Rostral growth of commissural axons requires the cell adhesion molecule MDGA2
- Pascal Joset†1,
- Andrin Wacker†2,
- Régis Babey†1,
- Esther A Ingold1,
- Irwin Andermatt2,
- Esther T Stoeckli2 and
- Matthias Gesemann1, 2Email author
© Joset et al; licensee BioMed Central Ltd. 2011
Received: 26 October 2010
Accepted: 4 May 2011
Published: 4 May 2011
Long-distance axonal growth relies on the precise interplay of guidance cues and cell adhesion molecules. While guidance cues provide positional and directional information for the advancing growth cone, cell adhesion molecules are essential in enabling axonal advancement. Such a dependence on adhesion as well as guidance molecules can be well observed in dorsal commissural interneurons, which follow a highly stereotypical growth and guidance pattern. The mechanisms and molecules involved in the attraction and outgrowth towards the ventral midline, the axon crossing towards the contralateral side, the rostral turning after midline crossing as well as the guidance along the longitudinal axis have been intensely studied. However, little is known about molecules that provide the basis for commissural axon growth along the anterior-posterior axis.
MDGA2, a recently discovered cell adhesion molecule of the IgCAM superfamily, is highly expressed in dorsolaterally located (dI1) spinal interneurons. Functional studies inactivating MDGA2 by RNA interference (RNAi) or function-blocking antibodies demonstrate that either treatment results in a lack of commissural axon growth along the longitudinal axis. Moreover, results from RNAi experiments targeting the contralateral side together with binding studies suggest that homophilic MDGA2 interactions between ipsilaterally projecting axons and post-crossing commissural axons may be the basis of axonal growth along the longitudinal axis.
Directed axonal growth of dorsal commissural interneurons requires an elaborate mixture of instructive (guidance) and permissive (outgrowth supporting) molecules. While Wnt and Sonic hedgehog (Shh) signalling pathways have been shown to specify the growth direction of post-crossing commissural axons, our study now provides evidence that homophilic MDGA2 interactions are essential for axonal extension along the longitudinal axis. Interestingly, so far each part of the complex axonal trajectory of commissural axons uses its own set of guidance and growth-promoting molecules, possibly explaining why such a high number of molecules influencing the growth pattern of commissural interneurons has been identified.
For its function the mammalian central nervous system depends on precisely organized neuronal circuits. Synaptic connections between the cells of a circuit are established during development when axonal growth cones grow along specific pathways, reaching even very distant targets with exceptionally high precision. A combination of cell adhesion molecules, surface receptors and axon guidance molecules enables the growth cone to invade permissive areas and grow along specific molecular gradients [1, 2]. Long distances are covered by splitting the entire trajectory into smaller segments with intermediate targets . Such intermediate targets, also called choice points, mark the end of one segment and the beginning of another. At choice points the growth cone morphology as well as the axonal trajectory change dramatically, often leading to temporary stalling and a decrease in growth rate . Choice points have first been described in invertebrates such as grasshopper or Drosophila, where these intermediate targets are represented by specific cells called guidepost cells, whose ablation leads to axon stalling and miss-projections .
One of the best studied choice points in vertebrates is the ventral midline, where specialized cells called floor-plate cells selectively regulate axon crossing in bilaterally symmetric animals [2, 5]. While some axons are attracted by the floor plate, others are selectively repelled. Cell populations whose axons are attracted by the floor plate are dorsolateral commissural interneurons (dI1 and dI2) . Upon reaching the midline, commissural axons cross the floor plate to reach the contralateral side, where they turn orthogonally into the longitudinal axis, growing either along the floor-plate or extending laterally to join the ventral or lateral funiculus [2, 6, 7].
The role of the floor-plate as an important choice point for commissural axons has been clearly demonstrated in several studies [8–10]. The floor-plate-derived molecule netrin-1 was identified as the major chemoattractant for dorsolaterally located commissural axons . Inactivation of either netrin-1 or its receptor, DCC (Deleted in colorectal cancer), causes severe miss-projections of commissural axons, leaving only few axons reaching the midline correctly [8, 9]. No axons reached the midline when netrin-1 -/- mice were treated with the Shh inhibitor cyclopamine, demonstrating a role of Shh not only as a morphogen but also as a guidance molecule that cooperates with the chemoattractant netrin-1 .
While both netrin-1 and Shh are responsible for attracting commissural axons towards the ventral midline, other short-range guidance cues and adhesion molecules govern midline crossing. The best-studied molecules in this context are cell adhesion molecules of the immunoglobulin superfamily, such as axonin-1/TAG-1, NgCAM/L1, NrCAM, nectins and SynCAMs/Nectin-like molecules (Necls) [11–13]. Axonin-1 is highly expressed by commissural interneurons, whereas NrCAM, Nectin3, and SynCAM2/Necl3 are strongly up-regulated in floor-plate cells during the period of axonal midline crossing. Direct evidence for a role of these molecules in commissural axon outgrowth came from in vivo perturbation assays demonstrating that, in the absence of axonin-1/NrCAM, heterophilic nectin or heterophilic SynCAM interactions, axons either failed to enter and cross the floor plate or had problems turning into the longitudinal axis [11–13].
Aberrant pathfinding at the ventral midline was also found when ephrinB/EphB signalling was perturbed [14, 15] or in the absence of F-spondin function . While F-spondin seemed to regulate the turning angle of commissural axons, the morphogens Shh and Wnt were shown to be required for post-crossing commissural axon guidance [17–19]. In the mouse, Wnt4 is expressed in a decreasing anterior-to-posterior gradient in the floor plate and attracts post-crossing commissural axons rostrally in a Frizzled3-dependent manner . Shh was found to be expressed in an opposite gradient, with the highest expression levels in the caudal spinal cord. In vivo loss- and gain-of-function studies demonstrated that Shh was required for the correct navigation of post-crossing commissural axons in the chicken spinal cord and that this effect was mediated by hedgehog-interacting protein (Hhip) . Intriguingly, in chicken, Wnts were not expressed in a gradient along the anteroposterior axis. Rather, a Wnt activity gradient was formed by the graded expression of the Wnt antagonist Sfrp1 (secreted frizzled related protein 1), which was shaped by Shh . Both a Wnt activity gradient and a Shh expression gradient provide directional information for post-crossing commissural axons. However, it is less clear whether they also affect neurite elongation directly. Interestingly, Avraham and colleagues  have recently shown that the axons of contralaterally and ipsilaterally projecting dI1 interneurons intermingle during longitudinal growth, suggesting that adhesive interaction between different fascicles might be a driving force for longitudinal growth.
Recently, we have isolated two novel cell adhesion molecules of the immunoglobulin superfamily, called MDGA1 and MDGA2, which are exclusively expressed in the peripheral and central nervous system . Rat MDGA1 shows high expression levels in developing dI1 interneurons, whereas rat MDGA2 is predominantly expressed in spinal motoneurons and some subpopulations of spinal interneurons . In chicken, MDGA1 has been shown to be highly expressed in motoneurons and in the floor plate . In contrast to the rat counterpart, expression of chicken MDGA1 in commissural interneurons is rather low and can be observed only at later stages, raising the question of whether some functions of the mammalian MDGA1 are covered by the MDGA2 homolog in chicken. In order to study MDGA2 function in vivo, we have cloned the chicken MDGA2 ortholog. Chicken MDGA2 RNA is highly expressed in commissural interneurons and dorsal root ganglia (DRG) neurons. Using RNA interference (RNAi) and function-blocking antibodies, we were able to show that MDGA2 plays a crucial role in the growth of commissural axons along the longitudinal axis, suggesting that MDGA2 is required for axonal elongation, whereas Wnt and Shh signalling controls the growth direction of commissural axons after midline crossing.
With the development of in ovo RNAi as a tool for specific gene silencing, the chicken embryo became a powerful system to study gene function during development . We thus decided to use the chicken embryo for studies addressing MDGA function. Database searches indicated that the chicken genome, as observed for other vertebrate species, contains two MDGA genes. The domain organization of the chicken MDGA proteins is identical to that of the corresponding rat ortholog: six immunoglobulin C-2 type repeats are combined with a fibronectin type III repeat and a MAM domain. Amplification of MDGA2 resulted in the identification of two different cDNA transcripts. One transcript shows an identical organization to rat MDGA2, and the other transcript has an insertion of 48 bp at position 2,032. Conservation between chicken and rat MDGAs is in the range 80% to 90%, suggesting that rat and chicken proteins might have conserved functions. For more details on the conservation and the phylogenetic relation of MDGAs, see Additional files 1 and 2.
MDGA2 transcripts are highly expressed in spinal interneurons
MDGA2 is present on growth cones and neurites of commissural and dorsal root ganglia neurons
Down-regulation of MDGA2 causes pathfinding errors of commissural axons
The phenotype observed after MDGA2 knockdown could be phenocopied when MDGA2 antibodies were injected into the central canal (Figure 4H). As observed for the RNAi experiments, injections of MDGA2 antibodies did not affect the circumferential growth of commissural axons nor did they prevent them from crossing the midline (Figures 4 and 5). However, as seen after RNAi-mediated knockdown of MDGA2, post-crossing commissural axons of embryos treated with MDGA2 antibodies did not turn into the longitudinal axis. The majority of the axons stalled at the floor-plate exit site. In contrast to embryos treated with MDGA2 antibodies, embryos injected with IgG control antibodies did not show any perturbation of commissural axon growth, demonstrating that high concentrations of IgGs diffusing from the central canal did not influence axonal growth and pathfinding of commissural neurons unspecifically (Figure 4I).
MDGA2 interacts homophilically
Ipsilaterally and contralaterally projecting axonal tracts intermingle in the longitudinal axis
Contralateral knockdown of MDGA2 causes similar turning phenotypes
Commissural interneurons located in the dorsal part of the chicken spinal cord send out axons along very specific, highly stereotypic pathways . In the lumbosacral spinal cord, outgrowth of commissural axons starts as early as stage 19, and by stage 22 commissural axons have reached the ventral midline and start crossing it . By stage 25, most commissural axons have turned into the longitudinal axis and extend rostrally within the ventral funiculus before deviating from the ventral midline to join more dorsally located fibre tracts. Our experiments have shown that MDGA2 is strongly expressed in pre- as well as post-crossing commissural axons and that RNAi-mediated knockdown and antibody perturbation causes severe pathfinding defects in commissural axons.
At this point it is worth mentioning that MDGA2 is also transiently expressed by floor-plate cells during the time when commissural axons cross the ventral midline. This most likely explains how perturbation of MDGA2 function could also interfere with midline crossing of commissural axons. About 25% of the commissural axons failed to cross the midline. However, due to the redundancy of IgCAMs with growth-promoting function, 75% of the axons reached the contralateral side normally. Previous studies demonstrated a growth-promoting effect of NrCAM and NgCAM . Like NrCAM, MDGA1 and MDGA2 are also expressed by floor plate cells [12, 34] (MG and PJ, unpublished observation). Expression of these molecules may enable commissural axons to enter and cross the ventral midline area. In this respect it will be interesting to study the binding properties of MDGA2 in more detail. While we have clearly demonstrated homophilic interactions between MDGA2 molecules, heterophilic interaction partners have not yet been identified. Putative candidates might be other members of the Ig superfamily, especially MDGA1, axonin-1, F11, NrCAM and NgCAM. Binding sites for MDGA1 within the developing spinal cord have been studied by Fujimura and colleagues . Interestingly, MDGA1 binding sites only partially overlap with MDGA1 or MDGA2 expression sites, suggesting that MDGA1 does not or only weakly interacts homophilically or heterophilically with MDGA2. Using chemical cross-linkers we indeed found neither homophilic MDGA1 interactions nor did we observe the formation of MDGA1-MDGA2 heterodimers (Additional file 7).
While chicken MDGA2 is highly expressed in dorsal commissural interneurons, its rat counterpart seems to be absent or only weakly expressed in the corresponding dI1 interneurons . In rat, however, MDGA1 is highly expressed in dI1 interneurons, suggesting that functions might have been shifted between rat and chicken MDGA1 and MDGA2 during evolution. Such functional shifts have already been observed for a number of proteins, such as NgCAM/L1  and Wnt proteins . While such species differences might initially lead to some confusion about protein function, it will ultimately help to better understand and predict functional divergence between different species, including humans.
An essential mechanism enabling axonal growth is the generation of neuron-substrate interactions via cell adhesion molecules. In contralaterally and ipsilaterally projecting spinal interneurons the cell adhesion molecule MDGA2 is expressed during the period of growth along the longitudinal axis. Axons of these neuronal populations intermingle during elongation in the ventral funiculus. Elongation of dorsal commissural neurons after midline crossing is impaired when MDGA2 is knocked down in this neuronal population; a defect that is phenocopied when MDGA2 is downregulated on the contralateral side. Hence, for longitudinal growth of post-crossing commissural axons a homophilic interaction of MDGA2 is required.
Materials and methods
Cloning of chicken MDGA2
Reverse transcription was done on total RNA isolated from stage 26 spinal cord using either random hexamers or oligodT primers. A MDGA2 fragment covering the sequence between 1,966 and 2,541 was amplified using the following two degenerated primers based on the rat MDGA-2 sequence: cartggacrcaratgaa (sense) and tgrtgrccrtacatrtg (antisense).
The 3' end of MDGA2 was isolated using a PCR RACE (rapid amplification of cDNA ends) strategy. Using this strategy, a MDGA2 fragment covering the area between positions 2,226 and 3,332 could be isolated, having a designated stop codon at position 2,917. Template cDNA was generated using a specific 3' RACE primer (cccgaattctagaagcttctcgag[T]18V). PCR primers for the first reaction were as follows: gaggcatatgaagtccg (sense; 2,179 to 2,195) and cccgaattctagaagcttc (antisense) followed by a nested reaction with the sense primer ggactccactattcgggt (2,226 to 2,243).
The 5' end was cloned by PCR using the sense primer atgttcatgttcacgtgaag atg (based on the rat sequence) and specific antisense primer ggagcactatacttgatg (2,244 to 2,261) on a cDNA template that has been reverse transcribed from total RNA isolated from stage 26 spinal cords with a specific reverse transcription primer, aggactgacaag, corresponding to sequence 2,491 to 2,502. The atg sequence given in bold represents the putative atg start codon.
MDGA2 expression constructs
Expression constructs for soluble (ΔGPI) and full-length MDGA2 were cloned. An EcoRI restriction site and a partial Kozak consensus sequence were added at the 5' site of the sense primer aaaaagaattcaccatggatgtagcgatcggg, allowing easy cloning and optimal expression. The antisense primer aaaatgcggccgctgatcgtaaattgttggc contains a Not restriction site at the 5' end, allowing in-frame expression. The resulting PCR fragments were cloned into the Topo pCR2 vector (TOPO® TA Cloning kit; Invitrogen, Carlsbad, CA, USA) and verified by sequencing. ΔGPI fragments were subcloned in-frame into the expression vectors pcDNAI or a derivate of pMES containing either a FLAG tag (Sigma-Aldrich, St Louis, MO, USA) or a myc tag at the 3' end. Full length MDGA2 containing a FLAG tag introduced 3' of the signal sequence was subcloned into pcDNAI.
In situ hybridization
Linearised templates spanning the MDGA2 sequences 1 to 1,284 and 1,285 to 2,920 were used to obtain Dig-labelled sense and antisense RNA probes (DIG RNA Labelling Kit; Roche Diagnostics GmbH, Mannheim, Germany) with T3 and T7 RNA polymerase (Roche Diagnostics).
Chicken embryos were staged as described by Hamburger and Hamilton . Fixation times (4% paraformaldehyde (PFA) fixation) varied between 20 minutes (stages 14 to 20) and 1 hour (stages 36 and older). Subsequently, tissues were embedded in Tissue-Tek® compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands), frozen on dry ice and stored at -80°C. Sections cut at a thickness of 25 μm were collected on SuperFrost Plus slides (Menzel GmbH & Co. KG, Braunschweig, Germany) and immediately dried for 2 to 4 hours. Sections were hybridized with two RNA probes for MDGA2 (1 to 1,284; 1,285 to 2,920) using previously published protocols . After hybridization, sections were developed in colouring solution (240 μg/ml levamisole, 35 μg/ml nitro blue tetrazolium, 17.5 μg/ml 5-bromo-4-chloro-3-indolyl phosphate in TBS buffer) in the dark until the desired intensity of reaction product was achieved.
Dorsal root ganglia outgrowth
DRGs from stage 30 chicken were dissected and placed on polylysine-coated dishes and incubated at 37°C for 2 to 3 days in growth medium (DMEM/F12, 10% FCS, 0.36% methocel solution, AraC, 100 ng/ml nerve growth factor (NGF), 50 μg/ml gentamycin).
DRGs were fixed in 4% PFA for 15 minutes and subsequently incubated for 30 minutes in PBS containing 2% goat serum and 0.2% fish skin gelatine. MDGA2 peptide antibodies were added for 1 h at room temperature following several wash steps with PBS and the application of the secondary antibody for 30 minutes.
Dissociated commissural neurons
The dorsal most 25% of stage 26 spinal cords were dissected. After trypsin treatment and trituration, the dissociated neurons were cultured on polylysine-coated (10 μg/ml;Sigma) eight-well LabTek slides (Nalgene-Nunc, Thermo Fisher Scientific, Rochester, NY 14625, USA) for 24 h in growth medium (MEM with GlutaMAX-I (Invitrogen), 1 mM pyruvate, 4 mg/ml Albumax (Invitrogen), N3 (100 μg/ml transferring, 10 μg/ml insulin, 20 ng/ml triiodothyronine, 40 nM progesterone, 200 ng/ml corticosterone, 200 μM putrescine, 60 nM sodium selenite; reagents from Sigma). Cells were fixed with 4% PFA for 15 minutes, washed in PBS and blocked in PBS containing 10% FCS for 20 minutes. The cells were subsequently incubated with the primary antibodies (rabbit anti-axonin-1, rabbit anti-MDGA2) in 10% FCS/PBS for 90 minutes and washed several times with PBS. After incubation with the secondary antibody (goat anti-rabbit-Cy3; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA 19390, USA), the cells were washed well in PBS and subsequently analyzed by confocal microscopy.
Total RNA was isolated from the electroporated and the unelectroporated side of stage 26 MDGA2 knockdown embryos and reverse transcribed using oligodT primers. Real-time PCR was performed with two different sets of MDGA2 primers amplifying the regions 826 to 995 and 2,436 to 2,607 and one primer pair for an actin control (916 to 1,089) as well as a GAPDH control (770 to 880). Amplification of a MDGA1 fragment (2,439 to 2,584) on the above described cDNAs was used to demonstrate the specificity of the MDGA2 downregulation (MDGA1 levels in the MDGA2 dsRNA electroporated side were 91.8 ± 6.3% compared to the control non-electroporated side).
Transfection of HEK293T cells was done using the PolyFect transfection reagent (Qiagen GmbH, 40724 Hilden, Germany) according to the manufacturer's instruction. Serum-free conditioned media containing recombinant MDGA2 carrying either a flag or a myc tag were concentrated to approximately 20 μg/ml using the Centricon centrifugation device (Milipore-Amicon, Billerica, MA 01821, USA). Chemical cross-linkers (bis(sulfosuccinimidyl) suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), 3,3'-dithiobis(sulfosuccinimidylproprionate)) were added at a ten-fold molar excess (in respect to the present free amino groups) and incubated at room temperature for 30 minutes. The reaction was terminated by adding 10 mM Tris-buffered saline and the formed protein complexes were analyzed by western blot. Beads (Fluorosbrite PolyFluor Microspheres; Polysciences, Inc., Warrington, PA 18976, USA) were coupled according to the manufacturer's instructions with either BSA or conditioned medium containing different forms of recombinant MDGA2. Prior to the aggregation, assay beads were briefly sonicated. Aggregation was performed in a rotating shaker for exactly 30 minutes and the formed aggregates were immediately analysed by fluorescent microscopy.
Long double-stranded RNA synthesis
Two long dsRNA fragments covering different parts of the designated cDNA sequence (-15 to 1,248 and 1,975 to 2,746) were used to down-regulate MDGA2 functions. Linearised plasmids containing the different fragments were transcribed with T3 or T7 polymerase according to the manufacturer's protocol (Roche Diagnostics). Following transcription, the plasmid DNA was digested with RNase-free DNase for 1 h. Single-stranded RNA molecules were then extracted once with phenol-chloroform-isoamylalcohol (25:24:1; pH 4.5) and once with chloroform-isoamylalcohol (24:1) before precipitating with ethanol. Equal amounts of sense and antisense RNA were mixed, heated for 5 minutes to 95°C and subsequently allowed to cool down to room temperature overnight. Formation of dsRNA was confirmed by non-denaturing agarose gel electrophoresis.
In ovo RNAi and antibody injection
Hisex chicken embryos obtained from a local supplier were used according to regulations of the Veterinäramt des Kantons Zürich. Electroporation was done as described previously . Between 0.1 and 0.5 μl dsRNA (300 ng/μl) mixed with a YFP control plasmid (20 ng/μl) and Trypan Blue (0.04% v/v; Invitrogen) were injected into the central canal of 3-day-old chick embryos (stages 18 to 20) using glass capillaries. Five pulses of 50-ms duration at 25 V were given using platinum electrodes (BTX, Genetronics, San Diego, CA, USA) of 4 mm length with a distance of 4 mm between anode and cathode. MDGA2 antibodies were raised against three different peptides whose sequences are indicated in Figure 2A. A standard immunisation protocol offered by Eurogentec was used for the antibody production (Eurogentec, 4102 Seraing, Belgium). Antibodies from the final bleed were affinity purified against the corresponding peptides. Peptide antibodies against MDGA2 (3 to 5 μg/μl) were injected into the central canal of stage 18 chicken embryos as previously described . For control injections, purified IgGs from non-immunized rabbit were used at identical concentrations.
Tracing of contra- and ipsilateral axons
Five-day-old chicken embryos were decapitated and fixed in 4% PFA/1× PBS for 1.5 h at room temperature. A 2-mm thick transverse section of the embryo (at the lumbar level of the spinal cord) was injected with lipophilic dye (FastDiI and FastDiA, 5 mg/ml in methanol; Molecular Probes, Eugene, OR, USA) at the caudal surface as indicated in Figure 7A. Sections were cryoprotected overnight with 25% sucrose in 0.1 M sodium phosphate buffer (pH 7.4), embedded in OCT Tissue-Tek and frozen on dry ice. For analysis, the tissue was cut into 25-μm thick sections on a cryostat (Leica CM1850).
Open-book preparations of stage 26 chicken spinal cords were prepared as described previously . Briefly, FastDiI (Molecular Probes) at a concentration of 5 mg/ml was injected into the region of dorsally located interneurons. After 2 days of incubation to allow for the diffusion of the dye, spinal cords were mounted in PBS between coverslips and analyzed by confocal microscopy. Confocal sections were taken with a step size of approximately 0.6 μm for a maximal projection of approximately 50 μm.
Fluorescence intensities at different locations along the trajectory of commissural axons (see schematic representation in Figure 5: A, ipsilateral; B, floor plate; C, floor-plate exit; D, rostral longitudinal axis; E, caudal turn; F, no turn; G, 45° turn; H, -45° turn) were quantified using the NIH ImageJ program . Background fluorescence levels A' to H' at corresponding locations were subtracted from the values obtained at the different sites of measurement (Atot(A - A'), Btot(B - B')...). The raw data are given in the table in Figure 5A. Subsequently, fluorescence intensities were normalized to the control values, with the control representing 100% at each given location. Injection sites (n between 40 and 60) were quantified using the program Prism 4 (GraphPad Software Inc., La Jolla, CA 92037, USA) and the obtained data were converted into a histogram (Figure 5B) showing the percentage of commissural axon intensity being present at particular locations compared to control values (floor-plate, floor-plate exit, rostral turn).
bovine serum albumin
Dulbecco's modified Eagle's medium
dorsal root ganglia
expressed sequence tag
foetal calf serum
green fluorescent protein
yellow fluorescent protein.
This work was supported by the Swiss National Science Foundation, TH Research Grants from the ETH Zurich and the EMDO Foundation Zurich.
- Chilton JK: Molecular mechanisms of axon guidance. Dev Biol. 2006, 292: 13-24. 10.1016/j.ydbio.2005.12.048.View ArticlePubMedGoogle Scholar
- Kaprielian Z, Runko E, Imondi R: Axon guidance at the midline choice point. Dev Dyn. 2001, 221: 154-181. 10.1002/dvdy.1143.View ArticlePubMedGoogle Scholar
- Bentley D, Caudy M: Pioneer axons lose directed growth after selective killing of guidepost cells. Nature. 1983, 304: 62-65. 10.1038/304062a0.View ArticlePubMedGoogle Scholar
- Palka J, Whitlock KE, Murray MA: Guidepost cells. Curr Opin Neurobiol. 1992, 2: 48-54. 10.1016/0959-4388(92)90161-D.View ArticlePubMedGoogle Scholar
- Stoeckli ET: Molecular mechanisms of commissural axon pathfinding. Prog Brain Res. 1998, 117: 105-114.View ArticlePubMedGoogle Scholar
- Moon MS, Gomez TM: Adjacent pioneer commissural interneuron growth cones switch from contact avoidance to axon fasciculation after midline crossing. Dev Biol. 2005, 288: 474-486. 10.1016/j.ydbio.2005.09.049.View ArticlePubMedGoogle Scholar
- Stoeckli ET: Longitudinal axon guidance. Curr Opin Neurobiol. 2006, 16: 35-39. 10.1016/j.conb.2006.01.008.View ArticlePubMedGoogle Scholar
- Keino-Masu K, Masu M, Hinck L, Leonardo ED, Chan SS, Culotti JG, Tessier-Lavigne M: Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell. 1996, 87: 175-185. 10.1016/S0092-8674(00)81336-7.View ArticlePubMedGoogle Scholar
- Serafini T, Colamarino SA, Leonardo ED, Wang H, Beddington R, Skarnes WC, Tessier-Lavigne M: Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell. 1996, 87: 1001-1014. 10.1016/S0092-8674(00)81795-X.View ArticlePubMedGoogle Scholar
- Charron F, Stein E, Jeong J, McMahon AP, Tessier-Lavigne M: The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell. 2003, 113: 11-23. 10.1016/S0092-8674(03)00199-5.View ArticlePubMedGoogle Scholar
- Okabe N, Shimizu K, Ozaki-Kuroda K, Nakanishi H, Morimoto K, Takeuchi M, Katsumaru H, Murakami F, Takai Y: Contacts between the commissural axons and the floor plate cells are mediated by nectins. Dev Biol. 2004, 273: 244-256. 10.1016/j.ydbio.2004.05.034.View ArticlePubMedGoogle Scholar
- Stoeckli ET, Landmesser LT: Axonin-1, Nr-CAM, and Ng-CAM play different roles in the in vivo guidance of chick commissural neurons. Neuron. 1995, 14: 1165-1179. 10.1016/0896-6273(95)90264-3.View ArticlePubMedGoogle Scholar
- Niederkofler V, Baeriswyl T, Ott R, Stoeckli ET: Nectin-like molecules/SynCAM are required for post-crossing commissural axon guidance. Development. 2010, 137: 427-435. 10.1242/dev.042515.View ArticlePubMedGoogle Scholar
- Imondi R, Kaprielian Z: Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons. Development. 2001, 128: 4859-4871.PubMedGoogle Scholar
- Kadison SR, Makinen T, Klein R, Henkemeyer M, Kaprielian Z: EphB receptors and ephrin-B3 regulate axon guidance at the ventral midline of the embryonic mouse spinal cord. J Neurosci. 2006, 26: 8909-8914. 10.1523/JNEUROSCI.1569-06.2006.View ArticlePubMedGoogle Scholar
- Burstyn-Cohen T, Tzarfaty V, Frumkin A, Feinstein Y, Stoeckli E, Klar A: F-Spondin is required for accurate pathfinding of commissural axons at the floor plate. Neuron. 1999, 23: 233-246. 10.1016/S0896-6273(00)80776-X.View ArticlePubMedGoogle Scholar
- Lyuksyutova AI, Lu CC, Milanesio N, King LA, Guo N, Wang Y, Nathans J, Tessier-Lavigne M, Zou Y: Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling. Science. 2003, 302: 1984-1988. 10.1126/science.1089610.View ArticlePubMedGoogle Scholar
- Bourikas D, Pekarik V, Baeriswyl T, Grunditz A, Sadhu R, Nardo M, Stoeckli ET: Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat Neurosci. 2005, 8: 297-304. 10.1038/nn1396.View ArticlePubMedGoogle Scholar
- Domanitskaya E, Wacker A, Mauti O, Baeriswyl T, Esteve P, Bovolenta P, Stoeckli ET: Sonic hedgehog guides commissural axons directly and indirectly by regulating Wnt activity. J Neurosci. 2010, 30: 11167-11176. 10.1523/JNEUROSCI.1488-10.2010.View ArticlePubMedGoogle Scholar
- Avraham O, Hadas Y, Vald L, Zisman S, Schejter A, Visel A, Klar A: Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1. Neural Dev. 2009, 4: 21-10.1186/1749-8104-4-21.PubMed CentralView ArticlePubMedGoogle Scholar
- Litwack ED, Babey R, Buser R, Gesemann M, O'Leary DD: Identification and characterization of two novel brain-derived immunoglobulin superfamily members with a unique structural organization. Mol Cell Neurosci. 2004, 25: 263-274. 10.1016/j.mcn.2003.10.016.View ArticlePubMedGoogle Scholar
- Fujimura Y, Iwashita M, Matsuzaki F, Yamamoto T: MDGA1, an IgSF molecule containing a MAM domain, heterophilically associates with axon- and muscle-associated binding partners through distinct structural domains. Brain Res. 2006, 1101: 12-19. 10.1016/j.brainres.2006.05.030.View ArticlePubMedGoogle Scholar
- Pekarik V, Bourikas D, Miglino N, Joset P, Preiswerk S, Stoeckli ET: Screening for gene function in chicken embryo using RNAi and electroporation. Nat Biotechnol. 2003, 21: 93-96. 10.1038/nbt770.View ArticlePubMedGoogle Scholar
- Hamburger V, Hamilton HL: A series of normal stages in the development of the chick embryo. J Morphol. 1951, 88: 49-92. 10.1002/jmor.1050880104.View ArticlePubMedGoogle Scholar
- Sonderegger P: Axonin-1 and NgCAM as "recognition" components of the pathway sensor apparatus of growth cones: a synopsis. Cell Tissue Res. 1997, 290: 429-439. 10.1007/s004410050950.View ArticlePubMedGoogle Scholar
- van der Merwe PA, Barclay AN: Transient intercellular adhesion: the importance of weak protein-protein interactions. Trends Biochem Sci. 1994, 19: 354-358. 10.1016/0968-0004(94)90109-0.View ArticlePubMedGoogle Scholar
- Shiga T, Oppenheim RW, Grumet M, Edelman GM: Neuron-glia cell adhesion molecule (Ng-CAM) expression in the chick embryo spinal cord: observations on the earliest developing intersegmental interneurons. Brain Res Dev Brain Res. 1990, 55: 209-217.View ArticlePubMedGoogle Scholar
- Silos-Santiago I, Snider WD: Development of interneurons with ipsilateral projections in embryonic rat spinal cord. J Comp Neurol. 1994, 342: 221-231. 10.1002/cne.903420206.View ArticlePubMedGoogle Scholar
- Yaginuma H, Shiga T, Oppenheim RW: Mechanisms of axonal guidance used by interneurons in the chick embryo spinal cord. Perspect Dev Neurobiol. 1993, 1: 205-215.PubMedGoogle Scholar
- Yaginuma H, Shiga T, Homma S, Ishihara R, Oppenheim RW: Identification of early developing axon projections from spinal interneurons in the chick embryo with a neuron specific beta-tubulin antibody: evidence for a new 'pioneer' pathway in the spinal cord. Development. 1990, 108: 705-716.PubMedGoogle Scholar
- Parra LM, Zou Y: Sonic hedgehog induces response of commissural axons to Semaphorin repulsion during midline crossing. Nat Neurosci. 2010, 13: 29-35. 10.1038/nn.2457.View ArticlePubMedGoogle Scholar
- Nawabi H, Briancon-Marjollet A, Clark C, Sanyas I, Takamatsu H, Okuno T, Kumanogoh A, Bozon M, Takeshima K, Yoshida Y, Moret F, Abouzid K, Castellani V: A midline switch of receptor processing regulates commissural axon guidance in vertebrates. Genes Dev. 2010, 15: 396-410.View ArticleGoogle Scholar
- Fitzli D, Stoeckli ET, Kunz S, Siribour K, Rader C, Kunz B, Kozlov SV, Buchstaller A, Lane RP, Suter DM, Dreyer WJ, Sonderegger P: A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons. J Cell Biol. 2000, 149: 951-968. 10.1083/jcb.149.4.951.PubMed CentralView ArticlePubMedGoogle Scholar
- Stoeckli ET, Sonderegger P, Pollerberg GE, Landmesser LT: Interference with axonin-1 and NrCAM interactions unmasks a floor-plate activity inhibitory for commissural axons. Neuron. 1997, 18: 209-21. 10.1016/S0896-6273(00)80262-7.View ArticlePubMedGoogle Scholar
- Kadmon G, Altevogt P: The cell adhesion molecule L1: species- and cell-type-dependent multiple binding mechanisms. Differentiation. 1997, 61: 143-150. 10.1046/j.1432-0436.1997.6130143.x.View ArticlePubMedGoogle Scholar
- Liu Q, Dwyer ND, O'Leary DD: Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR. J Neurosci. 2000, 20: 7682-7690.PubMedGoogle Scholar
- Perrin FE, Stoeckli ET: Use of lipophilic dyes in studies of axonal pathfinding in vivo. Microsc Res Tech. 2000, 48: 25-31. 10.1002/(SICI)1097-0029(20000101)48:1<25::AID-JEMT4>3.0.CO;2-F.View ArticlePubMedGoogle Scholar
- Image J. [http://rsb.info.nih.gov/ij/index.html]
- Gesemann M, Lesslauer A, Maurer CM, Schönthaler HB, Neuhauss SCF: Phylogenetic analysis of the vertebrate Excitatory/Neutral Amino Acid Transporter (SLC1/EEAT) family reveals lineage specific subfamilies. BMC Evol Biol. 2010, 10: 117-10.1186/1471-2148-10-117.PubMed CentralView ArticlePubMedGoogle Scholar
- MUSCLE. [http://www.ebi.ac.uk/Tools/msa/muscle/]
- Gblocks. [http://molevol.cmima.csic.es/castresana/Gblocks.html]
- PredGPI. [http://gpcr2.biocomp.unibo.it/predgpi/pred.htm]
- GPI-SOM. [http://gpi.unibe.ch/]
- Hooper NM, Low MG, Turner AJ: Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C. Biochem J. 1987, 244: 465-469.PubMed CentralView ArticlePubMedGoogle Scholar
- White IJ, Souabni A, Hooper NM: Comparison of the glycosyl-phosphatidylinositol cleavage/attachment site between mammalian cells and parasitic protozoa. J Cell Sci. 2000, 113: 721-727.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.