|Home | About | Journals | Submit | Contact Us | Français|
During mitotic division in the telencephalic proliferative ventricular zone (VZ), the nuclei of the neural precursors move basally away from the ventricular surface for DNA synthesis, and apically return to the surface for mitotic division; a process known as interkinetic migration or “to-and-fro” nuclear translocation. The cell, which remains attached to the ventricular surface, either continues cycling, or exits the cycle and migrates to the subventricular zone (SVZ) or the developing cortical plate. While gap junctions/hemichannels are known to modulate DNA synthesis via Ca2+ waves, the role of Ca+ oscillations and the mechanism of nuclear translocation in the VZ precursors are unclear. Here we provide evidence that during apical nuclear migration, VZ precursors display dynamic spontaneous Ca2+ transients, which depend on functional gap junctions/hemichannels via ATP release and Ca2+ mobilizing messenger diffusion. Furthermore, we found that blocking gap junctions/hemichannels or shRNA mediated knockdown of connexin 43 (Cx43) retards the apically directed interkinetic nuclear migration accompanied with changes in the nuclear length/width ratio. In addition, we demonstrated that blocking functional gap junctions/hemichannels induces phosphorylation of small GTPase cdc42 in the VZ precursors. The basal phase of interkinetic migration is much slower and appears to be mediated passively by mechanical forces after cell division. Our findings indicate that functional interference with gap junctions/hemichannels during embryonic development may lead to abnormal corticogenesis and dysfunction of the cerebral cortex in adult organisms.
During forebrain development, the cortical projection neurons are generated from the neural precursors in the embryonic proliferative VZ/SVZ and migrate radially to the overlaying cortical plate. This process includes DNA synthesis, nuclear translocation, cell division, phenotypic commitment and migration to its final destination (Rakic, 1988b). Although oscillatory, alternating interkinetic (to-and-fro) nuclear migration during cell division, which requires the rearrangement of cytoplasmic tubular and filamentous organelles (Xie et al., 2007; Del et al., 2008; Alexandre et al., 1989; Solecki et al., 2004, 2009), has been known for more than 70 years (Watterson, 1966), the significance and molecular mechanism orchestrating this dynamic process is not well understood (Sidman and Rakic, 1973; Metin et al., 2008).
Similar to other developing pseudostratified epithelia, such as the neural tube or lens placode, in which mitotic division occurs at the surface, cell nuclei are thought to be passively displaced away from the surface during their DNA synthesis phase before returning actively to the surface to divide again (Langman, et al., 1965; Zwann et al., 1969). The significance of this widespread nuclear translocation is not clear, apart from being necessary to avoid overcrowding of the multiplying nuclei at the surface. Since the formation of the laminar cerebral cortex depends on temporarily coordinated production of neurons in the VZ/SVZ, which requires a precise control of cell cycles. Thus, it is particularly important to learn more about the mechanism of apical nuclear translocation. So far, it has been well established that neuronal precursors in the VZ are extensively coupled in radial columnar clusters via gap junctions (Loturco and Kriegstein, 1991; Bahrey and Moody, 2003), which enables the coupled proliferative unit to act as a syncytium via direct metabolic and electrical communication. Consistent with these observations, connexins (Cxs), the subunits of the connexons (hemichannels) and gap junctions, are expressed in the embryonic cerebral cortex (Oyamada et al., 2005; Nadarajah et al., 1997; Bittman et al., 1997; Buniello et al., 2004; Belliveau and Naus, 1995). More specifically, their expression varies according to phase of the cell cycle and developmental stage (Oyamada et al., 2005; Nadarajah et al., 1997; Bittman et al., 1997). Recently, it has been demonstrated that both the extracellular adhesive domain and the intracellular C terminal of Cx43 play an essential role in cortical neuronal migration (Cina et al., 2009; Elias et al., 2007). Since Cxs are predominantly expressed within the proliferative VZ during the period of neurogenesis, it is expected that they would play an important role in maintaining the neural progenitor pool by controlling its self-renewal.
It is also well established that functional gap junctions/hemichannels are involved in the propagation of Ca2+ waves in the embryonic proliferative zones (Weissman et al., 2004) and that their pharmacological blocking abolishes these Ca2+ waves, thereby preventing DNA synthesis in the neural precursors (Bittman et al., 1997; Weissman et al., 2004; Liu et al., 2008). Furthermore, disorganization of the VZ/SVZ in the GFAP-Cre/floxed Cx43 conditional knockout mice indicates the involvement of gap junctions/hemichannels in neurogenesis and neuronal migration (Wiencken-Barger et al., 2007). After each round of DNA replication, the nuclei of the VZ precursors are pulled down by the assembled microtubules attached to the centrosome located at the ventricular surface (Xie et al., 2007). This nuclear movement in apical direction is associated with changes in the cytoskeleton controlled by small molecular Rho-GTPases (Heasman and Ridley, 2008; Cappello, et al., 2006), which are, in turn, regulated by intracellular Ca2+ signaling (Jin et al., 2005; Price et al., 2003a). In the present study, we have examined the specific role of gap junctions/hemichannels in regulation of Ca2+ signaling and their effect on the apically directed interkinetic nuclear migration of the precursors in the VZ during cortical neurogenesis.
The ACSF contains (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 NaHCO3, and 10 Glucose. The osmolarity is around 310 mOsm/L and pH is 7.4 when bubbled with 95% O2/5% CO2. 0 mM Ca2+ ACSF was prepared by replacing the Ca2+ with equal mM EGTA. The HEPES solution contains (in mM): 156 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 15 Glucose, and 10 HEPES, pH was adjusted to 7.4 with 1N NaOH. The following drugs were used: ATP (0.1–100 µM), suramin (100 µM), MFA (60, 100 µM), carbenoxolone (100 µM), 2-Methylthioadenosine 5'-triphosphate (Mes-ATP, 100 µM), 2-APB (50 µM), 18-alpha-glycerrhetinic acid (40 µM) and indomethacin (100 µM); all are Sigma products except Hoechst (10 µM), NP-EGTA AM (5 µM) and BAPTA-AM (50 µM, Molecular Probes, respectively).
All experimental procedures were in accordance with the animal welfare guidelines of Yale University on the ethical use of animals for experimentation. Timed pregnant CD-1 mice (Charles River) at embryonic days E14 to E16 were sued in this study. Following rapid decapitation, the embryo brain was removed and put in cold balanced Hank’s solution and then placed into 3% Agarose (Type VII, Sigma). Agarose was allowed to solidify on ice (2–3 min), and coronal sections were made at 300 µm in cold ACSF using the vibrotome (Vibrotome, USA). Slices were placed in oxygenized ACSF and recovered at room temperature for 30 min.
Time lapse live imaging and calcium imaging was performed as previously described (Weissman et al., 2004; Pearson et al., 2005; Demello, 1983; Komuro and Rakic, 1996; Liu et al., 2008). Slices were transferred to oxygenized ACSF containing calcium indicator Fluo-4 (Fluo-4 AM, 10 µM, Molecular Probes) and incubated at 37 °C for 30 min. The mean fluorescence of individual cells was calculated and normalized to its initial value at time 0 (F/F0). A change in fluorescence in excess of a criterion level of 10% above baseline was accepted as a response or Ca2+ transients. The changes of average frequency and amplitude (F/F0) of Ca2+ transients in the same view field were used to determine the effect of drugs before and during drug application. Power spectral analysis was performed in the normalized Ca2+ signaling (F/F0) with the software provided by Microcal Origin. The sampling interval was set at 3s and Hanning window was used as a filter. The nuclear motility an migration was detected nuclear labeling with Hoechst (10 µM, Molecular Probes) at 37 °C for 30 min.
Patch clamp recording is as previously described (Liu et al., 2005). Whole-cell patch-clamp recordings were obtained as previously described (Liu et al, 2005, 2006). Patch pipettes were pulled from thin-walled borosilicate glass (outer diameter, 1.55 mm; internal diameter, 1.2 mm; WPI, TW150F-40) on a PP- 83 puller (Narishige, Japan). Pipettes had resistances of 3–6 MX when filled with the intracellular solution. For intracellular labeling, 0.1% Lucifer yellow (LY, dilithium salt) was added to the pipette solution. Recordings were performed using an Axopatch- 200B amplifier (Axon Instruments, CA). Current signals were low-pass filtered at 2–5 kHz and digitized on-line at 5–20 kHz using a Digidata 1320 digitizing board (Axon Instruments) interfaced with a DELL computer.
Slices were loaded with NP-EGTA AM (5 µM) for 30 min at 37°C, then transferred to the chamber perfused with oxygenized ACSF. One hemisphere of the neocortex was then subjected to UV bleaching for 2s with 10s interval for 5 times. During bleaching, the perfusate was replaced by HEPES solution containing Lucifer yellow (5mg/ml). For dye uptake blocking experiments, MFA (100 µM) was included in the perfusate before and during UV bleaching. Some slices were fixed in 4% PFA and then resectioned in 20 µm for fluorescence detection. Lucifer yellow fluorescence was detected in both hemispheres under bleaching and un-bleaching; normalized pixel densities were used for quantification.
ATP released from the perfused slices was measured using ATP bioluminescence detection kit (ENLITEN ATP assay system, Pormega). Frontal embryonic brain slices containing the neocortex were perfused with oxygenized ACSF in a self made perfusion system (1–1.2ml/5min). The perfusate was continuously collected every 5min. 50 µl perfusate and 50 µl ATP assay agent were mixed and the luminescence was measured in the ENLLITEN 20 luminometer. Standard curve showed high sensitivity and good correlation at 0−12 to 10−7 M ATP (r=0.9999, supplemental data FigS1A).
To detect spontaneous ATP release from the VZ/SVZ cells of the neocortex, low density cultured GL261 cells on coverslips were labeled with Hoechst (1µg/ml) and loaded with Ca2+ indicator Fluor-4 and then put in the recording chamber mounted on the invert confocal microscope (LSM 510, Zeiss). Coronal sectioned slices from the embryonic brain (E15) were overlaid on the coverslips. After a recovery period of at least 30 min, Ca2+ signaling was recorded in GL261 cells corresponding to the region of the VZ/SVZ identified under the DIC.
Fluo-4 loaded embryonic brain slices were transferred to a recording chamber on the stage of inverted Zeiss LSM 510 confocal microscope and continuously perfused with oxygenized ASCF. The stimulation target cells under the slice surface (≥50 µm) were identified by using Fluo-4 fluorescence under 488 nm excitation and the Ca2+ signaling of the slices was continuously recorded (1Hz) before, during and after the targeted cells were stimulated with a short period (<15s) of two-photons (870 nm) delivery. Pre-experiments were used to adjust and set the energy intensity of the two photon beam to get a point to lyse the targeted cells within 10 to 15s. The Ca2+ responses in the adjacent cells were analyzed as described above.
BrdU (50mg/kg, i.p.) was administered to pregnant mice at E15. After 1 hour, embryonic brain slices (300 µm) were prepared as described above. The slices were placed onto Millicell-CM culture inserts (Millipore, Bedford) in 6-well culture trays (Corning, Inc., Corning) with neuronal base medium containing 1% N2, 2% B27, 1% Glutamax, 2.5 % HEPES buffer (1M) and 1% Pen/Strep (GIBCO). Vehicle (DMSO, <0.1%), MFA (60 µM), carbenoxolone (100 µM), BAPTA-AM (50 µM) or indomethacin (100 µM) was added to the culture medium, respectively. Slices were then incubated for 3 hours at 37°C in 5% CO2 incubator. The slices were then fixed in 4% PFA and then cryoprotected with 30% sucrose. 20µm cryosections were made and used for BrdU staining and other immunostaining.
We used the shRNA Target finder at Genscript Corp to design shRNA sequences against the mouse Cx43, and the shRNA fragment was inserted into the pRNAT-U6.2/Lenti vector. The targeting sequences are as follows;
The scrambled shRNA control sequence is 5’-GGAATACAATAAGGAACGATA-3’
The full length Cx43 DNA structure MC205621 is provided by OriGene Technologies Inc. To analyze the effect of Cx43 shRNA in vivo, we used in utero electroporation. We also tested the effect of another structure of Cx43 shRNA, MusCx43SiRNACT2 (Iacobas et al., 2008).
Embryo brains (E15–18) were fixed overnight in 4% PFA. Brains were cryostat sectioned (20 µm) by microtome. The cultured embryonic neocortical slices were first fixed in 4% PFA and then cryostat resectioned to 20 µm. The primary antibodies include rat monoclonal anti-BrdU (1:50; Accurate Chemical, Westbury), Mouse anti neuronal class III –tubulin (Tuj-1, 1:500, Covance, Berkeley), pH3 and Ki67 (1:500 respectively, Invitrogen), Rabbit anti-pcdc42 and -cdc42 (1:500 respectively, Cell Signaling Technology), and chicken anti-GFP (1:2000, Sigma).
N2a cell culture, protein extraction and immunoblotting were performed using a standard protocol. The antibodies used for immunoblots are anti-Cx43 (1: 200, Invitrogen), GFP (1:2000, Santa Cruz) and GAPDH (1:200; chemicon). ECL plus system (Amersham Biosciences) was used to detect horseradish peroxidase-conjugated (1:5000; Bio-Rad). Streptavidin-biotinylated HRP was used at 1:20000 (GE Healthcare). To reprobe the membrane, Restore Western blot stripping buffer (Pierce) was used.
Statistical data are present as mean ± s.e.m. Statistical analyses were performed using student’s t test or X2 test in appropriate experiments, p<0.05 is considered significant.
We examined the intracellular Ca2+ signals in acute embryonic brain slices in situ at near physiological condition (2 mM Ca2+ ACSF) and found that individual Ca2+ transients are the predominant Ca2+ fluctuations in the VZ/SVZ (Fig1A, also see the movie, supplemental data movie 1). A distinguishing characteristic of the spontaneous Ca2+ transients is their high dynamics, which display an oscillating frequency as high as 3 peaks/min and last for the whole period of observation (>10 min). The Ca2+ transients randomly appear and disappear with varied duration from 4 to 20s. The percentage of cells showing spontaneous Ca2+ transients varies from 60% to 80% in the VZ/SVZ cells. According to the patterns of Ca2+ transients, these cells can be classified into two groups. The cells in the first group (marked by circles with numbers in Fig1A1) display fast repeated Ca2+ transients with short duration (traces indicated by circles with numbers in Fig1A2). The cells in the second group (marked by squares with numbers in Fig1A1) display slow frequency of Ca2+ transients with long duration (traces indicated by squares with numbers in Fig1A2). The mean frequencies for the fast and slow spontaneous Ca2+ transients are 16.5±4.6/10min and 3.2±2.6/10min, respectively (n=6 slices) and their average durations are 14.0±1.4s and 31.1±3.6s, respectively (n=6 slices). To our knowledge, these dynamic Ca2+ transients have not been described in the embryonic VZ/SVZ cells.
As shown in Fig1A1, both types of Ca2+ transients occur in cells across the whole range of the VZ. However, careful examination revealed that there are more cells in the upper strata of the VZ which show fast spontaneous Ca2+ transients than in the lower strata of the VZ (Fig1A1, also see the movie, supplemental data movie 1). In order to identify the properties of these Ca2+ transients in frequency domain, we used power spectrum analysis to analyze the high and low frequency Ca2+ transients. As expected, the high frequency Ca2+ transients display peaks of power spectrum extending to relatively higher frequency (FigB1), while the peaks of power spectrum of low frequency Ca2+ transients usually locates below 0.02Hz (FigB2). The percentages of power spectral density (PSD) at lower (0.005–0.02Hz) and higher (0.02–1.2Hz) frequency bands in both the high and low frequency Ca2+ transients were quantified in Fig1B3 (52.13 ± 6.22 vs. 79.89±2.13, p<0.01 and 48.95±6.05 vs. 22.59±2.46, p<0.01, n=10, respectively), which demonstrates the difference of distribution of power spectrum between the high and low frequency Ca2+ transients. Interestingly, the power spectra of both high and low frequency Ca2+ transients did not display multiple harmonic peaks, suggesting the chaotic property of these Ca2+ transients.
While Ca2+ waves were previously reported to exist in rat embryonic cerebrum in 0 mM Ca2+ ACSF (Weissman et al., 2004), we have rarely observed such cohort spontaneous Ca2+ waves in the mouse embryonic VZ/SVZ under our experimental conditions (2 mM Ca2+ ACSF. Only one Ca2+ wave was observed in the total 126 trials). However, Ca2+ waves are always evoked by electrical focal stimulation as well as single cell two photon stimulation (see below) in our experiment, which is consistent with the previous reports (Weissman et al., 2004; Liu et al., 2006).
To demonstrate the existence of functional gap junction in the embryonic VZ/SVZ cells, we first examined the electrical coupling in the mouse VZ cells at E16 using whole cell patch clamp recordings. When step voltages (−120 to 100 mV) were applied, 26% of the recorded cells display passive currents (Fig2A1) and lower input resistance (RIN, 343±92 MΩ, n=12) with a mean resting membrane potential (VR) of −76 ± 6 mV (n=12), suggesting that these cells are coupled via gap junctions (Loturco and Kriegstein, 1991). In contrast, other cells showed higher RIN (1–3 GΩ) with relatively lower VR (−40 – −60mV). About 60% of the recorded cells with low RIN were radial glia, which can be recognized by their morphology following filling with Lucifer yellow. More detailed analysis of the biophysical property of the VZ cells further suggests that these cells display variability in their VR and RIN. A plot of RIN as a function of VR illustrates a slight correlation between RIN and VR (r=0.594, FigS2A). The VZ cells could be separated into two groups based on their RIN as illustrated in FigS2B. Meclofenamic acid (MFA) had been used to block gap junctions/hemichannels in the embryonic neocortex (Weissman et al., 2004) and retina (Pearson et al., 2005); and was not accompanied by changes of intracellular calcium, pH, protein kinase C activity (Harks et al., 2001). Furthermore, we showed that the purinergic signaling cascade was intact in glioma cells treated with gap junction blockers (supplemental data FigS1B). We then checked the effect of MFA (100 µM) on gap junction coupling currents in the VZ/SVZ precursors. As shown in Fig2A1,2 the passive currents were significantly reduced by MFA (100 µM) and the RIN was increased to the level of the uncoupled cells (1.8±0.3 GΩ, n=7). After washout, the passive current was completely recovered. The MFA sensitive current reverses near the VR of the recorded cells (Fig2A2), which further suggests that they are mediated by gap junction coupling (Liu et al., 2006).
As a next step, we tested if functional gap junctions/hemichannels are involved in the spontaneous Ca2+ signal oscillation in the embryonic VZ/SVZ precursors. We found that the spontaneous Ca2+ transients were significantly reduced after blocking gap junctions/hemichannels with MFA (Fig2B1). Their mean frequency was reduced to 20±7.7% (p<0.01, n=4, Fig2B2). Similar results were observed with another type of gap junction blocker, carbenoxolone (100 µM), the frequency of Ca2+ transients was reduced to 19.1±4.6% (p<0.01, n=4, Fig2B2). We then analyzed the power spectrum of these remaining Ca2+ transients. Surprisingly, the power spectra usually display harmonic peaks with a dominant frequency at about 0.015Hz (Fig2C). These results suggest that gap junctions/hemichannels not only provide cues for the occurrence of dynamic Ca2+ transients in the VZ/SVZ, but also disturb the intrinsic harmonic Ca2+ signaling oscillations into an irregular chaotic condition, which might contribute to the biological behaviors of the VZ precursors.
Gap junction/hemichannel blockers may also affect the anion channels and P2X receptor channels (Spray et al., 2006). We, therefore, tested the effect of anion channel blocker DIDS (Eskandari et al., 2002) and P2X receptor blocker iso-PPADS (Zhao, 2005). As shown in Fig2B2, neither DIDS (10 uM, n=5) nor iso-PPADS (50 uM, n=6) has an effect on the spontaneous Ca2+ transients in the VZ precursors. Altogether, our results suggest that functional gap junctions/hemichannels are prerequisites for the dynamic spontaneous Ca2+ oscillations in the neural precursors of the VZ/SVZ.
VZ precursors express P2Y1 receptors and display Ca2+ increase in response to extracellular ATP (Liu et al., 2008; Weissman et al., 2004). In addition, we found that a low concentration of extracellular ATP induces Ca2+ oscillation in cultured glioma cells (data not shown). To provide direct evidence for ATP release in the embryonic cerebrum, we detected the ATP in the perfusate using bioluminescence assay (Lazarowski et al., 2000; De Vuyst et al., 2006) (Supplemental data FigS1A). ATP release from the embryonic neocortical slice in control perfusate (2 mM Ca2+ ASCF) was decreased with first order exponential decay (Fig3A1), which is similar to ATP release from cultured astrocytes via gap junctions/hemichannels (Lazarowski et al., 2000). The decay half time (t1/2) is 6.3 min and the remaining ATP concentration after 45 min perfusion is 0.06±0.01 nM. Opening the hemichannel by reducing extracellular Ca2+ (0 mM Ca2+ ACSF), which has been routinely used in cultured cells as well as in acute slices (De Vuyst et al., 2006; Weissman et al., 2004), significantly increased ATP release in the perfusate (Fig3A1, 2). The t1/2 is prolonged to 36.3 min (p<0.01, n=6) and the remaining ATP concentration is increased to 0.29±0.02 nM (p<0.01, n=6). We further tested if blocking of gap junctions/hemichannels could affect the ATP release. As expected, blocking gap junctions/hemichannels with carbenoxolone (100 µM) reduced the ATP release induced by 0 mM Ca2+ (t1/2=4.2 min, p<0.01, remaining concentration 0.12 ± 0.01 nM, p<0.01, n=6, Fig3A1, 2). Similar results were observed with another gap junction blocker, 18-alpha-glycerrhetinic acid (t1/2 = 3.7 min, p<0.01).
To examine the existence of spontaneous ATP release in the VZ precursors, we employed a bio-detection technique using Glioma cells (GL261) (Haas et al., 2006). At low cell density, the GL261 cells showed no obvious spontaneous Ca2+ transients, but responded to ATP at the nanomolar level (supplemental data FigS1B, C). We then overlaid the GL261 cells onto the surface of embryonic brain slices (E16) and checked the Ca2+ signaling in these GL261 cells locating at the VZ. As expected, small spontaneous Ca2+ transients were detected in those cells in 4/10 of the slices tested (Fig3B1). The mean frequency of the Ca2+ transients was 1.2 ± 0.1 peaks/10 min (n=12) and the mean amplitude was 0.17 ± 0.07 (F/F0, n=11). These Ca2+ transients showed varied rise time from 10 to 30 s and a mean duration of 74.9 ± 10.1 s (n=10). As shown in Fig3C1, application of P2Y receptor blocker suramin (100 µM) significantly reduced the spontaneous Ca2+ transients (mean frequency: 0.17 ± 0.4 peaks/10 min, p<0.01, n=12). For the slices that showed no spontaneous Ca2+ transients in the GL261 cells, we induced ATP release by perfusion with 0 mM Ca2+ ACSF. As shown in Fig3B2, the silent GL261 cell began to show Ca2+ transients and the basal line was elevated. Blocking gap junctions/hemichannels with MFA (100 µM) completely abolished these Ca2+ transients and reduced the basal line (Fig3B2). As a control, we did not observe similar Ca2+ transients in GL261 cells alone in 0 mM Ca2+ ACSF (supplemental data FigS1B).
To demonstrate the involvement of hemichannel-mediated ATP release in the occurrence of Ca2+ signal oscillation in the VZ precursors, we tested the effects of La3+ and Gd3+, both of which block the hemichannels but have no effect on gap junctions (Contreras et al., 2002, Stout et al., 2002). The Ca2+ transients were significantly reduced in the neural precursors after La3+ (100 µM) or Gd3+ (60 µM) application (Fig3C1, 2). ATP may also be released via the transporter system (Pangrsic et al., 2007). We then tested the effect of ATP-binding cassette transporter inhibitor Verapamil (10 µM) (Zhao, 2005), which showed no effect on the spontaneous Ca2+ oscillation in the VZ precursors (Fig3C2). Taken together, our results indicate that hemichannel-mediated ATP release plays a major role in the occurrence of Ca2+ signaling in the VZ precursors.
It has been shown that intracellular Ca2+ increase triggers the hemichannel opening in cultured cells (De Vuyst et al., 2006).Thus, we tested whether elevating intracellular Ca2+ could induce hemichannel opening in the VZ/SVZ cells. Embryonic cortical slices were first loaded with Ca2+ caging agent (5 µM NP-EGTA AM) and then one hemisphere was subjected to UV bleaching in HEPES perfusate containing Lucifer yellow (5mg/ml). As shown in Fig3D1, Lucifer yellow positive cells were detected in the bleached hemisphere, which are predominantly located at the upper (superficial) strata of the VZ. In contrast, very few Lucifer yellow positive cells were detected in the unbleached hemisphere. To demonstrate that the Lucifer yellow uptake is mediated by gap junctions/hemichannels, MFA (100 µM) was included in HEPES perfusate during bleaching. As expected, the uptake of Lucifer yellow was significantly reduced (Fig3B2). Similar results were also observed with carbenoxolone (100 µM, Fig3D2). Our results provide evidence that intracellular Ca2+ transients might trigger the opening of hemichannels, which reveals a regenerating mechanism for Ca2+ signal oscillations in the VZ precursors.
Previous work has shown that Ca2+ signaling in the embryonic cortical cells mainly depends on intracellular Ca2+ release from the ER (Owens and Kriegstein, 1998). We tested if the Ca2+ mobilizing messenger IP3 is involved in the spontaneous Ca2+ oscillation in the VZ precursors. As shown in Fig4A, blocking the IP3 receptor with 2-APB (100 µM) abolished the Ca2+ transients, which suggests that IP3 is the major Ca2+-mobilizing messenger responsible for the Ca2+ oscillation in the VZ precursors.
Although IP3 diffusion via gap junction has been reported in cultured cells (Finkbeiner, 1992) as well as in the Corti organ (Anselmi et al., 2008), it is not clear that it plays a role in the occurrence of Ca2+ signaling in the cortical VZ precursors. Therefore, we examined the synchronized-pairs of Ca2+ transients in adjacent VZ cells in situ (Fig4B). Following the Ca2+ transients in the presumable radial glia, simultaneous Ca2+ transients occurred in the adjacent cell (Fig4B1, B2). Unlike the propagated Ca2+ waves involving multiple cells, the synchronized Ca2+ transients occurred only in the adjacent cells with little phase delay (Fig4B3), indicating the existence of direct Ca2+ mobilizing messenger transfer among the VZ precursors.
To dissect the mechanism for Ca2+ signaling propagation in the VZ precursors, we induced ATP release from one single cell by using two-photon stimulation. The typical Ca2+ signaling propagation in the VZ/SVZ is shown in Fig5A (for the movie, see the supplemental data movie 2a). Three types of cells can be identified. Type I cell is the stimulated cell characterized with Ca2+ increase and decrease, which indicate the lysis of the cell and will be the initial major source of ATP. Type II cells are cells directly adjacent to the stimulated type I cell, also called first order cells (Anselmi et al., 2008). The Ca2+ responses in these cells could be caused by the ATP released from the type I cell. Type III cells, also called the propagated cells, represent the Ca2+ signaling propagation in the VZ (Fig5A1,A2). The mean values for the first order cells, the propagated cells and the maximum propagation distance in control are 3.1±0.3 cells, 11.5±3.3 cells and 46.7±4.58 µm, respectively (n=19, Fig5C1,C2). After blocking both gap junction and hemichannel, the remaining Ca2+ signaling propagation would represent the extracellular diffusion of ATP released from the stimulated cell. We first tested the effect of blocking gap junctions/hemichannels with MFA. We found that the numbers of the propagated cells and the maximum propagated distance were significantly reduced (2.5±0.6 cells and 17.7±2.8 µm, n=8, respectively), while the numbers of the first order cells showed no significant changes (Fig5C1, for the movie see the supplemental data movie 2b). After co-blocking of the P2Y receptor with suramin, the mean values for the propagated cell numbers and the maximum propagated distance were further reduced to 0.3 ± 0.3 cells and 1.9 ± 1.3 µm (n=9, respectively) (Fig5C1, C2). In addition, our previous work had shown that suramin alone abolished forcal stimulation evoked Ca2+ wave propagation in the VZ precursors (Liu et al., 2008). These results suggest that extracellular ATP diffusion can induce Ca2+ responses in the adjacent cells but could not account for the whole distance of Ca2+ signal propagation in the VZ. We further dissected the role of functional hemichannels in Ca2+ signal propagation. As shown in Fig 5C1, C2, selective blocking of the hemichannels with Gd3+ (100 µM) significantly reduced the maximum propagated distance, but the numbers of the propagated cells and the maximum propagated distance are still higher than those altered by gap junctions/hemichannel blocker MFA. Altogether, our results suggest that Ca2+ signaling propagation depends on functional gap junctions/hemichannels via both extracellular ATP release and intracellular Ca2+ mobilizing messenger diffusion in the neural precursors in the VZ.
To identify the role of functional gap junctions/hemichannels in interkinetic nuclear migration, the nuclei of the VZ cells were labeled with Hoechst and their movement was observed in situ by time lapse live imaging. In the control ACSF, a large number of VZ cells displayed dynamic nuclear motility characterized with multiple directional movements, which were indicated by a red cross in Fig6A1. The cells showing nuclear motility accounts for 61% of the total cells in the VZ/SVZ (1340 VZ/SVZ cells in 6 slices observed in 10 min, Fig6A2). After blocking gap junctions/hemichannels with MFA (100 µM), the nuclear motility was gradually reduced and most of them became “frozen” (Fig6A2). The percentage of VZ/SVZ cells showing nuclear motility was reduced to 6.7% (1241 cells in 6 slices, p<0.01, Fig6A2). We further examined if the nuclear motility depends on the intracellular Ca2+ signaling. After incubation with cell-permeable Ca2+ chelator BAPTA-AM (50 µM) for 30 min, the percentage of cells displaying nuclear motility was reduced to 13.7 % (727 cells in 3 slices, p<0.01, Fig6A2).
We then extended the time lapse recording time, so that we could analyze the interkinetic nuclear migration. In the control slices, the neural precursors displayed both up and down nuclear migration along the radial axis. However, the nuclear translocation toward the ventricular surface is more frequent and faster, suggesting that the apically directed migration is a more dynamic process (Xie et al., 2006). Fig6B shows the representative migration of a nucleus to the ventricular surface. As indicated by the dashed outline, the nuclear shape becomes elongated radially during the migration, suggesting that the nucleus is pulled down by the associated microtubules. We further detected the nuclear migration before and after blocking gap junctions/hemichannels. In Fig6C1, the arrows show the nuclear migrations in the VZ. The heads of arrows indicate the direction of nuclear movement and their lengths indicate the translocated distances during 60 min. Five nuclei with the fastest translocations were selected for statistics, which gives an average translocation distance of 15.2 µm and an average translocation speed of 1.8µm/min (n=4 slices, Fig6C2). After blocking gap junctions/hemichannels with MFA, the nuclear migration was inhibited and the average translocation distance and speed of five nuclei with the fastest translocations were reduced to 6.6 ± 2.3 µm and 0.9 ± 0.4µm/min, respectively (n=3 slices, Fig6C2). Furthermore, we found that after abolishing the intracellular Ca2+ with BAPTA-AM (50 µM), the interkinetic nuclear translocation was also significantly reduced (translocation distance: 6.56 ± 2.1 µm, speed: 0.87 ± 0.36 µm/min, n=3 respectively, Fig6C2). Thus, functional gap junctions/hemichannels may affect the interkinetic nuclear migration via intracellular Ca2+ signaling in the neural precursors in the VZ.
The role of functional gap junctions/hemichannels in interkinetic nuclear migration was further tested in cultured slices. We first detected the interkinetic nuclear migration in the VZ precursors in vivo. The cells in the S-phase were labeled by BrdU injection (50mg/kg i.p., at E15.5) and the distribution of BrdU+ nuclei was examined at one and four hours, respectively, after injection. As shown in Fig7A, BrdU+ nuclei mainly locate in the upper portion of the VZ/SVZ one hour after BrdU injection. Three hours later, many BrdU+ nuclei migrated to the lower portion of the VZ/SVZ, and some reached the ventricular surface. The interkinetic nuclear migration index, i.e., the ratio of BrdU staining density between the lower and upper half portion of the VZ/SVZ (Cina et al., 2009), is shown in Fig7C. To test the effect of gap junctions/hemichannels on the interkinetic nuclear migration, we incubated these BrdU labeled slices (1 hour after BrdU injection) with vehicle or gap junction/hemichannel blockers. As shown in Fig7B, many BrdU+ nuclei migrated apically to the lower portion of the VZ/SVZ in the control slices after incubation for 3 hrs. In slices treated with MFA (60 µM), the migration of the BrdU+ nuclei was delayed, and many of them stayed in the upper portion of the VZ/SVZ. Similar results were obtained with another gap junction/hemichannel blocker, carbenoxolone (100 µM), and with BAPTA-AM (50 µM, Fig7B, C). MFA is also known as a non-stereo anti-inflammatory drug that reduces the PGE2 production by inhibition of cyclooxygenase. Thus, we examined the effect of indomethacin (100 µM), another non-stereo anti-inflammatory drug which has no effect on gap junctions/hemichannels. As shown in Fig7B, C, the migration of the BrdU+ nuclei to the lower portion of the VZ/SVZ was not affected. Altogether, our results suggest that functional gap junctions/hemichannels are essential for the apically directed interkinetic nuclear migration in the VZ precursors.
To exclude the possibility that the migration defect was due to cellular apoptosis, we performed TUNEL staining. In slices treated with MFA (60 µM), the number of TUNEL+ cells showed no significant difference compared to the control (supplemental data FigS3).
During the G2 phase, the nuclei of the VZ precursors are pulled down by the assembled microtubules to the ventricular surface, which is associated with changes in nuclear shape (Xie et al., 2007). We analyzed the nuclear shape by measuring the ratio between nuclear length and width in the VZ precursors. The nuclei were labeled with Hoeschst and imaged by Z-stack confocal scanning. As expected, many VZ precursors displayed olive-like elongated nuclei in the control slices. In contrast, the nuclei of VZ precursors lost their olive-like shape and became less elongated after short term MFA treatment (60 µM, 60 min) (FigS4). To identify the G2 phase precursors in the VZ, co-immunostaining for Ki67 and BrdU in the embryonic brain labeled with BrdU (3 hrs after BrdU injection i.p. at E15) was performed. All the BrdU+ nuclei in the lower portion of the VZ were co-localized with Ki67 and displayed elongated olive-like shape (Fig8A). The nuclear length/width ratio in Ki67+/BrdU+ VZ cells was quantified, which gave mean values of 1.82±0.06 (n=48, in 4 slices) in the control slices (in ACSF for 60 min) and 1.33±0.06 (p<0.01, n=56 in 4 slices) in MFA treated slices (60 µM, 60 min) (Fig8A, C). These results suggested that the pulling force for the apically directed nuclear migration was compromised after blocking gap junctions/hemichannels. We further detected the M phase cells using immunostaining for phosphohisotnH3 (pH3). In the control slices, almost all of the pH3+ cells in the VZ located at the ventricular surface (Fig8B). In contrast, in slices treated with MFA (60 µM, 3 hrs), some pH3+ cells appeared above the ventricle surface (Fig8B) and the distance of the pH3+ nuclei to the ventricular surface was significantly increased (Fig8C), suggesting that the apically directed interkinetic nuclear migration was inhibited in the G2 phase cells after blocking gap junctions/hemichannels.
Rho GTPases are key regulators of cytoskeletal dynamics and involved in many cellular processes, including cell motility and migration (Heasman et al., 2008). It has been shown that small Rho-GTPase cdc42 plays an essential role in the apically directed interkinetic nuclear migration in the VZ precursors (Cappello et al., 2006). Here we tested if blocking gap junctions/hemichannels could affect the phosphorylation of cdc42 (p-cdc42), the inactivated form of cdc42 (Uddin et al., 2000). In the control slices, the p-cdc42 was mainly detected in the IZ and with little in the VZ precursors (Fig8D1). In contrast, in slices treated with MFA (60 uM, 60 min), the p-cdc42 was significantly increased in the VZ (Fig8D1 and the supplemental data). The ratios of p-cdc42 immunostaining density in the VZ against that in the IZ are shown in Fig 8D2. We also checked the expression of total cdc42 (t-cdc42) and found no obvious difference between the control and the MFA treated slices (Fig8D2 and also see the supplemental data FigS5). To test if Rho GTPase signaling is involved in the interkinetic nuclear migration in the VZ precursors, BrdU labeled embryonic brain slices (E14, 1 hr) were incubated with Rho GTPase inhibitor clostridium difficile Toxin B (1ng/ml). As shown in Fig 8E, Toxin B completely abolished the apically directed interkintic nuclear migration in the VZ. Similar to the effect of MFA, the BrdU+ nuclei mainly locate at the upper portion of the VZ in Toxin B treated slices and the interkinetic nuclear migration index was significantly reduced (Fig 8E1,2). Consistent with the fact that activities of Rho-GTPases are regulated by intracellular Ca2+ signaling (Jin et al., 2005; Price et al., 2003a), our results suggest that functional gap junctions/hemichannels might control the interkinetic nuclear migration by Rho-GTPase cdc42 signaling.
Cx43 is the major connexin in the mouse VZ precursors, including the radial glia, and is responsible for the gap junction coupling in the neuroepithelium (Loturco and Kriegstein, 1991). We tested if knockdown of Cx43 affects the interkinetic nuclear migration in the VZ precursors. We performed electroporation with EGFP plasmid and Cx43 or scrambled control shRNA in the VZ precursors at E15 and examined their nuclear shape and position at E16.5. As shown in Fig 9A, in the control brains, the EGFP+ VZ precursors identified by their foot-like processes to the ventricular surface, usually showed olive-like elongated cell bodies. The nuclei of these cells also displayed an olive-like shape and locate centrally within the cell bodies, which gave a mean length/width ratio of 2.11 ± 0.05 (n=21, p<0.01, Fig9C). In contrast, in the brains electroporated with Cx43 shRNA, the nuclei of EGFP+ VZ precursors lost their olive-like shape and became less elongated (Fig 9A1), which gave a mean length/width ratio of 1.72 ± 0.05 (n=37, Fig9C). Interestingly, these nuclei usually shifted to the upper portion of the cell bodies and made the cell shape like a thermo-balloon. These results strongly suggest that the pulling force for the apically directed interkinetic nuclear migration was compromised after knockdown of Cx43.
We further examined the nuclear positioning of the M phase cells in the VZ by immunostaining for pH3. In the control brains, the pH3+ cells mainly locate at the ventricular surface (Fig9B1). The mean value of the distances of the pH3+ nuclei to the ventricular surface is 8.3 ± 0.4 µm (n=90, Fig9C) and their count distribution is shown in Fig9B2. In Cx43 shRNA electroporated brains, some pH3+ nuclei in the VZ cells were observed above the ventricular surface (Fig9B1), suggesting that these cells enter into the M phase before they migrate to the ventricular surface. The mean distance of the pH3+ nuclei to the ventricular surface was significantly increased to 14.5 ± 0.6 µm (n=146, p<0.01, Fig9C) and their count distribution showed a secondary peak (Fig9B3) indicating the mispositioning of a sub-group of mitosis cells in the VZ. We also examined the number of pH3+ cells in the VZ and found no significant difference between the control and the Cx43 shRNA electroporated brain (data not shown), suggesting their overall entry into and exit out of mitosis were not affected. Finally, we tested if overexpession of Cx43 could rescue the effect of Cx43 shRNA. As expected, the number of the mispositioned pH3+ nuclei above the ventricular surface reduced and more pH3+ cells located at the ventricular surface in slices co-electroporated with Cx43 full length DNA (FigS6A1) and their average distance to the ventricular surface was significantly reduced (FigS6A2). Altogether, our results suggest that Cx43 is involved in the apically directed interkinetic nuclear migration in the VZ precursors.
To demonstrate the knockdown effect of Cx43 shRNA, we first transfected the mouse neuroblastoma cell line (N2a) with full length DNA for mouse Cx43 and then co-transfected with pCAG-EGFP and scramble or Cx43 shRNA. The expression of CX43 in N2a cells was significantly reduced when co-transfected with Cx43 shRNA (Fig9D1, D2, p<0.01, n=4, respectively), while the expression of EGFP was similar among each experiment (data not shown). The knockdown effect was further examined using immunostaining in the electroporated brain and the Cx43 expression was significantly reduced in Cx43 shRNA electroporated region (FigS6B1, B2). In addition, we observed that knockdown of Cx43 retarded migration of the postmitotic neurons into the cortical plate (data not shown), which is consistent with the previous observation (Cina et al., 2009; Elias et al., 2007).
In the present study we provide experimental evidence that gap junctions/hemichannels are involved in the apical phase of interkinetic nuclear migration in neural precursors. Our data suggest that regulation of apically directed interkinetic nuclear migration by intracellular Ca2+ signaling via both ATP release and Ca2+ mobilizing messenger diffusion may be an important mechanism by which functional gap junctions/hemichannels maintain the neural progenitor pool during their division.
Cytosolic Ca2+ signaling has previously been implicated in several aspects of nervous system development, including cell proliferation (Pearson et al., 2005; Weissman et al., 2004), differentiation (Gu and Spitzer, 1995), migration (Komuro and Rakic, 1996; Komuro and Rakic, 1993), neurite outgrowth and growth cone behavior (Gomez and Spitzer, 1999). In the present study, we confirmed the existence of Ca2+ signaling fluctuations in neural precursors of the VZ/SVZ (Owens and Kriegstein, 1998; Weissman et al., 2004), However, in addition, we have demonstrated the existence of highly dynamic spontaneous Ca2+ transients in situ at near physiological condition (2 mM Ca2+). Furthermore, our results indicate that Ca2+ transients, observed in slices in vitro, might underestimate their effect in vivo. P2Y receptor is expressed in the VZ precursors and extracellular ATP induces Ca2+ responses in these cells (Bittman et al., 1997; Weissman et al., 2004; Liu et al., 2008). We also demonstrated that ATP is spontaneously released via gap junctions/hemichannels and elevating cytosolic Ca2+ induces hemichannel opening in the VZ/SVZ precursors. In addition, Ca2+ mobilizing signaling can diffuse among the adjacent VZ cells. Thus, gap junctions/hemichannels are likely involved in the initiation, transfer and amplification of the Ca2+ signaling via ATP release and Ca2+ mobilizing messenger diffusion, and generate Ca2+ oscillation in the VZ/SVZ precursors. The Ca2+ transients in VZ/SVZ precursors mainly occur due to Ca2+ release from the ER via activation of IP3 receptor (Owens and Kriegstein, 1998; Weissman et al., 2004). Thus, various growth factors, hormones and extracellular signaling that act on G-protein coupled receptors may either alone or in combination contribute to the cytosolic Ca2+ signaling via the Ca2+-mobilizing second messenger IP3 in the neural precursors and thereby affect neurogenesis during cortical development.
Previous studies have shown that both actin- and microtubule-involved coupling between the nucleus and the centrosome are prerequisite for nuclear translocation during neuronal migration (Xie et al., 2007; Karfunkel, 1972; Messier and Auclair, 1974; Messier and Auclair, 1973; Webster and Langman, 1978; Metin et al., 2008; Splecki et al, 2009). The dynamics of actins and microtubules are modulated through a large number of cytoskeleton-associated proteins (Li and Gundersen, 2008) including cytoplasmic dynein and dynactin complex (Del et al., 2008; Dujardin and Vallee, 2002; Palazzo et al., 2001). The cytosolic Ca2+ signaling might modulate the interkinetic nuclear migration by affecting either the assembly of microtubules or the transcription of genes via activation of small GTPases. For example, elevation of intracellular Ca2+ potently induces Rac activation; and lamellipodia formation induced by intracellular Ca2+ elevation is blocked by inhibition of Rac signaling, which indicates that intracellular Ca2+ might regulate the cytoskeleton system by activation of Rac (Price et al., 2003b; Evers et al., 2000) including the small Rho-GTPase cdc42 (Cappello et al., 2006). In the present study, we demonstrated that blocking of gap junctions/hemichannels decreases nuclear motility and impairs the apically directed interkinetic nuclear migration in the VZ, which is accompanied by an increase of p-cdc42, the inactivated form of Rho-GTPase cdc42. In addition, pharmacologically blocking of Rho GTPase signaling completely abolished the interkinetic nuclear migration. Therefore, functional gap junctions/hemichannels may modulate the interkinetic nuclear migration via small Rho-GTPase.
How do gap junctions/hemichannels control the “to” and “fro” interkinetic nuclear migration during neurogenesis? Several reports suggested that gap junction coupling is predominant in the S/G2-phase cells and diminishes in the M-phase cells (Weissman et al., 2004; Bittman et al., 1997; Bittman and Loturco, 1999; Palazzo et al., 2001). Correspondingly, the neural precursors in the upper strata of the VZ seem to display more intensive spontaneous Ca2+ transients. We propose a model that gap junction/hemichannel-mediated Ca2+ signaling in the S/G2-phase cells enhances the interaction between the centrosome and the nucleus via microtubule assemble and promotes nuclear translocation toward the ventricular surface. In contrast, during the M- and G1-phase, reduction in gap junction/hemichannel coupling may de-polymerize the assembled microtubules and thus create conditions in favor of passive nuclear translocation, away from the ventricular surface. For example, mitotic divisions at the ventricular surface which increase cell density may displace the nuclei towards the upper strata of the VZ as suggested in the classical studies (Zwann et al., 1969). In contrast, migration in the apical direction is an active process. Consistent with this hypothesis, when the motor protein Dynactin-1 is disrupted in neuronal progenitors, their nuclei migrate more slowly toward the ventricular surface and more rapidly and at a longer distance away from the ventricular surface (Del et al., 2008). If the apical nuclear migration depends on gap junction/hemichannel-mediated Ca2+ signaling, interfering with gap junction’s/hemichannel’s activity would affect both the VZ cells reentry into as well as their exit out of the cell cycle. Indeed, our study demonstrated that pharmacological blocking or genetic knockdown of gap junctions/hemichannels retards the nuclear migration of the S/G2-phase cells in the upper strata of the VZ. In addition, disorganization of the VZ/SVZ and neuronal migration deficiencies in Cx43 genetic knockout mice (Wiencken-Barger et al., 2007) further confirms that gap junctions/hemichannels are involved in both cellular (nuclear) migration and neuronal progenitor production in the VZ/SVZ.
Our model is also consistent with the reports that functional gap junctions/hemichannels are sensitive to a large spectrum of chemical and biophysical factors, such as cations (Demello, 1983), pH, metabolite (Neyton and Trautmann, 1986), drugs (Harks et al., 2001), mechanical stimuli (Bao et al., 2004) and irradiation (Trosko et al., 1990). For instance, a low dose of ionizing radiation highly up-regulated the expression of connexins (Azzam et al., 2003); and, our group previously reported that exposure of the fetus to ionizing irradiation and ultrasound waves may cause misplacement of cortical neurons (Algan and Rakic, 1997; Ang et al., 2006). Abnormal neocortical development resulting from defective neurogenesis is associated with multiple neurological disorders, including autism, schizophrenia and mental retardation (Rakic, 1988a; Kriegstein, 2005; Gleeson and Walsh, 2000). Therefore, interference with gap junction/hemichannel-mediated Ca2+ signaling in neurogenesis may underlie the pathogenesis of neurological disorders caused by abnormal corticogenesis.
We thank the constructive discussion with Dr. Yuguo Yu and thoughtful comments provided by Drs., Josh Breunig, Albert E. Ayoub, Martin Dominguez, Mladen-Roko Rasin and Nenad Sestan. We are also grateful to Mariamma Pappy, Anita Begovic and Adi Begovic for the technical support. This work was supported by funding from the National Institutes of Health and the Kavli Institute for Neuroscience (P.R.), and a fellowship award from NAAR/Autism Speaks (X.L.).