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Involvement of IKAP in peripheral target innervation and in specific JNK and NGF signaling in developing PNS neurons.

Abashidze A, Gold V, Anavi Y, Greenspan H, Weil M - PLoS ONE (2014)

Bottom Line: Here we attempted to elucidate the role of IKAP in PNS development in the chick embryo and found that IKAP is required for proper axonal outgrowth, branching, and peripheral target innervation.Moreover, we demonstrate that IKAP colocalizes with activated JNK (pJNK), dynein, and β-tubulin at the axon terminals of dorsal root ganglia (DRG) neurons, and may be involved in transport of specific target derived signals required for transcription of JNK and NGF responsive genes in the nucleus.These results suggest the novel role of IKAP in neuronal transport and specific signaling mediated transcription, and provide, for the first time, the basis for a molecular mechanism behind the FD phenotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Neurodegenerative Diseases and Personalized Medicine, Department of Cell Research and Immunology, The Sagol School of Neurosciences, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
A splicing mutation in the ikbkap gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we attempted to elucidate the role of IKAP in PNS development in the chick embryo and found that IKAP is required for proper axonal outgrowth, branching, and peripheral target innervation. Moreover, we demonstrate that IKAP colocalizes with activated JNK (pJNK), dynein, and β-tubulin at the axon terminals of dorsal root ganglia (DRG) neurons, and may be involved in transport of specific target derived signals required for transcription of JNK and NGF responsive genes in the nucleus. These results suggest the novel role of IKAP in neuronal transport and specific signaling mediated transcription, and provide, for the first time, the basis for a molecular mechanism behind the FD phenotype.

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Related in: MedlinePlus

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.
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pone-0113428-g003: 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.

Mentions: 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).


Involvement of IKAP in peripheral target innervation and in specific JNK and NGF signaling in developing PNS neurons.

Abashidze A, Gold V, Anavi Y, Greenspan H, Weil M - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4237409&req=5

pone-0113428-g003: 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.
Mentions: 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).

Bottom Line: Here we attempted to elucidate the role of IKAP in PNS development in the chick embryo and found that IKAP is required for proper axonal outgrowth, branching, and peripheral target innervation.Moreover, we demonstrate that IKAP colocalizes with activated JNK (pJNK), dynein, and β-tubulin at the axon terminals of dorsal root ganglia (DRG) neurons, and may be involved in transport of specific target derived signals required for transcription of JNK and NGF responsive genes in the nucleus.These results suggest the novel role of IKAP in neuronal transport and specific signaling mediated transcription, and provide, for the first time, the basis for a molecular mechanism behind the FD phenotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Neurodegenerative Diseases and Personalized Medicine, Department of Cell Research and Immunology, The Sagol School of Neurosciences, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
A splicing mutation in the ikbkap gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we attempted to elucidate the role of IKAP in PNS development in the chick embryo and found that IKAP is required for proper axonal outgrowth, branching, and peripheral target innervation. Moreover, we demonstrate that IKAP colocalizes with activated JNK (pJNK), dynein, and β-tubulin at the axon terminals of dorsal root ganglia (DRG) neurons, and may be involved in transport of specific target derived signals required for transcription of JNK and NGF responsive genes in the nucleus. These results suggest the novel role of IKAP in neuronal transport and specific signaling mediated transcription, and provide, for the first time, the basis for a molecular mechanism behind the FD phenotype.

Show MeSH
Related in: MedlinePlus