IKAP is expressed in growing axons in the developing PNS

To evaluate ikbkap expression levels in the developing DRG in the context of PNS development, we studied the ikbkap expression together with other relevant genes in DRG of E6 to E18 embryos at the lumbar L4, L5, and sacral S1 levels. Quantitative real time PCR (QRT-PCR) analysis shows that ikbkap mRNA expression is at the highest level at E6, at the time of intensive neurite outgrowth and peripheral organ innervation (Fig. 1A). In this context, we compared the pattern of ikbkap expression to the neural specific βIII-Tubulin (TUBB3) and β-Actin (ACTB) genes, both are known key players in axonal outgrowth process [18]. Together with this, we related ikbkap expression to the homeodomain transcription factor Islet-1 (ISL1), one of the key regulators of sensory differentiation [19]. In this analysis, we found that by Pearson's correlation, ikbkap transcript levels are best correlated with the levels of ACTB (r = 0.97) and TUBB3 (r = 0.94), as well as with ISL1 (r = 0.86). In contrast, ikbkap expression is weakly correlated with a promoter of neural progenitor development Retinoic Acid Receptor β (RARB) (r<0.62), which was shown to induce neurite outgrowth in NGF-dependent DRG neurons [20]. A weak correlation with ikbkap expression (r<0.73) was found for the calcium channel CACNA1B gene, which is a representative for the primary calcium release electrophysiological activity that should appear at these early stages of PNS development [21]. Altogether, these results suggest that in the course of DRG development at the time of neuronal innervation ikbkap is expressed in a similar temporal pattern with cytoskeletal genes like actin and tubulin, and with a key regulator of sensory differentiation Islet-1.

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Figure 1. IKAP expression in the developing PNS.

(A) QRT-PCR analysis of expression levels of ikbkap, neural specific βIII-Tubulin (TUBB3), β-Actin (ACTB), Islet-1 (ISL1), Retinoic Acid receptor β (RARB), and CACNA1B in developing lumbar DRG of E6-E18 embryos. E10 was used arbitrarily as normalizing time point reference. Data from three independent experiments are presented as means ±SD, N = 5 embryos at each time point. Correlation data was obtained following Pearson's correlation test. (B–E) Tiling reconstruction showing IKAP expression in transverse sections of E3/HH18-E5/HH27 embryos at the hind limb level, stained with Hoechst 33342 to visualize nuclei (blue), and IKAP specific antibody (red) in combination with Tuj1 (B–C, green), HNK-1 (D, green), or with Islet-1 (E, green). Size bars 100 µm. NT- Neural tube, DRG – Dorsal Root Ganglia, SG – Sympathetic Ganglia, MN – motor neurons.

https://doi.org/10.1371/journal.pone.0113428.g001

At the protein level, IKAP is absent at E3 and E4 (Fig. 1B–C) but becomes apparent at E5 in the developing DRG neurons and in primary SG (Fig. 1D–E). At E5, the DRG is formed by a core of postmitotic differentiating sensory neurons, which send growing axons toward the spinal cord and to the peripheral targets, and of an outer layer of proliferating neural progenitors. As shown in Fig. 1D, IKAP (labeled in red) is abundantly expressed in growing neurites in the inner core of the DRG, but not in proliferating neural progenitors expressing the neural crest marker HNK-1 (labeled in green) in the outer layer. Confirming IKAP specific expression in postmitotic neurons, we show that IKAP is co-expressed with Islet-1 postmitotic transcription factor [22], (Fig. 1E). In the spinal cord, IKAP is detected in interneurons and motor neurons (MN) expressing Islet-1, and at the entry sites of growing DRG axons dorsally. Interestingly, IKAP is not expressed along the dorsoventral route in early forming PNS organs at E3 and E4 (Fig. 1B and 1C respectively), where the nascent neurons are stained with βIII-type neuron specific tubulin (Tuj1, green). Instead, IKAP is weakly expressed along the dorsolateral NCC migration pathway in distinct population of cells like melanocytes (outlined in red). We also investigated IKAP expression levels during the course of NCC migration and differentiation into neurons in vitro (Fig. 2). Neural tubes were explanted from the embryos just before the onset of NCC migration (E2/HH11), allowing the NCC to migrate (Fig. 2A–B) and to differentiate (Fig. 2C) in culture for the period of 24 and 72 hours. After 24 hours in culture, confocal images of NCC show low endogenous IKAP expression, partly co-localized with Tuj-1 (Fig. 2D–E). After 72 hours in culture, IKAP seems to be colocalized with Tuj-1 within the outgrowing neurites (Fig. 2C and 2H–L), but not with actin (Fig. 2G). Moreover, IKAP fluorescence intensity levels increase in the developing neurons at least 6 times compared to migrating NCC as shown by corrected total cell fluorescence (CTCF) quantification in Fig. 2I (explained in methods). Fig. 2J–L show IKAP intracellular localization in an extending neuron. Here IKAP shows a clear vesicular pattern and is distributed along Tuj-1 labeled extending filaments within the growing neurites (Fig. 2L), and to a lesser extent, IKAP is present at the soma region (Fig. 2K, indicated by an arrow). Interestingly, this vesicular pattern of IKAP localization is similar to one that we previously described in PNS neurons derived from human embryonic stem cells [23].

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Figure 2. Characterization of IKAP expression in differentiating NCC neuronal cultures.

Neural tubes were explanted from the embryos just before the onset of NCC migration (E2/HH11), allowing NCC to migrate and differentiate in culture. (A–B) Migrating NCC after 24 h in culture stained for Propidium iodide (PI) to visualize nuclei, specific NCC marker HNK-1 (A), neural specific tubulin (Tuj1, B). (C) Migrating NCC after 72 hours in culture stained Tuj1 and PI. (D–E) IKAP expression in migrating NCC (24 h in culture) show vesicular pattern and colocalized with Tuj1 at the leading edge of the cell (E, white arrow). Boxed area in D is magnified in E. (F–H) Immunofluorescence confocal images of quadruple stained cells show IKAP staining with Alexa-488 conjugated secondary antibody (green in G and H) either in combination with Phalloidin conjugated with TRITC for Actin labeling (red in G) or together with Tuj1 stained with Alexa 647 conjugated secondary antibody (red in H). Hoechst 33342 (blue) was added to stain nuclei in these images. (F) For IKAP fluorescence quantification in NCC versus developing neurons (I), NCC cell borders were selected using actin staining (upper panel), neuron cell borders were selected using Tuj1 staining (middle panel), and these cell borders were overlaid on IKAP stained picture (bottom panel). Then Area, Integrated Density, and Mean Gray Value were measured in the cells using ImageJ, and Corrected Total Cell Fluorescence (CTCF) was calculated as described in methods from a sample number of 15 neurons versus 15 NCC. (G–H) Two images of differentiating NCC showing the same confocal plane (72 h in culture). Note that IKAP levels increase in the outgrowing neurites of differentiating neurons, and that IKAP is colocalized with Tuj1 (H, red), but not with actin (G, red). (I). Corrected Total Cell Fluorescence (CTCF) in NCC and differentiating neurons. Data are represented as mean ±SD. (J–L) IKAP expression in NCC derived neurons show vesicular pattern and predominantly is localized along Tuj1 positive extending filaments within growing neurites (L), and to lesser extent at the soma region of the cell (K, indicated by an arrow). Boxed areas in J are magnified in K and L. (G–H) and (K–L) show a set of orthogonal slices, where the middle panel represent the xy plane, left panel represent the yz plane, and upper panel represent the xz plane.

https://doi.org/10.1371/journal.pone.0113428.g002

Ikbkap downregulation does not affect NCC migration

It was previously described that IKAP localizes to membrane ruffles and regulates cell migration in several cell types [24], but this molecular mechanism was not yet tested in migrating NCC. Here we show that in migrating NCC IKAP colocalizes with distinct Tuj1 rich structures at the leading edges of lamellipodia (Fig. S1B–D, white arrow), and in ikbkap siRNA treated NCC, such structures are disrupted (Fig. S1F–H, red arrow). However, despite this observed phenotype as consequence to ikbkap downregulation, no significant difference in NCC migration or neuronal numbers was detected either in vitro or in vivo (Fig. S1I–N). These results confirm a previous report by Hunnicutt and colleagues [17], and George and colleagues [16], which arrived to a similar conclusion that ikbkap downregulation does not affect NCC migration, pathfinding and DRG formation.

IKAP is involved in neurite outgrowth and peripheral target innervation in vivo

To test the IKAP role at the stages of neural outgrowth, the embryos were electroporated either with control or ikbkap specific siRNA at E2/HH11, and allowed to develop until E6. The transverse serial sections from these embryos were stained with Tuj1 antibody to display neuronal patterns (Fig. 3A–B). We found that in ikbkap downregulated embryos, the peripheral projections of the DRG neurons were markedly disturbed and axons were misguided at the ventral root exit from the spinal cord (Fig. 3B, white arrows), while in the control embryos the exit routes from the spinal cord and DRG were well defined (Fig. 3A). These neuronal guidance abnormalities were further confirmed in whole mount E6 embryo preparations. For these experiments, the embryos were electroporated at E2/HH11 with control siRNA or with ikbkap specific siRNA, supplemented with pCAAG GFP expressing plasmid to visualize the electroporared targets. The embryos showing strong GFP fluorescence were allowed to grow until stage E6. Figure 3C–D shows representative tiling reconstruction with serial z-planes composed of multiple images of GFP labeled nerves taken at the lumbar and hindlimb regions (outlined in white) in control (Fig. 3C), and ikbkap specific siRNA treated embryos (Fig. 3D), (n = 6 per group). Abnormal growth of lumbar nerves innervating the hind limb was observed in ikbkap downregulated compared to control embryos (see colored arrows). Higher magnification images in boxed regions at the proximal hind limb position (Fig. 3E–F) show anterior branches (labeled by white arrow), midline branches (labeled by blue arrow), and posterior branches (labeled by red arrow) and display diverse phenotypes between the control siRNA (Fig. 3E) and ikbkap siRNA (Fig. 3F) treatments. At the anterior position (white arrow), we can observe that the nerve fibers in ikbkap siRNA treated embryos are more ramified bearing more branches at the axon terminals. In contrast, at the medial region of the limb (blue arrow) a whole nerve branch seems to be absent in ikbkap siRNA (Fig. 3F) in comparison with control siRNA treated embryos (Fig. 3E). The red arrow indicates the site where a nerve branch shows fewer ramifications in ikbkap siRNA in comparison to the same branch at the same position in the control. Figure 3G and H show magnification of boxed areas at the most distal outgrowing nerve ends in hind limbs of control siRNA and ikbkap siRNA treated embryos respectively. The growing nerves innervate the hindlimb in a similar manner, but the axonal ends in ikbkap siRNA treated embryos seem to be less developed (Fig. 3G, light blue and purple arrows). Similar confocal microscopy analysis was performed in the dermis of the abdomen stained with Tuj1 antibodies to visualize the PNS network. Misguided and aberrant branching is clearly observed in the ikbkap downregulated neurons (Fig. 3K and L) compared to the control (Fig. 3I and J). Note the multiple emerging branching points observed in ikbkap downregulated axons (see arrows Fig. 3L). In conclusion, these results indicate that IKAP is involved in fine tuning of the innervation process involving branching and positioning of small processes, while the positioning and lengths of the main nerves seems to be unaffected by ikbkap downregulation. Supporting these findings, we show in DRG dissociated cultures that ikbkap downregulation affect neuronal network formation, increasing adhesion between cells and branching (Fig. S2).

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Figure 3. Ikbkap downregulation affects target innervation in vivo.

(A–B) The embryos were electroporated with control or ikbkap specific siRNA at E2/HH11, and allowed to develop until E6. The transverse serial sections were stained with Tuj1 antibody to display neuronal patterns and with Hoechst 33342 to visualize nuclei. In ikbkap downregulated embryos abnormal peripheral nerve projections are visualized at various positions (B, white arrows) compared to control embryos (A), n = 5 embryos per treatment. Size bar 100 µm. (C–H) To visualize growing nerves, the embryos were co-electroporated with control siRNA plus pCAAG GFP expressing vector or ikbkap specific siRNA plus pCAAG GFP expressing vector at E2/HH11, and allowed to develop until E6 (N = 6). Representative tiling reconstruction with serial z-planes composed of multiple images of GFP labeled nerves taken at the lumbar and hindlimb regions (outlined in white) of control siRNA treated embryos are shown in (C, E, G), and of ikbkap specific siRNA treated embryos in (D, F, H). Boxed areas in C and D are magnified in E–G and F–H respectively. Size bar 100 µm. (I–L) Close up of skin innervations in abdomen region of Tuj1 stained whole mount embryos from previous experiment. I, J – control siRNA treated embryos; K, L – ikbkap specific siRNA treated embryos. White arrows indicate abnormal branching points. Size bar 10 µm.

https://doi.org/10.1371/journal.pone.0113428.g003

Ikbkap downregulation affects tubulin structure in growth cones

We further aimed to investigate whether previously observed IKAP-dependent disturbances in target innervations can be explained by deregulation of the cytoskeleton. We demonstrate that in growth cones of control DRG cultures (Fig. 4A–D) IKAP is localized mostly along stable tubulin fibers (magenta arrows), and to less extent along dynamic tubulin fibers (light blue arrows) in filopodia and lamellipodia. In IKAP depleted growth cones (Fig. 4E–H), normal tubulin structures are disturbed and dynamic tubulin fibers seems to be fragmented and partly co-localized with IKAP (green arrows). To characterize changes in IKAP and tubulin localization in the growth cones, we compared IKAP and tubulin intensity and density distributions in a set of pictures from three independent experiments (Fig. 4I–L) using a custom algorithm as described in methods. In control growth cones (Fig. 4I and 4K), we observe large numbers of low intensity IKAP and tubulin pixels, whereas in ikbkap downregulated cultures (Fig. 4J and 4L) we observe low numbers of high intensity pixels. The same dynamics were observed in density analysis, confirming aggregation of IKAP and tubulin in ikbkap downregulated growth cones.

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Figure 4. Ikbkap downregulation affect β-tubulin structure in growth cone.

DRG from lumbar region of E10 embryos were electroporated with control or ikbkap specific siRNA, grown on laminin for 48 hours as described in methods, and stained with IKAP (red) and β-tubulin (green) antibodies. (A–H) Representative confocal images of the growth cone areas of control (A–D) and ikbkap siRNA treated (E–H) neurons. Boxed areas in A–B and E–F are magnified in C–D and G–H respectively. Colored arrows indicate IKAP localization along stable tubulin fibers (magenta arrows in C and D; red arrows in G and H), and along dynamic tubulin fibers (light blue arrows in C and D; green arrows in G and H). (I–L) Histograms representing the fluorescence intensities and densities of IKAP and β-tubulin signals at the growth cone area measured from multiple images by custom image analysis tool (see Methods).

https://doi.org/10.1371/journal.pone.0113428.g004

Ikbkap downregulation affects JNK and NGF signaling in DRG neurons

The results represented above show that ikbkap downregulation directly affects cytoskeletal morphology in axon terminals, which may have direct implications in microtubules dynamics, axonal growth, and axonal transport. Since IKAP is known to directly bind JNK [9], and JNK is known to regulate microtubules stability in neurons (reviewed in Sakakibara et al., [25]), we hypothesized that JNK functions might be affected in IKAP depleted neurons. In addition, we examined the possibility that IKAP is involved in retrograde transport by direct association with the motor protein dynein. To this end, we performed analysis of IKAP colocalization with active phosphorylated JNK (pJNK) and dynein in explanted DRG cultures taken from E10 embryos, which were electroporated with control or ikbkap specific siRNA and grown for 48 hours as described in methods (Fig. 5). Confocal analysis of a total depth of 2.66 µm divided in three fixed serial z-sections (of 0.88 µm z-step interval) at the growth cone area of axon terminals was performed in all images of three independent experiments. In control siRNA treated growth cones, specific JNK activation was mostly observed at the base z1 and middle z2 sections (Fig. 5A and B), but not at the top section z3 (Fig. 5C). p-JNK colocalization with IKAP mostly occurs at the z2 section (Fig. 5B and D). Similar colocalization pattern was detected for dynein and IKAP (Fig. 5I–L). In contrast, ikbkap siRNA treated growth cones show a spatial change in pJNK and dynein signals manifested by an increased ectopic pJNK aggregation colocalized with IKAP at all the 3 z-section levels (Fig. 5E–H), as well as aggregation of dynein with IKAP at the z-1 and z2 section levels (Fig. 5M–P). The colocalization between the signals was quantified using a colocalization image analysis tool (JACoP, ImageJ software) (Fig. 5Q) as described in methods. Interestingly, the results show that most of dynein and about a half of pJNK measured at the growth cone area in control culture conditions are associated with IKAP (93.4 and 52.7% respectively), but not vice versa for IKAP. As a result of ikbkap downregulation, a significant increase of about two folds in the proportion of IKAP which is colocalized with pJNK (IKAP/pJNK) was observed together with a significant increase (33%) in the proportion of pJNK which is colocalized with IKAP (pJNK/IKAP) at the growth cone area. The proportion of dynein co-localized with IKAP also appears to be affected (18% reduction) by ikbkap downregulation. Moreover, abnormal dynein accumulation along the developing neurites was observed in ikbkap downregulated cultures (Fig. 5S, white arrows) compared to control (Fig. 5R), suggesting a disturbance in dynein dependent transport in these cells. Overall, these results show that IKAP is spatially colocalized with pJNK and dynein and the localization pattern of expression in the axon terminus of both proteins is dependent of IKAP expression levels that may affect concomitantly axonal transport. To evaluate the ikbkap downregulation effect on potential dynein-dependent axonal transport disturbances and transcriptional regulation, we performed QRT-PCR analysis of known JNK responsive AP-1 genes c-jun, c-fos, and fosl2; along with known NGF induced immediate early transcription factors MEF2D, SRF and EGR1 [26], [27], [28] in control and ikbkap downregulated DRG cultures (Fig. 6A). We observed significant reduction in the expression levels of the pJNK and NGF signaling dependent genes as a consequence of 54% reduction in ikbkap mRNA levels after 48 hours in culture. In parallel, we performed analysis of the expression of cytoskeletal genes Tuj1 and β-Actin, regulators of proliferation SOX10 and SOX11, and calcium signaling genes SCN9A and CACNA1B, but their expression was not affected in respect to ikbkap downregulation (Fig. 6B). These results confirm our assumption that IKAP may be involved in transport of specific signals and demonstrate that ikbkap downregulation affects the expression of pJNK and NGF-inducible transcription factors in developing DRG neurons, establishing a mechanism that could explain the in vivo innervation phenotype described above (see Fig. 3 and 4), as well as increased neuronal cell death in FD neurons.

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Figure 5. Ikbkap downregulation affects pJNK and dynein localization in growth cones.

DRG from lumbar region of E10 embryos were electroporated with control or ikbkap specific siRNA, grown on laminin for 48 hours as described in methods, and stained for IKAP, phosphorylated JNK (pJNK) (A–H), or dynein (I–P, R, S). Confocal analysis of three serial z-sections of the growth cone was performed in images from three independent experiments. The total depth of the image z-stacks is 2.66 µm, z1 represents 0–0.88 µm, z2 represents 0.88–1.77 µm, and z3 represents 1.77–2.66 µm slices. (Q) Mander's overlap coefficient of IKAP-pJNK and IKAP-dynein localization was performed using ImageJ colocalization plugin (JACoP) as described in methods. Data are presented as mean ±SD. (R, S) Dynein accumulation along the developing neurites is observed in ikbkap downregulated cultures (S, arrows) compared with control neurites (R).

https://doi.org/10.1371/journal.pone.0113428.g005

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Figure 6. Ikbkap downregulation affects expression of pJNK and NGF responsive genes in DRG neurons.

DRG from the lumbar region of E10 embryos were electroporated with control or ikbkap specific siRNA, grown on laminin for 48 hours, and processed for QRT-PCR as described in methods. Data are presented as relative gene expression levels of mean ±SD.

https://doi.org/10.1371/journal.pone.0113428.g006

siRNA treatment

The embryos were staged according to Hamburger and Hamilton [36]. Four chick ikbkap specific or control scramble siRNA duplexes (with or without Cy3 label) were designed and purchased at Sigma Genosys (Table S1). Ikbkap specific siRNA duplexes were used in mix or interchangeably in different experiments. The siRNAs (0.5 µg/µl final concentration) in Fast Green dye solution (Sigma) were microinjected into the neural tube lumen at stage E2/HH11 embryos, and electroporated (BTX830 system, Molecular Delivery Systems) with square pulse (8 pulses of 25 mV for 50 msec with a 1 sec interval) using homemade tungsten electrodes placed at the two sides of the embryo. The efficiency of electroporation was assessed by siRNA labeled Cy3 fluorescence, and efficiency of ikbkap downregulation was evaluated by quantitative real-time PCR (QRT-PCR). Electroporation of dissected DRG was performed similarly in 24 well dish plates in 1 ml of fresh egg albumin, to reduce siRNA leakage.

Neural tube explants

After electroporation, the embryos at the 13 somite stage were selected. Neural tubes were excised in 2% pancreatin solution (Sigma) at the defined trunk region (Fig. S1I, red rectangle). The neural tubes were grown on fibronectin coated (15 µg/ml, Sigma) coverslips in DMEM supplemented with 10% FBS, 2 mM Glutamax, 1 mM sodium pyruvate, 1% of non-essential amino acid stock, 100 µg/ml penicillin, and 100 mg/ml streptomycin.

DRG cultures

Electroporated whole DRG or dissociated single cell neural progenitors (obtained from DRG with gentle trituration in 0.25% trypsin solution) were grown on laminin (5 µg/ml) coated coverslips in neurobasal medium (Invitrogen), supplemented with 2% B27 supplement (Invitrogen), 2 mM Glutamax (Invitrogen), 1 mM sodium pyruvate, 1% of non-essential amino acid stock, 100 µg/ml mg/ml penicillin, 100 mg/ml streptomycin, and 20 ng/ml rhNGF (R&D systems).

Immunofluorescence of cells, embryos, and frozen sections

The embryos or cell cultures were fixed with 4% PFA, washed thoroughly with PBT (PBS/0.01%Triton X-100), permeabilized with PBS/1% Triton X-100, blocked with PBS/2%BSA/10%FBS/0.05%Triton X-100 for 1 hour, and incubated with appropriate primary antibodies in blocking solution at 4°C overnight (whole mount embryos were incubated at 4°C for 48 hours with agitation). The specimens were washed several times with PBT and incubated with appropriate secondary antibodies (for 3–6 hour with agitation for whole mount embryos; 1 hour for cultures). Hoechst 33342 dye (0.02 mg/ml) was added to the secondary antibody solution for nuclear staining. The specimens were rinsed and prepared for visualization. For staining in frozen section, the embryos were snap frozen in OCT-embedding medium. Transverse sections of the embryos (16 µm thick), were immediately fixed with ice cold methanol for 5 minutes, washed in PBS, and stained as described above. Antibody sources are listed in Table S1.

Quantitative Real Time PCR

RNA from at least 3 biological replicates was extracted using RNeasy Kit (Qiagen) and reverse transcribed with Applied Biosystem Kit. QRT-PCR was performed in technical triplicates using Cyber green mix (AB gene, Surrey, UK) and Rotor-Gene 6000 (Qiagen) workstation. HPRT1 expression was taken to normalize gene expression levels. Primer sequences are listed in Table S1.

Image analysis

For IKAP fluorescence levels quantification (see Fig. 2I), CTCF (Corrected Total Cell Fluorescence) was calculated using ImageJ software as follows:

CTCF  =  Integrated Density - (Area of selected cell X Mean fluorescence of background readings).

Briefly, NCC cell borders were selected using actin staining as shown in Fig. 2F, upper panel), Neuron cell borders were selected using Tuj1 staining as shown in Fig. 2F, middle panel. These cell borders selections were overlaid on the IKAP stained pictures (Fig. 2F, bottom panel). Then to calculate CTCF the Area, Integrated Density, and Mean Gray Value as well as the background measurements of IKAP expression were measured in NCC cells and neurons using ImageJ, In the regions where neurons are located above the NCC, average NCC CTCF was substracted from total CTCF value to obtain neuronal CTCF value. Sample number N = 15 neurons versus 15 NCC.

For measuring IKAP and Tubulin expression levels in Fig. 4, custom MATLAB scripts were used to test intensity and density of the protein expression in the growth cones. Intensity values were defined as (Sum of intensities in user defined polygon area)/(Polygon area). Density values were defined as (Number of pixels above density threshold in user defined polygon area)/(Polygon area).

For measuring IKAP, pJNK and Dynein expression levels in Fig. 5, confocal images with resolution 1024×1024 pixels were used for analysis. The expression pattern of the stained proteins was assessed by making maximum intensity projections. For colocalization analysis, we used ImageJ Subtract Background function followed by Intensity Correlation Analysis (JaCoP plug-in) to obtain the Pearson's Correlation coefficient and Manders overlap coefficient (Bolte and Cordelieres, [37]).

Statistical analysis

Data are presented as means ±SD. Statistical significance was analyzed using one-way ANOVA and Post hoc analysis using Tukey follow up test. The differences with p≤0.05 were referred as significant.

Supporting information

Supporting information include Fig. S1, which describes experiments of ikbkap downregulation in migrating NCC and in early DRG in vitro and in vivo; Fig. S2, showing Ikbkap downregulation effect on the network formation in DRG dissociated cultures; Table S1, containing the list of siRNA and the primer sequences, and Table S2, containing list of antibodies used in this study.

Ethics Statement

In all our experiments, the embryos were euthanized by decapitation starting stage E4. According to the local guidelines, experiments with chick embryos at stages used in this study (between E2 and E18) do not require IACUC approval. The Tel Aviv IACUC is working according to the following rules: The state law of prevention of animal cruelty (animal experimentations) 1994; Guidelines for preventions of animal cruelty (animal experimentations) 2001 published by the council for animal experimentation and The NRC Guide for the Care and Use of Laboratory Animals.