NudC Is Required for Interkinetic Nuclear Migration and Neuronal Migration during Neocortical Development

In utero electroporation

Plasmids were transfected using intraventricular injection followed by in utero electroporation (Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001). In brief, pregnant Sprague Dawley rats (Hilltop) were used, and 1–2 μl cDNA (1–5 μg/μl) or 1 μg/μl siRNA were injected into the ventricle of embryonic brains at E16. A pair of copper alloy oval plates that were attached to the electroporation generator (Harvard Apparatus) transmitted five electric pulses at 50 V for 50 ms at 1-s intervals through the uterine wall. Animals were maintained according to protocols approved by the Institutional Animal Care and Use Committee at Columbia University. Each phenotypic analysis was done with at least 3 independent litters.

Western Blot

For western blotting HeLa cells and cortical cells were transiently transfected. HeLa Cells (40-50% confluent) cultivated in DMEM containing 10% FBS in 35mm dishes were transfected using effectene (Qiagen) for cDNA and oligofectamine (InVitrogen) for siRNA oligos as directed by the manufacturer. Primary cortical cells were obtained from E16 rat brains and cultured in Neurobasal/B27 medium. Briefly, cells were plated at a density of 1×10⁵ on 24 well plates in Neurobasal medium containing B27, L-glutamine (0.5mM), and FCS (2%). Cortical E16 cells were transfected using the nucleofector kit from Amaxa Biosystems, Gaithersburg, MD, following manufacturer’s protocol. Briefly, cortical cells were spun down (80 rpm) and mixed with 100 μl of nucleofector solution and 2 μg of plasmids. The cells/plasmid mixture was then nucleofected using program A-033.

Transfected cells were then plated at a density of 5×10⁶ per 60 mm culture dish. Cells were lysed 48 h after transfection in RIPA buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% NP-40, and 1 mM EGTA) with protease inhibitor cocktail for mammalian tissues (Complete, Roche). Proteins were separated in 4-20% gradient SDS–PAGE and transferred to PVDF membranes (Immobilon-P; Millipore). NudC was immunodetected using an antibody to mouse NudC that was raised in chicken against the full-length protein with AvesLabs, Inc. Lis1 was immunodetected with a monoclonal anti-Lis1 antibody (clone LIS1-338, Sigma-Aldrich) and Dynein Intermediate Chain with a monoclonal anti-Dynein Intermediate Chain antibody (MAB1618, Chemicon).

Immunoprecipitation assays

HeLa cells, seeded on 6 well plates, were co-transfected with GFP-NudC and pCDNA3.1 vector for control, NudC-myc, NudC-NT-myc or NudC-CT-myc. Cells were lysed 24h after transfection, in 150μl RIPA buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% NP-40, and 1 mM EGTA) containing protease inhibitor cocktail (Complete, Roche). Myc NudC was immunoprecipitated from 100 μl lysate using a mouse purified myc antibody (9E11) bound to protein-G sepharose beads (30 μl for each IP, GE Healthcare) for 2-4h at 4C. After IP, supernantants were saved and beads were washed 3 times in RIPA buffer and resuspended in 100 μl SDS sample buffer. Same volumes of Total, IP and supernatant were loaded on gel for western blotting. For quantification, the amount of co-immunoprecipitated GFP-NudC protein was normalized to the corresponding amount of myc protein immunoprecipitated. The wild-type GFP-NudC/NudC-myc ratios were set to 1 and all other values were calculated relative to it. Graph and statistical results were generated using GraphPad prism software.

Live cell imaging

Coronal slices were prepared 48–72 h after electroporation. Slices were placed on Millicell-CM inserts (Millipore) in culture medium containing 25% Hanks balanced salt solution, 47% basal MEM, 25% normal horse serum, 1× penicillin/streptomycin/glutamine (GIBCO BRL), and 0.66% glucose and were incubated at 37°C in 5% CO2. Multiple GFP-positive cells were imaged on an inverted microscope (Olympus) with a 40× objective. Time-lapse images were captured by camera (Hamamatsu Orca-ER) using MetaMorph software (Universal Imaging Corp.) at intervals of 10 min for 5–12 h.

Immunocytochemistry

Rat embryos were perfused transcardially with ice-chilled saline followed by 4% PFA (EMS) in 0.1 M PBS, pH7.4. Brains were postfixed in PFA overnight and sectioned on a Vibrotome (Leica). Slices were blocked at RT for 1 h with 10% serum, 0.5% Triton X-100 in PBS. Primary antibodies were applied overnight at 4° C in 10% serum, 0.5% Triton X-100 in PBS at the following concentrations: anti-PCNA (1:200, mouse, Dako); anti-TuJ1 (1:200, mouse, Covance); anti-Tbr2 (1:1000, Rabbit, Chemicon); anti-Phospho Histone 3 (1:200, Rabbit, Upstate). Sections were then washed with PBS and incubated for 1 hour at room temperature in 10% serum, 0.5% Triton X-100 in PBS with Cy3- or Cy5-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Confocal microscopy

Sections were imaged on an inverted laser-scanning confocal microscope (Zeiss LSM510 meta) with a 10, 20 and 40× water immersion objective (Zeiss). Excitation/emission wavelengths were 488/515 nm (GFP), 550/570 nm (Cy3), and 633/690 (Cy5). Z-series images were collected at 5 μm steps, and a projection of each stack was used for producing figures. In each brain slice, 100–500 cells could be found positive to GFP when electroporated with the constructs used. In experiments that involved colabeling or cell/process counting, images from individual optical sections were carefully examined.

Contribution of N- and C-terminal NudC domains to neuronal migration

NudC is well conserved among eukaryotes. However, Aspergillus NudC (22kDa) is much shorter than NudC in Drosophila, mouse, human and rat, all of which contain an extra N-terminal domain (Figure 2A). The homology between Aspergillus NudC and the C-terminal domain of mNudC is 78%, with 68% identity (Morris et al., 1997). Moreover it has been shown that the mammalian C-terminal domain of NudC can complement the NudC3 mutation in Aspergillus, demonstrating that the full Aspergillus function is contained within the C-terminal domain of mNudC (Morris et al., 1997). The extra N-terminal region contains a predicted coiled-coil domain that seems to be a late evolutionary addition that might mediate self-association. Indeed, we find that GFP-NudC coimmunoprecipitated either with full-length myc-NudC or a myc-NudC-N-terminal fragment (Figure 2B,C; cf. (Zheng et al., 2011)). However, the C-terminal domain of NudC was not able to co-immunoprecipitate with GFP-NudC full length, demonstrating that NudC interacts with itself via the N-terminal domain.

Overexpression of either full-length NudC or its fragments each affected neuronal migration in embryonic rat brain. The full-length protein produced a similar phenotype to that observed after NudC downregulation (Figure 3A,B and Supplemental Figure 1A,B). A reduced fraction of NudC-expressing progenitors reached the IZ by E19, none of which reached the CP (Figure 3A and Supplemental Figure 1A). By E21 NudC-expressing cells were mainly observed in the VZ/SVZ (Figure 3B and Supplemental Figure 1B), suggesting a loss of few further migrating cells. As observed with NudC RNAi, NudC overexpression resulted in a loss of RGPC processes by E21 (Figure ​(Figure3B3B and 4I-L). We also tested a mutant form of NudC L280P, based on the original Aspergillus NudC3 mutation (L146P) (Chiu and Morris, 1995; Chiu and Morris, 1997). Three days after electroporation a very mild phenotype was observed (Figure 3C and Supplemental Figure 1A) and the cellular distribution was very similar to the control (Figure 1C and Supplemental Figure 1A). However, two days later, at E21, the cellular distribution and morphology was completely normal (Figure 3D and Supplemental Figure 1B). Thus NudC-L280P largely reversed the effects of NudC overexpression, thereby serving as a control for the specificity of the NudC overexpression phenotype.

Open in a separate window

Figure 3

Dominant Negative Effects of NudC constructs on neuronal redistribution in the neocortex

(A–H) Micrographs of coronal sections of the cerebral cortex at E19 and E21, electroporated as indicated.

CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. The apical and pial surfaces are at the boundaries of the VZ and CP, respectively. Scale bars: 100 μm.

Three days after electroporation of NudC N-terminus in the embryonic brain, a migration phenotype (Figure 3E and Supplemental Figure 1A) was observed compared to control (Figure 1A and Supplemental Figure 1A), which was more pronounced two days later (Figure ​(Figure3F3F vs. ​vs.1D1D and Supplemental Figure 1B). Overexpression of the C-terminus of NudC resulted in a somewhat weaker phenotype (Figure 3G,H and Supplemental Figure 1A,B).

Effect of altered NudC expression on interkinetic nuclear migration in RGPCs

Recent interest in INM has led to several models for its underlying mechanism. Our own work has implicated cytoplasmic dynein in apical INM and kinesin-3 in basal INM (Tsai et al., 2007; Tsai et al., 2010). To test how NudC might participate in this process, we first examined the effect of NudC RNAi and overexpression on nuclear position in RGPCs.

Quantitation of nuclear distance from the ventricular surface was determined at E19 (Figure 4). Average distance with control plasmid was 21.8 ± 1.78 μm (Figure 4A-D). Similar to Lis1 downregulated cells (34.6 ± 2.6 μm, Figure 4M-P), the average distance for NudC RNAi electroporated cells was increased to 36.3 ± 2.9 μm (Figure 4E-H) and for cells overexpressing NudC to 40.7 ± 1.0 μm (Figure 4I-L), almost twice that of the control. These data suggested a role for NudC in regulating apically-directed nuclear movement in radial glial cells.

To test this possibility directly, control RNAi and NudC RNAi were introduced in E16 brains by in utero electroporation, and live imaging of E18 rat brain was performed two days later. Control cells (n=6 in 4 different slices) exhibited clear evidence of nuclear migration (Figure 5A) in both apical and basal directions (Figure 5D,E, green lines) at rates comparable to those in our previous studies (Tsai et al., 2005; Tsai et al., 2010). In contrast, cells electroporated with NudC RNAi showed no nuclear movement toward the ventricular surface (apically-directed movement) during the 8-9 h duration of observation (n=3; Figure 5B and 5D, blue lines). Basally-directed movement was, however, normal (n=3; Figure 5C, F, blue lines).

Open in a separate window

Figure 5

Live cell imaging of neural progenitor cell behavior within the VZ

(A) Cell body of a control progenitor cell at the radial glial stage migrates away from and then toward the ventricular surface, where it divides. Tracings of cell body positions for six typical controls are shown in panels D,E (green lines). (B,C) Cell body cells transfected with NudC RNAi. Note that the nuclei migrate away from the ventricular surface (C) but they do not move toward the ventricular surface (B) over a 9-h time period. Tracings show these behaviors in D,E (blue lines). Times are shown in hours/minutes. VS, ventricular surface. Scale bar: 20 μm.

Effect on mitosis and differentiation

NudC as been reported to participate in mitosis in cultured non-neuronal cells (Aumais et al., 2003; Nishino et al., 2006; Zhou et al., 2003). To test whether altered mitotic behavior contributed to the NudC neuronal distribution defects we used antibodies to phosphohistone H3 (pH3) or phosphovimentin. By 3 days NudC RNAi resulted in a substantial decrease in mitotic cells (Figure 6A,B,E,F,K,L) whereas the other conditions examined showed little effect (Figure 6C,D,G,H,K,L).

Open in a separate window

Figure 6

Mitotic effects of NudC RNAi and Dominant Negatives

(A–J) Fluorescent micrographs depict immunostainings with pH3 of E17, E18 and E19 coronal sections from brains electroporated with GFP (A,E), NudC RNAi (B,F), NudC (C,G), Lis1 RNAi (D,H), NudC-AA (I) and NudC-EE (J)

(K-M) Percentage of cells in mitosis at E17, E18 and E19 in the VZ/SVZ after electroporation with GFP, NudC RNAi, NudC, Lis1 RNAi, NudC-AA and NudC-EE. Error bars represent s.e.m. Scale bar: 20 μm. Student’s t-test, *P≤0.05, ***P≤0.0001.

NudC is phosphorylated by the mitotic kinase plk1 on two conserved sites (Ser275 and Ser327 in mouse). These phosphorylations have been shown to be important for mitosis and cytokinesis in tissue cultured cells (Aumais et al., 2003; Nishino et al., 2006; Zhou et al., 2003). We tested whether these phosphorylations were also important in our brain system. A phosphomutant form of NudC (NudC AA), in which the amino acids Ser 275 and Ser 327 are changed to alanine and a phosphomimetic form of NudC (NudCEE) in which the amino acids Ser 275 and Ser 327 are changed to glutamate were electroporated in E16 brains.

Almost no electroporated cells were detected after two days, due to either very weak expression of the constructs in brain or, as we suspect, to a loss of transfected cells. In favor of this hypothesis many transfected cells were observed at one day after electroporation. At this stage a very substantial increase in mitotic cells was observed in cells overexpressing the phosphomutant form of NudC (Figure 6I,M) while no effect was detected in cells overexpressing phosphomimetic NudC (Figure 6J,M).

We would like to thank Wei-Nan Lian, Shahrnaz Kemal and Garrett E. Seale for critical discussion and technical help, Tania Nayak and Daniel Hu for critical reading of the manuscript. This work was supported by NIH grant HD40182 to RBV, an EMBO Postdoctoral fellowship to SC, an American Heart Association Postdoctoral Fellowship to PM, and a March of Dimes grant to RBV.