlap at the region between the basal plate and the alar plate in r3 of the st-12-13 embryos (Dixon and Lumsden, 1999). At this moment we do not know how this difference between mouse and chicken affects the selection of the NCC migration path.

As was shown in Figure 2, E and 2F, CNIL was expressed in migrating NCCs emigrating from r5. ErbB4 expression in these cells was not observed in the chick hindbrain (Dixon and Lumsden, 1999). At this moment we do not know what ligand-receptor system is supported by CNIL in these cells. Gene expression analysis of these CNIL-positive NCCs and forced expression of C-CNIL in r5-r6 may reveal CNIL function in r5-derived NCCs in the future.

We did not examine presently whether mouse HB-EGF has an expression pattern similar to that of the chicken in the developing hindbrain. Our present study suggests that the secreted form of HB-EGF has an important role in the selection of NCC migration path in the chick hindbrain. There was not much difference in the amount of cell-surface HB-EGF between the in the wild-type CNIL- and C-CNIL–expressing cells. Membrane-bound HB-EGF is known to transmit juxtacrine signals to neighboring cells (Iwamoto and Mekada, 2000). Because HB-EGF KO mice (Iwamoto et al., 2003) and mice in which the HB-EGF locus was replaced with an uncleavable form (HB^uc) or a transmembrane domain-truncated form (HB; Yamazaki et al., 2003) have already been generated, examination of the cranial nerve phenotypes of their embryonic hindbrain is anticipated. Such studies should clarify which form of the HB-EGF acts dominantly for the barrier function against NCC migration in mice.

As far as we have checked, we have found no hindbrain segmentation defects or identity alteration of r4; however, there remains a possibility that more detailed analysis using proper markers may reveal some defects in the C-CNIL–expressing neural tube.

There are many mouse gene mutants that show cranial nerve defects, including COUP-TF1 (Qiu et al., 1997), Hoxa3 (Manley and Capecchi, 1997; Watari et al., 2001), ErbB2 (Lee et al., 1995), ErbB3 (Erickson et al., 1997), ErbB4 (Gassman et al., 1995), nrg1 (Meyer and Birchmeier, 1995), Semaphorin III/D (SemaIII, collapsin-1; Taniguchi et al., 1997), Neuropillin-1 (Kitsukawa et al., 1997), and the Hes1 and Hes5 double mutant (Hatakeyama et al., 2006). Among these, ErbB2, ErbB3, nrg1 mutants have cranial ganglia of reduced size, suggesting that their products are necessary for the survival of the NCC precursors. But ErbB4, SemaIII, and Nrp1 mutants have ganglia of normal size. Recently, the Semaphorin/Neuropilin signaling system in the chick hindbrain was also shown to be necessary for the proper stream-like migration of neural crest cells (Osborne et al., 2005). Signals necessary for the survival or the migration path selection of the NCCs would seem to intermingle with each other. More information is still needed to unravel the complex interactions among different tissues, e.g., hindbrain neural tube, NCCs, ectoderm, and surrounding mesenchyme. Also, the downstream targets of transcription factors whose mutants exhibit cranial nerve defects should be investigated. Studies on the downstream signaling of ErbB4, activated by HB-EGF, would help to identify the molecules responsible for repulsive property of mesenchymal cells adjoining r3.

CNIL is expressed in many tissues, including cancer cells (our unpublished results). Further studies on the CNIL protein should reveal the relationship between cellular behavior and ErbB4 activity in other tissues, which relationship cannot be clarified by just analyzing the expression patterns of ErbB4 and its ligands.

	ACKNOWLEDGMENTS

 
We are grateful to N. Watari-Goshima, Y. Hayakawa, and members of M. Takeichi and T. Uemura's labs for their discussions regarding this study. We thank Drs. K. Umesono and R. Yu for providing the chicken cDNA library, T. Ichii for the modified pCA vectors, N. E. Hynes for ErbB4 cDNA, and S. Higashiyama for NTAK cDNA. This study was supported by grants from the Sumitomo Foundation, the Asahi Glass Foundation, and by a grant-in-aid (18570195) from the Ministry of Education, Science, and the Technology of Japan to O.C.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-08-0733) on January 17, 2007.

Address correspondence to: Osamu Chisaka (chisaka{at}lif.kyoto-u.ac.jp)

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