Correspondence to Richard B. Vallee: rv2025{at}columbia.edu
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Abbreviations used in this paper: CP, cortical plate; IZ, intermediate zone; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interference RNA; SVZ, subventricular zone; VZ, ventricular zone.
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Introduction |
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LIS1 orthologues are essential for nuclear migration and nuclear orientation in fungi (Xiang et al., 1995; Geiser et al., 1997). In dividing vertebrate cultured cells, LIS1 associates with kinetochores and the cell cortex (Faulkner et al., 2000). LIS1 also associates with centrosomes (Smith et al., 2000; Tanaka et al., 2004) and is located at the leading edge of migrating fibroblasts (Dujardin et al., 2003). Interference with LIS1 produces severe mitotic defects (Faulkner et al., 2000; Tai et al., 2002) and inhibits the redistribution of cerebellar granule cell soma within reaggregate cultures (Hirotsune et al., 1998) as well as the directed migration of fibroblasts (Dujardin et al., 2003; Kholmanskikh et al., 2003). How these cellular defects may contribute to the neuronal migration disorder and the agyric or pachygyric morphology of the lissencephalic brain is not completely understood.
Developmental analysis of LIS1 heterozygous mouse lines and of cells transfected with cDNA constructs for LIS1 RNA interference (RNAi) has shown abnormalities in the extent of neuronal redistribution (Hirotsune et al., 1998; Gambello et al., 2003; Shu et al., 2004). However, detailed analysis of the migration pathway has not been performed. Recent work on normal brain has indicated that this pathway is quite complex. Cortical neurons are generated directly from radial glial cells in the ventricular zone (VZ) or indirectly from intermediate progenitor cells in the subventricular zone (SVZ) that are themselves generated from radial glia (Noctor et al., 2001, 2004; Haubensak et al., 2004). Neural progenitor cells are now known to progress through a series of morphogenetic stages. After classic interkinetic nuclear oscillations and cell division at the ventricular surface, newborn neurons ascend to the SVZ, where they convert to a multipolar nonmigratory phase (Rakic et al., 1974; Tabata and Nakajima, 2003; Noctor et al., 2004). After about a day, during which they begin to extend axonal processes, they convert to a bipolar stage and resume glial-directed radial migration. The importance of this complex progression in cortical development, how it is regulated, and how defects in this pathway may contribute to developmental diseases such as lissencephaly is not yet well understood.
This study was undertaken to gain insight into the specific role of LIS1 in neural progenitor behavior and neuronal cell migration. We conducted the first in situ live cell imaging analysis of neural progenitor cells with reduced LIS1 expression, and we followed the behavior of these cells throughout the migratory pathway. Surprisingly, migration and morphogenesis were blocked at multiple distinct stages, each of which has important implications for the biological function of LIS1 and for the physiological mechanisms underlying normal neurogenesis.
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Results |
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In brains that were transfected with the LIS1 shRNA construct, however, the majority of transfected cells were still distributed in the VZ/SVZ at day 4 (84.9 ± 2.0%, n = 5 embryos), with only a subset having reached the lower region of the IZ (10.4 ± 0.8%, n = 5 embryos). By day 6, cells expressing LIS1 shRNA had an almost identical distribution to that observed at days 2 and 4 (80.1 ± 4.0% in VZ/SVZ; 16.2 ± 3.3% in IZ; n = 3 embryos). Thus, the knockdown of LIS1 by RNAi in neurons appeared to have a direct effect on radial redistribution to the CP. A virtually identical migration arrest was produced by LIS1 siRNA oligonucleotide at days 2 and 4 (Fig. 1 C). However, the signal of the fluorescent siRNA oligonucleotide was too weak to detect by day 6, probably as a result of degradation or dilution of the fluorochrome or RNA. We note that the redistribution of LIS1 shRNAexpressing cells was inhibited not only at the center of the neocortex but also at its lateral boundaries. In these regions, the radial glial fibers are themselves distorted (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1; Misson et al., 1991), and migration along these tracks likely contributes to cortical expansion.
We also introduced full-length LIS1 (GFP-LIS1), an NH2-terminal LIS1 fragment (GFP-LIS1N), and the full-length dynamitin subunit of the dynactin complex (GFP-p50), each of which produce dominant inhibitory effects on dynein function in cultured cells (Echeverri et al., 1996; Faulkner et al., 2000; Tai et al., 2002). No clear effect on overall cell distribution was observed 2 d after the expression of each of these constructs, but a marked decrease in transfected cell number was observed by day 4. These results suggest that prolonged overexpression of these cDNA constructs may be lethal.
Accumulation of LIS1 and dynactin inhibited cells at the multipolar stage
As noted previously, newborn neurons have been shown to move from the VZ to the SVZ and assume an immobile multipolar morphology before converting to a bipolar morphology and recommencing their radial migration (Tabata and Nakajima, 2003; Noctor et al., 2004). To investigate the involvement of LIS1 in the specific stages in this pathway, we performed morphological analysis of the transfected cells. Individual radial glial, multipolar, and bipolar cells in different regions were readily distinguishable from the analysis of serial confocal images of brain slices (Fig. 2 A). In control brains, many multipolar cells were produced by day 2, constituting a majority of the population in the SVZ (control shRNA: 72.2 ± 1.7%, n = 4 embryos; empty vector: 68.3 ± 1.5%, n = 3 embryos; Fig. 2 B). Some cells in the lower SVZ and VZ still showed a radial glial morphology (control shRNA: 10.9 ± 2.1%, n = 4 embryos; empty vector: 11.4 ± 1.0%, n = 3 embryos). Some cells in the upper SVZ and a few cells that had reached the IZ exhibited prominent leading processes extending toward the CP (Fig. 2 A) as expected for bipolar migratory neurons (control shRNA: 9.0 ± 1.4%, n = 5; empty vector: 8.3 ± 1.1%, n =3 ; Fig. 2 B). By days 4 and 6, the numbers of cells with radial glial and multipolar morphology were decreased, the majority of control cells had become bipolar (control shRNA: 38.6 ± 1.4%, n = 3 embryos and 72.6 ± 1.8%, n = 3 embryos; empty vector: 44.6 ± 3.3%, n = 5 embryos and 76.8 ± 3.9%, n = 3 embryos, respectively; Fig. 2 B), and many had migrated to the CP.
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To evaluate the differentiation state of SVZ cells, sections were stained with the neuronal marker TuJ1 (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1) and progenitor cell markers nestin (Fig. 2 A) and vimentin (not depicted). Multipolar and bipolar cells that were transfected with LIS1 shRNA, siRNA oligonucleotides, GFP-LIS1N, GFP-LIS1, or GFP-p50 all expressed the neuronal marker TuJ1 but not nestin and vimentin, as is the case for cells transfected with control plasmids or infected with retrovirus (Noctor et al., 2004). These results provided further support that cells with reduced LIS1 expression were arrested in a stage corresponding to the multipolar stage of postmitotic neurons in normal brain.
LIS1 has been implicated in cell division by its localization to mitotic kinetochores and the mitotic cell cortex of nonneuronal cells. Dominant negative cDNAs, antibody microinjection, and antisense oligonucleotides all resulted in a pronounced accumulation of cells in prometaphase (Faulkner et al., 2000; Tai et al., 2002). To test for changes in neural progenitor cell cycle progression, brains that were transfected with the empty vector, LIS1 shRNA, or GFP-LIS1N constructs were stained on day 2 using the antiphosphovimentin antibody 4A4, which labels M-phase neural progenitor cells. In control brains that were transfected with empty vector, 3.7 ± 0.9% (n = 4) of GFP-expressing cells in the VZ/SVZ were 4A4 positive (Fig. 3, top). To our surprise, very few LIS1 shRNA and GFP-LIS1Ntransfected cells were 4A4 positive (0.68 ± 0.33%, n = 4; and 0.67 ± 0.35%, n = 3; respectively). We also immunostained cells for the nuclear transcription factor Ki67, which is expressed in proliferating cells from S-phase through M-phase of the cell cycle. In day 2 control cells, which were primarily located within the VZ/SVZ, 32.0 ± 4.3% (n = 4) of the cells were Ki67 positive (Fig. 3, bottom). In contrast, the percentages of Ki67-positive cells in LIS1 shRNA (5.4 ± 1.2%, n = 4) and GFP-LIS1N (7.2 ± 1.5%, n = 3)transfected brains were dramatically decreased (P < 0.01).
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Finally, we examined videos of control and shRNA-transfected cells for the frequency of mitotic events. Consistent with our immunohistochemical analysis, we observed that 8/73 control cells divided during time-lapse periods of 1218 h (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1). In contrast, 0/86 LIS1 shRNAtransfected cells were observed to divide during a comparable period.
Block in interkinetic nuclear oscillations and cell division in radial glial cells
Despite the accumulation of multipolar cells in the SVZ, we still found that a small percentage of radial glial cells remained in the VZ of LIS1 shRNAtransfected brains (Figs. 1 B and 2 B). To test whether these cells were still motile, live cell imaging of brain slices was again performed. In control shRNAtransfected cells, nuclei were observed to oscillate, albeit in a discontinuous fashion: periods of immobility were followed by relatively rapid directed movements toward (n = 13/28) or away (n = 11/28) from the ventricular surface (Fig. 5 A and Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1). LIS1 shRNAtransfected radial glial cells appeared to be morphologically normal, exhibiting their characteristic long bipolar radial processes. Surprisingly, however, nuclear movement was almost completely abolished among all of the 23 cells from 3 embryos that were monitored. Random movements of very limited range (15 µm) were still observed, but directed nuclear migration over substantial distances was eliminated (Fig. 5 B and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1). Nuclei were stalled at various distances from the ventricular surface and maintained their positions over substantial periods of time.
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Impaired radial migration of bipolar cells
Our results identified two distinct forms of neural progenitor cell behavior that were completely inhibited by LIS1 RNAi. Nonetheless, some cells with a bipolar morphology reached the lower IZ (Fig. 1 B). To test whether these cells represented a motile subpopulation of LIS1 shRNA transfectants, live cell imaging was again performed. In control brains, migrating bipolar cells in the IZ were observed between days 3 and 4. The cells extended a leading process toward the CP of relatively constant length (Fig. 6 A, top; and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1). Movement of the cell bodies was discontinuous as the cell underwent locomotion toward the pial surface.
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Altered axonal extension
In developing neocortex, most migrating neurons put out a trailing process (Fig. 1 B, arrows), which is thought to represent the developing axon (Schwartz and Goldman-Rakic, 1991; Noctor et al., 2004). These processes persist as the cells become bipolar and move toward the CP. Despite the accumulation of LIS1 shRNAtransfected cells in the SVZ, tangential axonlike processes were observed at days 4 and 6 (Fig. 7, A and B). These processes extended medially in the same direction as seen for control cells. However, the axonlike processes in LIS1 shRNAtransfected cells were shorter and somewhat curved and branched (Fig. 7 A). The role of LIS1 in this aspect of neural progenitor behavior has not been previously studied. For this reason and to gain further support for the origin and character of these processes, we monitored their behavior in living slices.
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Discussion |
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A long-standing issue in the field of brain development is the functional significance of interkinetic nuclear oscillations: does nuclear position merely correlate with cell cycle stage or does it control it? We observed no mitotic events in cells expressing LIS1 shRNA, which is in dramatic support of the latter possibility. This evidence could relate to the defects in cell division resulting from the expression of dominant negative cDNAs, antibody injection, and antisense oligonucleotides in our previous work (Faulkner et al., 2000; Tai et al., 2002). However, each of these treatments caused cells to accumulate in prometaphase with a markedly increased mitotic index overall. This contrasts dramatically with the current situation in which no mitotic events were observed in VZ and SVZ progenitor cells, and mitotic index was substantially reduced. We argue, therefore, that the current observed decrease in mitotic cells must have a different cause.
In the case of radial glial cells in the VZ, an appealing possibility is that known or unknown mitogenic factors that are enriched at the ventricular surface are required not only in controlling neurogenesis but also in controlling mitotic entry. In this case, the inability of nuclei to reach the ventricular surface in our experiments would limit their access to mitogenic agents. This possibility is supported by a decrease in the percentage of LIS1 shRNAtransfected radial glial cells that are positive for phosphovimentin or that exhibit condensed chromosomes as judged by DAPI staining (unpublished data). Intriguingly, the few mitotic cells we did see in the latter analysis were all located at the ventricular surface. These may represent rare cells not yet detected by our live cell imaging, which retained sufficient LIS1 to enter mitosis. However, their exclusive location at the ventricular surface further supports a spatial control mechanism for mitotic entry.
We also observed a reduced number of mitotic figures within the SVZ in fixed brain tissue and in the number of Ki67-positive multipolar cells (Fig. 3). Thus, it appears that the number of SVZ intermediate progenitor cells, which are still capable of division (Noctor et al., 2004), was significantly reduced by interference with LIS1 expression or function. It is unclear whether the reduced numbers of mitotic cells that we observed within the VZ and SVZ result from related mechanisms. We suspect that those transfected cells that reach the SVZ cells must, nonetheless, have experienced some effect of reduced LIS1 expression (e.g., a slower journey to their final destination). Conceivably, such a delay could alter fate determination for these cells, allowing premature exit from the cell cycle.
In a previous study with an LIS1 heterozygote mouse, cell divisions were reported to increase in the SVZ but decrease in the VZ (Gambello et al., 2003). Although these divisions were said to be in part "ectopic," it was not possible to distinguish whether they involved radial glial or SVZ intermediate progenitor cells. These results, therefore, cannot be readily related to our study. However, based on our current and previous results, we predict that cell division should proceed under conditions of haploinsufficiency, albeit at a reduced frequency. Division itself, however, should be prolonged. The net result may have different effects on mitotic index in the VZ and SVZ, but the precise quantitative outcome is difficult to predict. We note that increased ectopic divisions were recently reported in mice that were heterozygous null for NudE, which is another protein in the LIS1 and dynein pathway (Feng and Walsh, 2004). Thus, as predicted from in vitro studies (Faulkner et al., 2000), brain developmental disorders arising from mutations in the dynein pathway now appear to involve defects in cell proliferation as well as in migration.
LIS1 is necessary for progression from the multipolar to the migratory stage
The most striking effect of LIS1 RNAi and dominant negative LIS1 and dynactin cDNAs on the overall distribution of transfected neural progenitors was the accumulation of cells within the SVZ (Fig. 2), as was previously observed in RNAi analysis of the lissencephaly genes doublecortin (LoTurco, 2004) and LIS1 (Shu et al., 2004). Whether this pattern reflected a general migration delay or interference with specific steps in the migratory pathway was unknown. Our results demonstrate the latter to be the case. Although the previous study involving the use of LIS1 RNAi reported the bipolar morphology to be predominant in the SVZ (Shu et al., 2004), our evidence using both static and live cell imaging showed an absolute block in progression from the multipolar to the migratory stage (Fig. 4).
A specific block in exit from the SVZ is of considerable interest in understanding lissencephaly. This condition is characterized by the presence of a broad ectopic neuronal lamina within the white matter of the newborn neocortex (Dobyns and Truwit, 1995). Based on our results, we propose that this and, perhaps, other lamination defects reflect changes in specific rate-limiting steps in the neuronal migration pathway, predominantly involving exit from the SVZ.
The specific role of LIS1 in the conversion of multipolar to bipolar cells is uncertain. The first detectable event in this process was a thickening of the presumptive migratory process, which the nucleus subsequently enters (Fig. 4). This observation suggests that LIS1 and its regulatory target dynein are involved in the shift in cytoplasmic contents into the differentiating migratory process. We have previously observed that dynein and LIS1 are associated with the leading edge of migrating fibroblasts (Dujardin et al., 2003). More recently, we have observed a similar accumulation of dynein and LIS1 at the tips of rapidly growing laminin-induced neurites in primary chick dorsal root ganglia neurons (unpublished data). We suspect that LIS1 and dynein may be playing a related role in the initiation of the migratory process, although, not surprisingly, in its subsequent growth (see next section). Our results also suggest that at the reduced levels attained in this study, LIS1 is not required for entry into the multipolar state or in process maintenance. Whether the observed changes in process dynamics and morphology reflect the effects of residual LIS and whether further reduction in LIS1 would eliminate processes entirely remains to be seen.
Uncoupling of nucleokinesis from migratory process growth
The number of LIS1 siRNA, shRNA-, and dominant negative LIS1- and dynactin cDNAtransfected cells within the IZ and CP were greatly reduced relative to controls, which is consistent with a small probability of escape from the SVZ (Fig. 1). Those few cells that reached this region were not yet connected with the pial surface of the developing brain. Migration should, therefore, occur by locomotion (Nadarajah et al., 2001), which involves forward extension of the migratory process and somal translocation to keep up. The shRNA-expressing cells had a bipolar morphology, which is comparable in low magnification images with control cells. However, migration was completely abolished.
In striking contrast to normal bipolar cells, however, the soma were virtually immotile (Fig. 6). Nonetheless, growth of the migratory process persisted. Together, these results provide the first indication that these aspects of locomotion can be uncoupled. As is the case for SVZ cells, the migratory processes of LIS1 shRNAtransfected IZ cells exhibited numerous short, highly dynamic extensions. Whether these represent branches that are similar to those observed in axons or whether they are more similar to filopodial extensions is unclear. Curiously, we found net axonal growth to stop in LIS1 knockdown cells, whereas the leading migratory processes of bipolar cells maintained their ability to extend. One possible explanation for these differences is that the migratory process, but not the axon, is guided and supported by radial glial fibers, where dynein and LIS1 might be less essential. In any case, our results indicated that the basic roles of dynein and LIS1 may differ between process types.
Other studies have found that reduced expression of LIS1 and its interacting proteins NudE and NudEL or dynein inhibited somal translocation in dissociated neuronal cell cultures (Hirotsune et al., 1998; Gambello et al., 2003; Shu et al., 2004; Tanaka et al., 2004). Clear effects on somal movement were observed, but it is uncertain whether they represent nuclear migration within processes or traction-mediated translocation of the entire somal region. Recent evidence has suggested a role for LIS1, dynein, and NudEL in generating tension between the nucleus and centrosome in migratory neural progenitor cells (Shu et al., 2004; Tanaka et al., 2004), which could contribute to somal translocation (Solecki et al., 2004). Based on evidence from other systems, this behavior is likely to be mediated by cortically associated dynein and LIS1, with which centrosome-tethered microtubules interact (Palazzo et al., 2001; Dujardin et al., 2003; Etienne-Manneville and Hall, 2001). We suspect that the observed defect in somal movement within bipolar IZ cells that were transfected with LIS1 shRNA may, in part, involve a similar pool of cortical dynein that is unable to engage cytoplasmic microtubules.
LIS1 is important in axonal extension
We could detect a single long axon extending from both multipolar and bipolar cells based on outgrowth length, caliber, and direction of the process. The axon remained clearly identifiable in cells that were subjected to LIS1 RNAi, demonstrating the persistence of underlying polarity in multipolar and bipolar cells. The persistence of axons, despite clear interference with their continued extension, may indicate that growth was initiated before LIS1 levels had become severely reduced. These considerations suggest that LIS1 is not required for maintenance of processes once they have formed.
How LIS1 and dynein function to control process dynamics has been the subject of recent investigation. Inhibition of cytoplasmic dynein function using dynamitin was reported to induce myosin-mediated neurite retraction (Ahmad et al., 2000). We have more recently found that both LIS1 and dynein become concentrated at sites of nascent process formation at the leading edge of chick sympathetic and dorsal root ganglia growth cones in response to laminin and that they persist at the tips of rapidly growing processes (unpublished data). Acute interference with either LIS1 or dynein by antibody injection eliminated growth cone remodeling. We suspect that related effects occur in the axons of SVZ cells that were transfected with LIS1 shRNA. Why the extension of axons would be more sensitive to reduced LIS1 expression than the growth of migratory processes (see previous section) is uncertain. However, our results suggest that elongation of the two types of process is controlled by substantially different mechanisms.
The function of early appearing axonal processes characterized in this study is not well established, and the effects that we observed on their extension had not been anticipated from prior studies. Use of retrograde tracing has shown connectivity of migrating neurons in the fetal monkey cerebrum to the opposite cerebral hemisphere (Schwartz and Goldman-Rakic, 1991). The processes that we observed (Fig. 7) exhibited organized and directed growth toward the midline of the cerebrum, strongly suggesting that they correspond to those involved in connectivity to other brain regions. The truncated axons that result from reduced LIS1 expression seem unlikely to be capable of reaching their targets. Our results, therefore, raise the novel possibility that similar, albeit less pronounced, effects may be involved in lissencephaly. Previous studies in hippocampus of LIS1 heterozygous mice and fruit flies showed stunted dendritic branches and abnormal synaptic transmission (Fleck et al., 2000; Liu et al., 2000). The effects on axon outgrowth that are identified in this study could well result in connectivity defects (Ross, 2002). These, in turn, may have severe consequences for brain function and could contribute to the loss of higher brain function, the frequency of epilepsy, cerebral palsy, and seizures that are characteristic of classical lissencephaly, and dramatically decreased life span.
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Materials and methods |
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In vitro RNAi assay
For transient transfection with pRNAT constructs, cells were plated on six-well dishes to 7080% confluency and were transfected using LipofectAMINE 2000 (Invitrogen). For transient transfection with siRNA oligonucleotides, cells were plated on six-well dishes to 4050% confluency and were transfected using OligofectAMINE (Invitrogen). Cells were lysed 4548 h after transfection in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, and 1 mM EGTA with protease inhibitor cocktail for mammalian tissues (Sigma-Aldrich). pAb anti-LIS1 (Santa Cruz Biotechnology, Inc.) and mAb antitubulin (Sigma-Aldrich) were used for Western blotting.
In utero electroporation
Plasmids or oligonucleotides were transfected using intraventricular injection followed by in utero electroporation (Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001). In brief, pregnant Sprague Dawley rats (Taconic) were used, and 12 µl cDNA (15 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.
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 (Ted Pella). Slices were blocked at RT for 1 h with 10% serum, 0.1% Triton X-100, and 0.2% gelatin in PBS. Primary antibodies were applied overnight at the following concentrations: anti-LIS1, 1:200 (gift from M. Mizuguchi, University of Tokyo, Tokyo, Japan; Mizuguchi et al., 1995); anti-TuJ1, 1:40 (Babco); antinestin, 1:200 (Chemicon), antivimentin, 1:40 (CBL); 4A4, 1:1,000 (MBL International Corporation); and anti-Ki67, 1:200 (Chemicon). Sections were then washed with PBS and incubated in Cy5-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).
Confocal microscopy
Sections were imaged on an inverted laser-scanning confocal microscope (FluoView 300; Olympus) with a 40x NA 0.8 water immersion objective (Olympus). Excitation/emission wavelengths were 488/515 nm (GFP), 568/590 nm (DsRed), and 633/690 (Cy5). Z-series images were collected at 23-µm steps in FluoView, and a projection of each stack was used for producing figures. Images were contrast enhanced, assembled into montages, and false color was applied using Photoshop (Adobe). In each brain slice, 100500 cells could be found positive to GFP when electroporated with the constructs used. When Cy3-labeled synthetic siRNA oligonucleotides were used, 3080 siRNA-positive cells per slice could be detected. These cells were all counted for statistic analysis (Table S1). In experiments that involved colabeling or cell/process counting, images from individual optical sections were carefully examined.
Live cell imaging
Coronal slices were prepared 2448 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, 1x penicillin/streptomycin/glutamine (GIBCO BRL), and 0.66% glucose and were incubated at 37°C in 5% CO2. Multiple GFP-positive cells (Table S2) were imaged on an inverted microscope (model DMIRB; Leica) with a 40x NA 0.55 objective. Time-lapse images were captured by camera (CoolSNAP HQ; Roper Scientific) using MetaMorph software (Universal Imaging Corp.) at intervals of 10 or 15 min for 1018 h. Epifluorescence images from several focal planes were deconvolved using AutoDeblur software (AutoQuant Imaging, Inc.) to produce sharp images.
Online supplemental material
Fig. S1 shows that neuronal migration occurs radially but also along curved radial glial fiber tracks. Fig. S2 shows that multipolar morphology is predominant in neural progenitor cells that were transfected with Cy3-LIS1 siRNA oligonucleotides or dominant negative LIS1 and dynamitin constructs. Fig. S3 shows that multipolar cells expressing LIS1 shRNA are positive for the neuronal marker TuJ1. Tables S1 and S2 summarize the number of embryos that were used and cells that were counted in this study. Videos S1S10 demonstrate the behavior of neural progenitor cells at different stages and areas in live preparations of normal and LIS1 shRNAtransfected brains. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200505166/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health (NIH) grant 40182, a grant from the March of Dimes Birth Defects Foundation to R.B. Vallee, and NIH grants NS21223 and NS35710 to A.R. Kriegstein.
Submitted: 26 May 2005
Accepted: 3 August 2005
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