Article Figures & Data
Figures
- Figure 1.
KIDLIA is expressed in neurons and is localized in the nucleus. A, Immunohistochemistry in brain slices of P0 mouse cortex. KIDLIA was colocalized with the nuclear marker Hoechst. Scale bar = 10 μm. B, C, Immunohistochemistry of KIDLIA at E17. KIDLIA expression began in the SP region of the upper IZ and throughout the cortical plate and was restricted to the cortical plate at P0. Scale bars = 20 μm. D, E, KIDLIA was expressed only in cells positive for the neuronal marker NeuN at P0 (left) and P14 (right). F, KIDLIA expression was not observed in cells expressing the glia marker GFAP at P14. Scale bars = 20 μm (full picture); 10 μm (enlarged area). UpCP, upper cortical plate; LoCP, lower cortical plate.
- Figure 2.
In utero electroporation of KIDLIA shRNA disrupts neuronal migration but does not affect the multipolar-to-bipolar transition. A, Schematic of the IUE procedure. Pups were injected with shRNA-GFP DNA into the lateral ventricle (LV) at E15, and the anode of the Tweezertrode was placed above the dorsal telencephalon. Pups were the returned to the mother to mature until the times indicated. B, Representative images of scrambled and KIDLIA shRNA electroporated neurons at E17. Immunostaining of KIDLIA shows a clear reduction of KIDLIA expression compared with scrambled controls and nearby nonelectroporated neurons. Scale bar = 10 μm. C, Brain slices taken at E17 after IUE of KIDLIA shRNA-GFP or scrambled shRNA-GFP at E15. Scale bar = 50 μm. D, KIDLIA knockdown caused a greater percentage of neurons in the upCP and a smaller fraction in the IZ. More than 1500 GFP+ neurons from five brains were analyzed in each group. E, Brain slices taken at P0 after IUE of KIDLIA shRNA-GFP or scrambled shRNA-GFP at E15. Scale bar = 50 μm. F, Analysis of neuronal migration at P0 showed that more neurons were in the upCP and less in the IZ compared with scrambled controls. More than 1200 GFP+ neurons from four brains were analyzed in each group. G, H, Analysis of the multipolar-to-bipolar transition. In E17 brain, multipolar neurons were observed in the upper IZ (magnified lower right) before CP entry, and neurons showed a normal transition back to a bipolar morphology after CP entry (magnified upper right) in both KIDLIA and scrambled shRNA electroporated neurons. Scale bars = 10 μm. **p < 0.01, ***p < 0.001. Error bars = SEM. Yellow dashed line indicates the pia. UpCP, upper cortical plate; LoCP, lower cortical plate.
- Figure 3.
Knockdown of KIDLIA affects apical dendrite growth and orientation in vivo. A, Images (left) and tracings (right) of P4 layer II/III cortical neurons after electroporation of GFP-labeled shRNA constructs at E15. Yellow dashed lines indicate the pia; scale bar = 10 μm. B, No significant change was observed in the apical dendrite angle to the pia after knockdown of KIDLIA. C, An increase was observed in the length of the major apical dendrite (n = 6). D, Decrease in the distance of the soma to the pia (n = 6). E, No significant difference was observed in the number of dendrite tips reaching the pia. F, Images (left) and tracings (right) of P14 layer II/III cortical neurons after electroporation of GFP-labeled shRNA constructs at E15.5. Dashed lines indicate the pia; scale bar = 10 μm. G, Merged tracings of the major apical dendrites of P14 neurons after IUE of scrambled and KIDLIA shRNA showed that knockdown of KIDLIA disrupted the orientation of the apical dendrites toward the pia. Red bars show the average angle of the dendrites in relation to the pial surface (dashed line). H, Quantification of the angle of the major apical dendrite toward the pia showed a significant increase after loss of KIDLIA expression (n = 5). An angle of 0° indicates that a dendrite is growing directly toward the pia. I, A significant decrease in the length of the major apical dendrite. J, A decrease in the distance of the soma to pia. K, A significant decrease in the percentage of dendrite tips that reached the pia was observed after knockdown of KIDLIA in vivo. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars = SEM.
- Figure 4.
Knockdown of KIDLIA decreases dendritic growth and branching in vitro. A, Western blot after nuclear/cytoplasmic fractionation of primary rat hippocampal neurons shows clear nuclear expression of KIDLIA with no expression in cytoplasm. Neurons were treated with lentiviral KIDLIA shRNA or scrambled shRNA at DIV0 and collected at DIV6. B, Analysis of the Western blot data showed that the shRNA significantly reduced KIDLIA expression (n = 5). Nuclear loading control, HDAC1; cytoplasmic loading control, GAPDH. C, Immunostaining of KIDLIA in primary rat hippocampal neurons shows colocalization with the nuclear marker Hoechst. D, Immunostaining of dendrites (MAP2) and axons (tau1) at DIV12; scale bar = 10 μm. E, Sholl analysis of dendrite growth at DIV12 showed a significant change in branching (n = 14). F, Analysis of MAP2-positive dendrites showed a decrease in the longest dendrite segment (n = 14). G, Decrease in the number of dendrite branches (n = 14). (H) Significant decrease in the sum dendritic length (n = 14). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars = SEM.
- Figure 5.
Knockdown of KIDLIA leads to an increase in surface N-cadherin and the N-cadherin/δ-catenin association. A, Western blot from DIV6 neuronal lysates after treatment with KIDLIA shRNA virus (DIV0) showing an increase in total N-cadherin levels. Loading control, GAPDH. B, Quantification of the Western blot data represented in A (n = 3, each sample performed in duplicate and averaged). C, Knockdown of KIDLIA expression by shRNA lentivirus caused an increase in N-cadherin mRNA expression. Gene expression was normalized to the housekeeping gene, GAPDH (n = 3, each sample performed in triplicate and averaged). D, Surface biotinylation of neuronal lysates after treatment with scrambled or KIDLIA shRNA virus showed an increase in surface N-cadherin levels. E, Quantification of the Western blot data shown in C (n = 4). F, N-cadherin was co-immunoprecipitated with a dramatically larger fraction of δ-catenin after lentiviral shRNA knockdown of KIDLIA in neuronal lysates. The increased binding of δ-catenin to N-cadherin subsequently depleted the unbound, cytosolic fraction of δ-catenin. G, Images of neurons transfected with scrambled or KIDLIA shRNA with either GFP or δ-catenin–GFP; scale bar = 10 μm. H, Sholl analysis of the transfected neurons from E showed that δ-catenin overexpression could rescue the decreased dendrite growth and branching observed after knockdown of KIDLIA (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 Error bars, SEM.
- Figure 6.
Loss of KIDLIA disrupts actin dynamics at the neurite growth cone via δ-catenin. A, Immunocytochemistry of primary rat hippocampal neurons after transfection of scrambled or KIDLIA siRNA with GFP. Neurons were transfected at DIV0 and immunostained for KIDLIA (red) at DIV4. B, Analysis of the immunocytochemistry images showed that the siRNA significantly reduced KIDLIA expression (n = 10 for both groups). C, FRAP experiments after cotransfection of KIDLIA siRNA or scrambled siRNA with actin-GFP. Regions at the growing neurite tip were selected for photobleaching at 488 nm and imaged every 3 s. D, Analysis of the FRAP data showed that knockdown of KIDLIA produced a dramatic decrease in actin dynamics after photobleaching (n = 9). E, Neurons were cotransfected with KIDLIA siRNA or scrambled siRNA with actin-mCherry and δ-catenin–GFP or GFP alone, photobleached at 555 nm, and imaged every 3 s. F, Analysis of the FRAP data. Overexpression of δ-catenin rescued the actin dynamics after knockdown of KIDLIA (n = 7). G, The mobile fraction, calculated as the difference between the average level of bleaching and the level of recovery, was significantly decreased after knockdown of KIDLIA (n = 7). H, The immobile fraction of actin, calculated as the difference between the initial fluorescence and the level of recovery, was significantly decreased (n = 7). **p < 0.01, ****p < 0.0001. Error bars = SEM.
- Figure 7.
Increased RhoA activation leads to impaired actin dynamics. A, Western blot at DIV6 after treatment with KIDLIA shRNA or scrambled shRNA virus at DIV0. RhoA stimulation was performed with treatment with nocodazole (10 μm) for 30 min before collection of neuronal lysates. Activated RhoA-GTP was selectively immunoprecipitated using beads conjugated to the Rho binding domain of the Rho effector protein, rhotekin, and whole-cell lysates were probed for total RhoA levels. Loading control, GAPDH. B, Quantification of the RhoA assay showed a significant increase in the levels of activated RhoA-GTP after KIDLIA knockdown (n = 4). C, Images of neurons transfected with KIDLIA siRNA or scrambled siRNA with actin-mCherry and treated with CN06 or vehicle control 1 h before FRAP experiments. D, Analysis of the FRAP data showed that inhibition of the RhoA pathway could rescue the actin dynamics after knockdown of KIDLIA (n = 6). E, Images of neurons treated from DIV4 to DIV7 with CN06 or vehicle control after treatment with KIDLIA shRNA or scrambled control virus at DIV0; scale bar = 10 μm. F, Sholl analysis of the images in C showed that chronic inhibition of the RhoA pathway was sufficient to rescue dendrite outgrowth and branching (n = 11). G, Diagram depicting changes in the N-cadherin/δ-catenin signaling cascade after loss of KIDLIA expression. Increased surface N-cadherin sequesters δ-catenin after KIDLIA knockdown, thereby depleting the cytosolic free fraction of δ-catenin. Less inhibition on RhoA increases its activation and subsequent inhibition on neurite outgrowth via changes in actin dynamics. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars = SEM.
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