Proneural Transcription Factors Regulate Different Steps of Cortical Neuron Migration through Rnd-Mediated Inhibition of RhoA Signaling

Summary Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.


Ascl1 (-/-) CP vs WT
(F) Localization of Ascl1-type E boxes in the vicinity of the Rnd3 locus that are conserved in the mouse, rat and human genomes. Chromatin immunoprecipitation (ChIP) analysis of the binding of Ascl1 to Rnd3-associated conserved elements in chromatin prepared from the ventral telencephalon of E12.5 wild-type embryos. The control experiment used the same procedure without antibody. Four Rnd3 enhancers bound Ascl1 significantly, however the level of binding of E11 and E14 was low and only E1 and E5 were further analyzed. Student's t-test; *p<0.05, ***p<0.001; mean ± SEM of triplicate quantifications from at least two immunoprecipitations.
(C) Ex vivo electroporation of Rnd3 shRNA followed by 2 days of slice culture did not change the fraction of electroporated cells labelled with an antibody against activated caspase 3 indicating that Rnd3 silencing does not induce apoptotic cell death. mean ± SEM, n=9-10 sections in each experiment.
(B) Cortices were electroporated in utero at E14.5 with GFP and different shRNAs constructs as indicated and analyzed three days later. One way ANOVA followed by a Fisher PLSD post hoc test; **p<0.01, ***p<0.001 compared to control (ctrl) shRNA; ##p<0.01 compared to Rnd3 shRNA; +p<0.05, ++p<0.01 compared to Rnd2 shRNA. Scale bars represent 150 µm (B) and 200 µm (A).    Ascl1 induces expression of plasma membrane-localized Rnd3, which inhibits RhoA and induces F-actin depolymerization, resulting in stabilization and attachment of the leading process to a radial glial fiber. This in turn allows nucleokinesis and locomotion to proceed. Neurog2 induces early endosome-localized Rnd2, which may regulate the trafficking of membrane-associated molecules that promote neuronal polarization and extension of the leading process at the multipolar to bipolar transition. Rnd2-mediated inhibition of RhoA but not F-actin depolymerization is necessary for this process. Multiple extracellular signals may regulate these two pathways at different levels to coordinate neuronal migration.
Genotyping of Ascl1 conditional mutant allele was performed with the same forward primer as for the wild-type allele and the following reverse primer 5'-TAGACGTTGTGGCTGTTGTAGT- Ethell. pcDNA-hRhoA was obtained from Missouri S&T cDNA Resource Center and pAcGFP1-Mem vector was purchased from Clontech. The Cre expression plasmid pCIG2-Cre as well as the pNeuroD1-Rnd2*-IRES-GFP expression plasmid have been described previously (Heng et al., 2008).
The full length coding sequence for mouse Rnd2 was cloned by PCR using pCR-Rnd2 as template and then inserted into the EcoRI/HindIII sites of the pCMV-Flag vector to generate pCMV-Flag-Rnd2. Flag-Rnd3 was cloned by PCR using pCMV-Flag-Rnd3 as a template into XhoI/PstI sites of the pClG2 vector to generate pClG2-Flag-Rnd3. Flag-Rnd3 blunted fragment from pClG2-Flag-Rnd3 was cloned into blunted EcoRI sites of pNeuroD1-IRES-GFP expression plasmid to generate pNeuroD1-Rnd3-IRES-GFP.
Rnd2 Rnd3Cter , generated by three successive PCR, was inserted into the EcoRI/HindIII sites of the pCMV-Flag vector to generate pCMV-Flag-Rnd2 Rnd3Cter .
For luciferase reporter assays, Rnd3 E1 and E5 enhancers were cloned from mouse genomic DNA by PCR into the SalI/NheI sites of the luciferase reporter vector p-βglob-Luciferase (Heng et al., 2008 Rnd3 E1 and E5 enhancers were also cloned as 3' enhancer elements into the SalI/ApaI sites of a LacZ reporter vector harbouring a minimal β-globin promoter for the subsequent generation of transgenic reporter mice as previously described (Heng et al., 2008).
All of the above mentioned constructs were fully sequenced verified before their use in experiments.

In Utero Electroporation and Tissue Processing
In utero electroporation was performed as described previously  with minor modifications. Briefly, uteri of anaesthetized timed-pregnant mothers (14 days) with isoflurane in oxygen carrier were exposed through a 1 centimeter incision in the ventral peritoneum. The pregnant mouse was injected before the surgery with buprenorphine (Vetergesic; Alstoe Ltd).
Embryos were carefully lifted using ring forceps through the incision and placed on humidified gauze pads. DNA was prepared in endotoxin-free conditions (Qiagen) and was injected at the following concentrations: shRNA constructs, 1 µg/µl; rescue constructs, 1 µg/µl; pClG2-Cre, 1 µg/µl; pClG2-Centrin2-Venus, 2 µg/µl; RhoA probe, 0.25 µg/µl. Plasmid DNA solution mixed with 0.05% Fast Green (Sigma) was injected through the uterine wall into the telencephalic vesicle using pulled borosilicate needles (Harvard Apparatus) and a Femtojet microinjector (Eppendorf). Five electrical pulses were applied at 30V (50 msec duration) across the uterine wall at 1 sec intervals using 5 mm platinum electrodes (Tweezertrode 45-0489, BTX, Harvard Apparatus) connected to an electroporator (ECM830, BTX). The uterine horns were then replaced in the abdominal cavity and the abdomen wall and skin were sutured using surgical needle and thread. The pregnant mouse was warmed on heating pad until it woke up. The whole procedure was complete within 30 min. Three days following the surgery, pregnant mice were sacrificed by neck dislocation and embryos were processed for tissue analyses. Embryonic brains were fixed in 4% PFA overnight and then placed in 20% sucrose/PBS overnight. P2 brains were dissected from anesthetized pups subjected to intracardial perfusion of 0.9% NaCl, followed by 4% PFA in PBS.
Then brains were postfixed in 4% PFA and fixed samples were cryoprotected overnight in 20% sucrose in PBS at 4°C. Embryonic and postnatal brains were then embedded in OCT Compound and frozen before sectioning using a cryostat.
The different subregions of the cerebral cortex (VZ/SVZ, IZ and CP) were identified based on cell density and visualized with TOTO-3 iodide nuclear staining (Invitrogen). All images were acquired with a laser scanning confocal microscope (Radiance 2100, BioRad). For migration experiments, six sections were analyzed for each condition from three embryos from two or three litters obtained in parallel experiments. Cell counts were performed using MetaMorph software (Molecular Devices).
A statistical analysis was performed using either unpaired two-tailed Student's t-test between control and experimental condition, or one-way ANOVA followed by a PLSD Fisher post hoc test for multiple comparisons (StatView software, version 5).

Ex Vivo Cortical Electroporation and Dissociated Cell Cultures
Ex vivo electroporation was performed on injected mouse embryos' heads similarly to in vivo electroporation. The electrical parameters were the following: 50V, 50 msec length, 5 pulses, 1 sec interval. Following electroporation, brains were dissected in L15 (PAA Laboratories) and transferred into liquid 3% low melting agarose (Sigma) and incubated on ice for 1 hr. Embedded

Live Imaging
One day after ex vivo electroporation, GFP was imaged in live brain slices using 900nm multiphoton excitation (Spectraphysics DeepSee) with a Leica SP5 confocal scanner on a DM6000 CFS upright microscope. A 10x,0.4NA (dry) lens was used and reflected excitation collected with a non descanned PMT through a 525/50 filter (Chroma).

Fluorescence Resonance Energy Transfer (FRET)
Samples for analysis of FRET by acceptor photobleaching were imaged using a Zeiss LSM 510 META laser scanning confocal microscope and a 25 x NA 1.4 Ph2 or 63 x Plan Apochromat NA 1.4 Ph3 oil objective as specified. The CFP and YFP channels were excited using the 440 nm diode laser and the 514 nm argon line respectively. The two emission channels were split using a 545 nm dichroic mirror, which was followed by a 475-525 nm bandpass filter for CFP and a 530 nm longpass filter for YFP (Chroma). Pinholes were opened to give a depth of focus of 2 µm for each channel. Scanning was performed on a sequential line-by-line basis for each channel. The gain for each channel was set to approximately 75 % of dynamic range (12-bit, 4096 grey levels) and offsets set such that backgrounds were zero. Time-lapse mode was used to collect one prebleach image for each channel followed by bleaching with a minimum of 50 iterations of the 514 nm argon laser line at maximum power (to bleach YFP). A second post-bleach image was then collected for each channel. Control non-bleached areas were acquired for all samples in the same field of view as bleached cells to confirm specificity of FRET detection. Pre-and post-bleach CFP and YFP images were then imported into Image J for processing. Briefly, images were smoothed using a 3 x 3 box mean filter, background subtracted and post-bleach images fade compensated. A FRET efficiency ratio map over the whole cell was calculated using the following formula: (CFP postbleach -CFP prebleach )/CFP postbleach . Ratio values were then extracted from pixels falling inside the bleach region as well as an equally sized region outside of the bleach region and the mean ratio determined for each region and plotted on a histogram. The non-bleach ratio was then subtracted from the bleach region ratio to give a final value for the FRET efficiency ratio. Data from images were used only if YFP bleaching efficiency was greater than 70 %.

Cell Cultures
Primary cortical neuron cultures were prepared from E14.5 mouse cortices. Dissected cortices were pooled in complete Neurobasal medium and triturated with Pasteur pipettes. Dissociated cortical cells were cultured as just described.
HEK 293 cells and mouse embryocarcinoma cell line P19 were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 1% penicillin/streptomycin and transfected with lipofectamine according to the manufacturer's protocol (Invitrogen). Primary cortical neurons, HEK and P19 cells were cultured in humidified incubator at 37°C under 5% CO 2 atmosphere.

Western Blotting
After extraction of total proteins or nuclear proteins for Ascl1 in the presence of protease and phosphatase inhibitors (Sigma), proteins were separated on 4-12% gradient gels using the XCell in T-TBS, membranes were incubated for 1 h at room temperature with peroxidase-labelled secondary antibody. The immunoreactive bands were visualized using enhanced chemiluminescent detection reagents according to the manufacturer's instructions (ThermoScientific).

In Silico Search for Ascl1 Binding Sites and Chromatin Immunoprecipitation Assays
Conserved Ascl1 binding sites across mouse, rat and human genomes within the Rnd3 gene locus were identified using the UC Santa Cruz genome browser (http://genome.ucsc.edu/cgibin/hgGateway).
Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Castro et al., 2006) with chromatin prepared from E12.5 ventral telencephalon and with a monoclonal mouse anti-Ascl1 antibody or without antibody as a negative control. Immunoprecipitated chromatin was quantified using the Real Time PCR (AB Applied Biosystems) and a SYBR-Green based kit for quantitative PCR (iQ Supermix, BioRad). Quantities of immunoprecipitated chromatin were calculated by comparison to a standard curve generated by serial dilutions of input chromatin. Plotted data represent the mean of two independent assays and three independent amplifications. The primers used for amplification of the Rnd3 E1 and E5 enhancers are the following: E1 Fwd: 5'-TGCTGCTCTTGCTTTGTCTC-3' E1 Rev: 5'-CGTTCCCTGCTGCTCTAAT-3' E5 Fwd: 5'-AATTACTCAGCTTGGGCACAG-3' E5 Rev: 5'-CATTTCCTCCTACGGCTCAT-3'

Luciferase Assays
For luciferase assays, P19 cell transfections were performed in triplicate in 24-well plates by using Lipofectamine 2000. Each well was transfected with 250 ng appropriated expression plasmids, 125 ng luciferase reporter plasmid, and 250 ng CMV-β-gal plasmid as internal control.
Cells were lysed 24 hr after transfection (Passive Lysis Buffer, Promega), and extracts were assayed for luciferase and β-gal activities.