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• W2068384450 abstract "•oRG mitosis is preceded by MST, a rapid translocation toward the cortical plate•MST requires ROCK and myosin II activation, but not intact microtubules•MST is not dependent on centrosomal translocation into the leading process•Diseases attributed to disrupted neuronal migration may also involve oRG cell MST Evolutionary expansion of the human neocortex is partially attributed to a relative abundance of neural stem cells in the fetal brain called outer radial glia (oRG). oRG cells display a characteristic division mode, mitotic somal translocation (MST), in which the soma rapidly translocates toward the cortical plate immediately prior to cytokinesis. MST may be essential for progenitor zone expansion, but the mechanism of MST is unknown, hindering exploration of its function in development and disease. Here, we show that MST requires activation of the Rho effector ROCK and nonmuscle myosin II, but not intact microtubules, centrosomal translocation into the leading process, or calcium influx. MST is independent of mitosis and distinct from interkinetic nuclear migration and saltatory migration. Our findings suggest that disrupted MST may underlie neurodevelopmental diseases affecting the Rho-ROCK-myosin pathway and provide a foundation for future exploration of the role of MST in neocortical development, evolution, and disease. Evolutionary expansion of the human neocortex is partially attributed to a relative abundance of neural stem cells in the fetal brain called outer radial glia (oRG). oRG cells display a characteristic division mode, mitotic somal translocation (MST), in which the soma rapidly translocates toward the cortical plate immediately prior to cytokinesis. MST may be essential for progenitor zone expansion, but the mechanism of MST is unknown, hindering exploration of its function in development and disease. Here, we show that MST requires activation of the Rho effector ROCK and nonmuscle myosin II, but not intact microtubules, centrosomal translocation into the leading process, or calcium influx. MST is independent of mitosis and distinct from interkinetic nuclear migration and saltatory migration. Our findings suggest that disrupted MST may underlie neurodevelopmental diseases affecting the Rho-ROCK-myosin pathway and provide a foundation for future exploration of the role of MST in neocortical development, evolution, and disease. The human neocortex is characterized by a marked increase in size and neuronal number as compared to other mammals. Neural stem cells called outer radial glia (oRG), present in large numbers during human, but not rodent, brain development, are thought to underlie this expansion (Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar, Lui et al., 2011Lui J.H. Hansen D.V. Kriegstein A.R. Development and evolution of the human neocortex.Cell. 2011; 146: 18-36Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar). oRG cells are derived from ventricular radial glia (vRG), the primary neural stem cells present in all mammals (LaMonica et al., 2013LaMonica B.E. Lui J.H. Hansen D.V. Kriegstein A.R. Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.Nat. Commun. 2013; 4: 1665Crossref PubMed Scopus (151) Google Scholar, Malatesta et al., 2000Malatesta P. Hartfuss E. Götz M. Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage.Development. 2000; 127: 5253-5263Crossref PubMed Google Scholar, Miyata et al., 2001Miyata T. Kawaguchi A. Okano H. Ogawa M. Asymmetric inheritance of radial glial fibers by cortical neurons.Neuron. 2001; 31: 727-741Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, Noctor et al., 2001Noctor S.C. Flint A.C. Weissman T.A. Dammerman R.S. Kriegstein A.R. Neurons derived from radial glial cells establish radial units in neocortex.Nature. 2001; 409: 714-720Crossref PubMed Scopus (1562) Google Scholar, Shitamukai et al., 2011Shitamukai A. Konno D. Matsuzaki F. Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors.J. Neurosci. 2011; 31: 3683-3695Crossref PubMed Scopus (337) Google Scholar, Wang et al., 2011Wang X. Tsai J.W. LaMonica B. Kriegstein A.R. A new subtype of progenitor cell in the mouse embryonic neocortex.Nat. Neurosci. 2011; 14: 555-561Crossref PubMed Scopus (359) Google Scholar). Both progenitor cell types display basal processes oriented toward the cortical plate, along which newborn neurons migrate (Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar, Misson et al., 1991Misson J.P. Austin C.P. Takahashi T. Cepko C.L. Caviness Jr., V.S. The alignment of migrating neural cells in relation to the murine neopallial radial glial fiber system.Cereb. Cortex. 1991; 1: 221-229Crossref PubMed Scopus (145) Google Scholar, Rakic, 1971Rakic P. Guidance of neurons migrating to the fetal monkey neocortex.Brain Res. 1971; 33: 471-476Crossref PubMed Scopus (488) Google Scholar, Rakic, 1972Rakic P. Mode of cell migration to the superficial layers of fetal monkey neocortex.J. Comp. Neurol. 1972; 145: 61-83Crossref PubMed Scopus (1701) Google Scholar). However, oRG cells reside primarily within the outer subventricular zone (oSVZ), closer to the cortical plate than vRG cells, and lack the apical ventricular contact characteristic of vRG cells (Chenn et al., 1998Chenn A. Zhang Y.A. Chang B.T. McConnell S.K. Intrinsic polarity of mammalian neuroepithelial cells.Mol. Cell. Neurosci. 1998; 11: 183-193Crossref PubMed Scopus (176) Google Scholar, Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar). While vRG cell behavior, mitosis, and lineage have been extensively studied (Bentivoglio and Mazzarello, 1999Bentivoglio M. Mazzarello P. The history of radial glia.Brain Res. Bull. 1999; 49: 305-315Crossref PubMed Scopus (119) Google Scholar, Hartfuss et al., 2001Hartfuss E. Galli R. Heins N. Götz M. Characterization of CNS precursor subtypes and radial glia.Dev. Biol. 2001; 229: 15-30Crossref PubMed Scopus (607) Google Scholar, Noctor et al., 2001Noctor S.C. Flint A.C. Weissman T.A. Dammerman R.S. Kriegstein A.R. Neurons derived from radial glial cells establish radial units in neocortex.Nature. 2001; 409: 714-720Crossref PubMed Scopus (1562) Google Scholar, Noctor et al., 2004Noctor S.C. Martínez-Cerdeño V. Ivic L. Kriegstein A.R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.Nat. Neurosci. 2004; 7: 136-144Crossref PubMed Scopus (1683) Google Scholar, Noctor et al., 2008Noctor S.C. Martínez-Cerdeño V. Kriegstein A.R. Distinct behaviors of neural stem and progenitor cells underlie cortical neurogenesis.J. Comp. Neurol. 2008; 508: 28-44Crossref PubMed Scopus (296) Google Scholar, Qian et al., 1998Qian X. Goderie S.K. Shen Q. Stern J.H. Temple S. Intrinsic programs of patterned cell lineages in isolated vertebrate CNS ventricular zone cells.Development. 1998; 125: 3143-3152PubMed Google Scholar, Taverna and Huttner, 2010Taverna E. Huttner W.B. Neural progenitor nuclei IN motion.Neuron. 2010; 67: 906-914Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), much less is known about regulation of oRG cell proliferation and the unique mitotic behavior of these cells (Betizeau et al., 2013Betizeau M. Cortay V. Patti D. Pfister S. Gautier E. Bellemin-Ménard A. Afanassieff M. Huissoud C. Douglas R.J. Kennedy H. Dehay C. Precursor diversity and complexity of lineage relationships in the outer subventricular zone of the primate.Neuron. 2013; 80: 442-457Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, Gertz et al., 2014Gertz C.C. Lui J.H. LaMonica B.E. Wang X. Kriegstein A.R. Diverse behaviors of outer radial glia in developing ferret and human cortex.J. Neurosci. 2014; 34: 2559-2570Crossref PubMed Scopus (90) Google Scholar, Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar, LaMonica et al., 2013LaMonica B.E. Lui J.H. Hansen D.V. Kriegstein A.R. Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.Nat. Commun. 2013; 4: 1665Crossref PubMed Scopus (151) Google Scholar, Pilz et al., 2013Pilz G.A. Shitamukai A. Reillo I. Pacary E. Schwausch J. Stahl R. Ninkovic J. Snippert H.J. Clevers H. Godinho L. et al.Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type.Nat. Commun. 2013; 4: 2125Crossref PubMed Scopus (151) Google Scholar). oRG cell cytokinesis is immediately preceded by a rapid translocation of the soma along the basal fiber toward the cortical plate, a process termed mitotic somal translocation (MST) (Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar). Due to the relative abundance of oRG cells in humans, it has been hypothesized that genetic mutations causing significant brain malformations in humans, but minimal phenotypes in mouse models, may affect oRG cell-specific behaviors such as MST (LaMonica et al., 2012LaMonica B.E. Lui J.H. Wang X. Kriegstein A.R. OSVZ progenitors in the human cortex: an updated perspective on neurodevelopmental disease.Curr. Opin. Neurobiol. 2012; 22: 747-753Crossref PubMed Scopus (101) Google Scholar). However, the molecular motors driving MST have not been identified, hindering exploration of the function of MST in human brain development and its possible role in disease. MST is reminiscent of interkinetic nuclear migration (INM) of neuroepithelial and vRG cells, in which nuclei of cycling cells migrate back and forth along the basal fiber between the apical and basal boundaries of the ventricular zone in concert with the cell cycle. INM is controlled by the centrosome, the microtubule motors kinesin and dynein, and associated proteins, with actomyosin motors playing an accessory role (Taverna and Huttner, 2010Taverna E. Huttner W.B. Neural progenitor nuclei IN motion.Neuron. 2010; 67: 906-914Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). As oRG cells are derived from vRG cells and display analogous nuclear movements, it has been hypothesized that MST requires similar molecular motors as INM (LaMonica et al., 2012LaMonica B.E. Lui J.H. Wang X. Kriegstein A.R. OSVZ progenitors in the human cortex: an updated perspective on neurodevelopmental disease.Curr. Opin. Neurobiol. 2012; 22: 747-753Crossref PubMed Scopus (101) Google Scholar). We find that MST requires activation of the Rho effector ROCK and nonmuscle myosin II (NMII), but not intact microtubules, centrosomal advancement into the leading process, or calcium influx. Conversely, oRG cell mitosis requires intact microtubules, but not NMII activation, demonstrating that MST and mitosis are mutually dissociable. We examine the expression profiles of genes implicated in the Rho-ROCK-myosin pathway that cause large developmental brain malformations when mutated in humans, but not in mice. Interestingly, several disease genes thought to primarily affect neuronal migration display expression profiles similar to known radial glial genes, consistent with expression in oRG cells. This observation suggests that defects in oRG behaviors such as MST may partially underlie cortical malformations currently attributed to defective neuronal migration. Together, these results increase our understanding of the cellular and molecular basis for human cortical evolution and have important implications for studying disease mechanisms that cannot be effectively modeled in mice. MST is thought to contribute to radial expansion of the oSVZ during human brain development (Lui et al., 2011Lui J.H. Hansen D.V. Kriegstein A.R. Development and evolution of the human neocortex.Cell. 2011; 146: 18-36Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar). Supportive of this idea, we imaged oRG cell divisions in human fetal cortical slices at the border of the upper oSVZ and intermediate zone (IZ) during peak neurogenesis and oSVZ growth (gestational weeks 15–20). We observed many divisions in which oRG cells translocated out of the oSVZ and into the IZ, thereby increasing oSVZ size (Movie S1). We found that MST trajectory in the human oSVZ was overwhelmingly toward the cortical plate (Figure 1A). Furthermore, MST frequency and translocation distances were greater in humans as compared to ferrets and mice, species displaying proportionally smaller oSVZ sizes (Figures 1B–1D). These observations are suggestive of a role for MST in human oSVZ expansion. However, in-depth exploration of the function of MST in development and disease first requires an understanding of the underlying molecular mechanisms, which have remained elusive. We initially hypothesized that MST depends on the same molecular machinery as INM. To determine the relative contributions of microtubule motors and actomyosin to MST, we applied inhibitors of microtubule polymerization and NMII (the most well-characterized myosin in brain development; Tullio et al., 2001Tullio A.N. Bridgman P.C. Tresser N.J. Chan C.C. Conti M.A. Adelstein R.S. Hara Y. Structural abnormalities develop in the brain after ablation of the gene encoding nonmuscle myosin II-B heavy chain.J. Comp. Neurol. 2001; 433: 62-74Crossref PubMed Scopus (96) Google Scholar, Vallee et al., 2009Vallee R.B. Seale G.E. Tsai J.W. Emerging roles for myosin II and cytoplasmic dynein in migrating neurons and growth cones.Trends Cell Biol. 2009; 19: 347-355Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) to human fetal cortical slice cultures. We performed time-lapse imaging of oRG cell behaviors and quantified translocation (MST) and division frequency in each slice before and after addition of inhibitors or DMSO (control) (Figures 1E–1I and S1). Treatment of slices with a high concentration (100 μM) of blebbistatin, a selective NMII inhibitor, nearly abolished both translocations and divisions (data not shown). However, treatment with a low concentration (5 μM) of blebbistatin caused a significantly greater reduction in translocations than in divisions, suggesting that NMII plays a relatively larger role in MST than in mitosis. Conversely, treatment with the microtubule depolymerizing reagent nocodazole (1 μM) reduced divisions significantly more than translocations. Additionally, nocodazole, but not DMSO or blebbistatin, decreased the proportion of translocations that ended in division. We found that oRG cells express two isoforms of NMII, NMIIa and NMIIb (Figures 1J, 1K, and S1), which have both been shown to play essential roles in neuronal migration (Vicente-Manzanares et al., 2009Vicente-Manzanares M. Ma X. Adelstein R.S. Horwitz A.R. Non-muscle myosin II takes centre stage in cell adhesion and migration.Nat. Rev. Mol. Cell Biol. 2009; 10: 778-790Crossref PubMed Scopus (1338) Google Scholar). These results demonstrate that MST and mitosis are mutually dissociable in oRG cells. MST requires NMII activation, but not intact microtubules, and thus, not microtubule motors. Conversely, mitosis is relatively more dependent on intact microtubules than on NMII activation. We asked whether inhibition of MST directly affects daughter cell fate. Blebbistatin treatment of human fetal cortical slices did not alter the ratio of TBR2+ to SOX2+ cells in the oSVZ as compared to DMSO (p = 0.38, unpaired Student’s t test), suggesting that MST does not control cell fate. Inhibition of MST may lead to cell crowding or have other indirect effects that could influence cell fate on a longer timescale than we could analyze using our slice culture system. To control for non-cell-autonomous effects and to enable examination of subcellular mechanisms, we used dissociated neural progenitor cell cultures. Blocks of gestational week 15–20 (GW15–20) dorsal neocortical tissue spanning the ventricle to the cortical plate were dissociated by papain treatment and trituration. We previously observed that oRG-like cells undergo MST in dissociated fetal human cortical cultures (LaMonica et al., 2013LaMonica B.E. Lui J.H. Hansen D.V. Kriegstein A.R. Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.Nat. Commun. 2013; 4: 1665Crossref PubMed Scopus (151) Google Scholar). To confirm oRG identity of oRG-like cells, we performed fate staining on daughter cells after MST division (Figures 2A–2C and S2). Similar to oRG cells in slice culture (Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar), daughters of MST divisions in dissociated culture expressed SOX2 (65 out of 65) and PAX6 (17 out of 17), usually expressed nestin (18 out of 24), rarely expressed TBR2 (2 out of 34), and never expressed βIII-tubulin (0 out of 20). As in slice culture, we observed expression of both NMIIa (14 out of 14 cells) and NMIIb (ten out of ten cells) in dissociated oRG cell daughters (Figures 2I and 2J). We concluded, based on morphology, behavior, and marker expression, that cells undergoing MST in dissociated culture are oRG cells, validating the use of dissociated cultures to study oRG cell behaviors. We quantified translocation (MST) and division frequency in dissociated culture after motor protein inhibition. Similar to results in slice culture, blebbistatin treatment reduced translocations significantly more than divisions, while nocodazole treatment reduced divisions without significantly affecting translocations (Figures 2D–2H and Movie S2). Upon drug washout, blebbistatin-treated cultures showed an increase in translocations, while nocodazole-treated cells that had remained rounded up after MST underwent cytokinesis, suggesting that the effects of inhibitors were reversible and not due to cell death (Movie S3; Figure S2). Furthermore, inhibitor-treated cultures did not display increased staining for cleaved caspase-3, confirming that the effects of inhibitor treatment could not be attributed to apoptosis (Figure S2). Thus, results in dissociated culture confirm observations in slice culture that MST and mitosis are mutually dissociable in oRG cells. Intact microtubules are required for oRG cell mitosis, but not for MST, while NMII activation is required for oRG cell MST and is relatively less important for mitosis. Though microtubule polymerization is not required for oRG cell MST in humans, nocodazole treatment significantly increased MST distance in both slice culture and dissociated cells (Figure 3A). Based on previous observations in rodent (Wang et al., 2011Wang X. Tsai J.W. LaMonica B. Kriegstein A.R. A new subtype of progenitor cell in the mouse embryonic neocortex.Nat. Neurosci. 2011; 14: 555-561Crossref PubMed Scopus (359) Google Scholar), we hypothesized that the centrosome migrates into the basal fiber prior to translocation, remains connected to the nucleus via a microtubule cage, and ultimately determines the location of translocation cessation and cytokinesis (Tsai et al., 2007Tsai J.W. Bremner K.H. Vallee R.B. Dual subcellular roles for LIS1 and dynein in radial neuronal migration in live brain tissue.Nat. Neurosci. 2007; 10: 970-979Crossref PubMed Scopus (351) Google Scholar). Nocodazole treatment would disrupt nucleus-centrosome coupling, eliminating the “stop” signal for translocation. To determine whether the centrosome precedes the nucleus into the basal process, we performed time-lapse imaging of centrosome behavior in dissociated human oRG cells after transfection with a construct encoding the centrosomal protein Centrin-2 (Cetn2) fused to the fluorescent reporter dsred (Figure S2) (Tanaka et al., 2004Tanaka T. Serneo F.F. Higgins C. Gambello M.J. Wynshaw-Boris A. Gleeson J.G. Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration.J. Cell Biol. 2004; 165: 709-721Crossref PubMed Scopus (360) Google Scholar). While centrosome location was variable during interphase, centrosomes consistently returned to the soma prior to MST and remained adjacent to the nucleus throughout translocation (Figures 3B and 3D; Movie S4). In contrast, during saltatory migration of other cells in dissociated culture, the centrosome often preceded the nucleus into the leading process prior to a migratory step (Figures 3C and 3D; Movie S4). Interestingly, somal translocation distances were much greater during MST than during saltatory migration steps, suggesting that an increased translocation distance may limit the role of the centrosome during MST (Figure 3E). We concluded that MST does not require centrosomal advancement into the leading process prior to nuclear translocation. Instead, microtubule polymerization may directly regulate actomyosin contractility (Schaar and McConnell, 2005Schaar B.T. McConnell S.K. Cytoskeletal coordination during neuronal migration.Proc. Natl. Acad. Sci. USA. 2005; 102: 13652-13657Crossref PubMed Scopus (280) Google Scholar). Our results thus far suggested that MST does not require intact microtubules or centrosomal translocation into the basal process, but that MST is dependent on NMII activation. To verify that the effects of blebbistatin on MST were specifically due to NMII inhibition, we tested whether inhibition of ROCK, an upstream activator of NMII, similarly blocked MSTs (Govek et al., 2011Govek E.E. Hatten M.E. Van Aelst L. The role of Rho GTPase proteins in CNS neuronal migration.Dev. Neurobiol. 2011; 71: 528-553Crossref PubMed Scopus (127) Google Scholar). Treatment of dissociated cultures with the ROCK inhibitor Y-27632 (10μM) mimicked the effects of blebbistatin treatment, greatly reducing translocations without significantly affecting divisions (Figure 4A). While blebbistatin had a small effect on mitosis, ROCK inhibition had no effect, likely due to myosin playing a bigger role in oRG cell cytokinesis than ROCK. Treatment with a second, more potent and selective ROCK inhibitor, dimethylfasudil (1 μM), mimicked blebbistatin and Y-27632 treatment, reducing translocations without significantly affecting divisions (Figure 4A). These results confirm that the effects of blebbistatin on MST are specifically due to NMII inhibition, and suggest that the Rho-ROCK-NMII pathway may control MST, as ROCK is activated by the GTPase RhoA (Heng and Koh, 2010Heng Y.W. Koh C.G. Actin cytoskeleton dynamics and the cell division cycle.Int. J. Biochem. Cell Biol. 2010; 42: 1622-1633Crossref PubMed Scopus (183) Google Scholar). ROCK- and NMII-dependent actomyosin contraction may occur throughout the soma and basal process, as we often observed shortening and thinning of the primary process during MST in dissociated oRG cells (Figure S2). This observation is consistent with NMII expression throughout oRG cell processes (Figures 2I and 2J). We asked whether calcium influx, a parallel activator of NMII, is required for MST. We treated human fetal progenitor cultures with ML-7, an inhibitor of myosin light chain kinase (MLCK). MLCK activates NMII and is downstream of Ca2+-calmodulin, but not ROCK. ML-7 (10 μM) had no effect on translocations or divisions (Figure 4A). We further verified these results by subjecting dissociated cultures to treatment with a panel of calcium channel inhibitors, including the nonspecific calcium channel blocker NiCl2 (50 μM), ryanodine receptor blocker ruthenium red (50 μM), and the IP3-gated calcium channel blocker 2-APB (50 μM). Calcium channel inhibition had no effect on either translocations or divisions (Figure 4A). These results suggest that calcium influx is not responsible for NMII activation leading to MST. We have demonstrated here that MST and mitosis can be uncoupled, and that MST requires ROCK and NMII activation, but not intact microtubules, centrosomal translocation into the leading process, or calcium influx. It is possible that in oRG cells, RhoA-activated ROCK either directly phosphorylates NMII, inhibits myosin phosphatase, or both, leading to actomyosin contraction and MST (Figure 4B). The expression and activity of known cell-cycle regulators support a role for the Rho-ROCK-myosin pathway in MST: RhoA is activated in a cell cycle-dependent manner by CDK1, and RhoA has been demonstrated to participate in the G2 to M transition (Heng and Koh, 2010Heng Y.W. Koh C.G. Actin cytoskeleton dynamics and the cell division cycle.Int. J. Biochem. Cell Biol. 2010; 42: 1622-1633Crossref PubMed Scopus (183) Google Scholar). Several evolutionary forces could have led to the unique dependence of MST on actomyosin motors. Nuclear translocation distance may dictate molecular motor dependence. Interkinetic nuclear migration and saltatory migration involve small nuclear translocation steps, limiting the distance between the centrosome and the nucleus. The larger translocation distances of MST could hinder maintenance of tension between the centrosome and a perinuclear microtubule cage, making a centrosome-based mechanism untenable. Actomyosin motors are also approximately 10-fold faster than microtubule motors, and may be better suited to drive the rapid, large-amplitude translocations of MST (Månsson, 2012Månsson A. Translational actomyosin research: fundamental insights and applications hand in hand.J. Muscle Res. Cell Motil. 2012; 33: 219-233Crossref PubMed Scopus (36) Google Scholar). Additionally, we observed chromosome condensation and establishment of a metaphase plate during MST using time-lapse transmitted light microscopy, suggesting that prophase and metaphase occur prior to the completion of MST (Figure 3B; Movie S4). Microtubule depolymerization occurs during prometaphase and may preclude dependence of MST on microtubule motors (Rusan et al., 2002Rusan N.M. Tulu U.S. Fagerstrom C. Wadsworth P. Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport.J. Cell Biol. 2002; 158: 997-1003Crossref PubMed Scopus (103) Google Scholar). A recent study suggested a broader diversity of progenitor cell types and behaviors within the macaque oSVZ than we observed in humans, including a larger proportion of apically directed MSTs (Betizeau et al., 2013Betizeau M. Cortay V. Patti D. Pfister S. Gautier E. Bellemin-Ménard A. Afanassieff M. Huissoud C. Douglas R.J. Kennedy H. Dehay C. Precursor diversity and complexity of lineage relationships in the outer subventricular zone of the primate.Neuron. 2013; 80: 442-457Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). While the definition of oRG cells used by Betizeau and colleagues is more ambiguous than ours, it is clear that at least a subset of oSVZ progenitor cells display basally directed MST that shifts the border of the oSVZ toward the cortical plate, appearing to expand oSVZ size by moving neural stem cells further away from the ventricle. Thus, while we observed that MST does not directly control cell fate, MST may accelerate fetal brain development by delivering oRG daughters, including intermediate progenitor cells and their neuronal progeny, closer to their destinations in the cortical plate (Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (914) Google Scholar, Wang et al., 2011Wang X. Tsai J.W. LaMonica B. Kriegstein A.R. A new subtype of progenitor cell in the mouse embryonic neocortex.Nat. Neurosci. 2011; 14: 555-561Crossref PubMed Scopus (359) Google Scholar). Apically directed MST, along with other oSVZ progenitor cell behaviors not described in our study, may also function to reduce cell crowding. It is possible that discrepancies in oRG behaviors observed in macaques and humans reflect species-specific differences in MST function or a labeling bias in one or both studies. Alternatively, Betizeau and colleagues may have interpreted apically directed progenitor cell migration followed by division as MST due to a lower sampling frequency (one frame per 1–1.5 hr) than ours (one frame per 8–20 min). We wondered whether MST and other oRG-specific behaviors are affected in human neurodevelopmental disorders. Several genetic mutations that target the Rho-ROCK-myosin pathway lead to cortical malformations in humans that have historically been attributed to defective neuronal migration (Figure 4; Table S1). However, our finding that MST depends on this pathway suggests that MST may also be affected. Indeed, the expression patterns within the fetal human cortex of several cortical malformation candidate genes resemble the expression patterns of known radial glial genes, and hence of oRG cells, more closely than those of immature neuronal genes (Figure S3). oRG cells comprise only a small proportion of neural progenitor cells in mice as compared to humans (Wang et al., 2011Wang X. Tsai J.W. LaMonica B. Kriegstein A.R. A new subtype of progenitor cell in the mouse embryonic neocortex.Nat. Neurosci. 2011; 14: 555-561Crossref PubMed Scopus (359) Google Scholar), and this difference could help explain why mouse models of cortical malformations such as microcephaly, periventricular heterotopia, and lissencephaly often display relatively mild phenotypes (Table S1). Future studies may reveal that mutations that affect the Rho-ROCK-myosin pathway and have minimal or altered phenotypes when reproduced in mouse models primarily target MST and not neuronal migration in human patients. Fetal brain tissue was collected from elective pregnancy termination specimens at San Francisco General Hospital, and was transported in ice-chilled artificial cerebrospinal fluid (ACSF) to the laboratory for further processing. Tissues were collected only with previous patient consent and in strict observance of legal and institutional ethical regulations. Research protocols were approved by the Gamete, Embryo, and Stem Cell Research Committee (institutional review board) at University of California, San Francisco. See Supplemental Experimental Procedures for further details. Blocks of tissue from GW15–20 fetal dorsal cortex were imbedded in agarose, and 300 μm vibratome slices were generated and transferred to cortical slice culture medium containing CMV-GFP adenovirus. After labeling, slices were imaged using an inverted Leica TCS SP5 with an on-stage incubator at 15–25 min intervals for up to 6 days. Maximum intensity projections of the collected stacks were compiled and generated into movies, which were analyzed using Imaris. See Supplemental Experimental Procedures for further details. Dorsal cortical tissue was subjected to papain-based dissociation, and dissociated cells were plated at a density of 500,000–1,000,000 cells per well in matrigel-coated 12-well cell culture plates. Cultures were maintained in a Dulbecco’s modified Eagle’s medium-based dissociated culture medium. For cell fate and inhibitor experiments, cells were labeled with CMV-GFP adenovirus. For centrosome imaging experiments, cells were transfected with dsred-Cent2 (Cetn2) plasmid (Addgene plasmid 29523). Cultures were transferred to an inverted Leica TCS SP5 with an on-stage incubator and imaged using a ×10 objective at 8 min to 20 min intervals. See Supplemental Experimental Procedures for further details. Stock solutions of inhibitors were as follows: blebbistatin (100 mM in DMSO), nocodazole (2 mM in DMSO), Y-27632 (10 mM in DMSO), dimethylfasudil (10 mM in water), ML-7 (10 mM in DMSO), NiCl2 (1 M in water), ruthenium red (10 mM in DMSO), and 2-aminoethoxydiphenyl borate (2-APB) (Sigma; 10 mM in DMSO). Control treatment was 0.5% DMSO, which was greater than or equal to the final DMSO concentration for each inhibitor. See Supplemental Experimental Procedures for further details. See Supplemental Experimental Procedures for a detailed description of methods, which were standard procedures. Embryonic day 27 (E27) timed-pregnant ferrets were obtained from Marshall BioResources and maintained according to protocols approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco. E39 pregnant dams were deeply anesthetized with ketamine followed by isoflurane administration. Ovariohysterectomy for fetus collection was then performed and embryonic brains, along with meninges, removed in ice-chilled ACSF bubbled with 95% O2/5% CO2. The dorsal cortex was dissected away from ventral structures, imbedded in 3% low-melting-point agarose in ACSF, and sectioned using a vibratome to obtain 250–300 μm slices. Slices were transferred to cortical slice culture medium and treated as described for human slices, including labeling with Adeno-GFP and imaging using an inverted Leica TCS SP5 microscope. Maximum-intensity projections of the collected stacks were compiled and generated into movies, which were analyzed using Imaris. MST was defined as a translocation of greater than or equal to 20 μm (approximately one cell diameter) of the soma along the basal process (slice culture) or the primary process (dissociated culture), with a velocity of greater than or equal to 20 μm/hr, coinciding with cell rounding, and ending either in immediate cytokinesis or in a prolonged, rounded state. Angle with respect to the ventricular surface was measured, and trajectories were grouped in increments of 30°. A vector sum was computed to determine the overall trajectory of all MSTs. See Supplemental Experimental Procedures for further details. To examine the expression across brain regions of genes associated with human neurodevelopmental diseases, we used the BrainSpan laser microdissection and microarray profiling data set made available by the Allen Institute (BrainSpan, 2011BrainSpan. (2011). Atlas of the Developing Human Brain. Funded by ARRA awards 1RC2MH089921-01, 1RC2MH090047-01, and 1RC2MH089929-01. http://developinghumanbrain.org.Google Scholar). The data set was generated from four brains of ages GW17, 18, 23, and 23.5, which were cryosectioned, microdissected, and subjected to mRNA profiling by hybridization to custom Agilent microarrays. See Supplemental Experimental Procedures for further details. All quantifications were performed blind, and p values < 0.05 were considered statistically significant. See Supplemental Experimental Procedures for a detailed description of statistical methods. B.E.L.O. and A.R.K. designed the experiments; B.E.L.O. and J.H.L. carried out all experiments except those with ferret tissue and performed quantifications and analyses; C.C.G. performed ferret experiments; B.E.L.O. and A.R.K. wrote the initial manuscript; and all authors edited and approved the final manuscript. We thank A. Alvarez-Buylla, J. Chan, and members of the Kriegstein laboratory for thoughtful discussions and critical reading of the manuscript. We thank W. Walantus, Y.Y. Wang, and S. Wang for technical support. We thank Dr. J. Gleeson for use of the dsred-Cent2 (Cetn2) plasmid. We thank the staff at the San Francisco General Hospital for providing access to donated fetal tissue. The research was made possible by a grant from the California Institute for Regenerative Medicine (grant number TG2-01153). Additionally, this work was supported by Bernard Osher and by award number R01NS075998 from the NINDS. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of CIRM or any other agency of the State of California or of the NINDS or the NIH. Download .pdf (5.26 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Figures S1–S3, and Table S1https://www.cell.com/cms/asset/c2c1ff30-aa5b-4ca0-a823-ed993f8cdfa6/mmc2.mp4Loading ... Download .mp4 (3.9 MB) Help with .mp4 files Movie S1. oRG Cell MST in the Upper oSVZ Is Directed towards the Cortical Plate, Leading to Radial oSVZ Expansionhttps://www.cell.com/cms/asset/aef25541-6c94-477c-8823-51b7dffed497/mmc3.mp4Loading ... Download .mp4 (5.18 MB) Help with .mp4 files Movie S2. Side-by-Side Comparisons of Individual oRG Cells in Dissociated Human Fetal Progenitor Cultures Treated with DMSO versus Motor Protein Inhibitorshttps://www.cell.com/cms/asset/10236dad-64ec-44ae-bb94-5c8e81fb3a58/mmc4.mp4Loading ... Download .mp4 (7.39 MB) Help with .mp4 files Movie S3. Effects of Nocodazole Treatment of Dissociated Human Fetal Cortical Progenitor Culture, followed by Washouthttps://www.cell.com/cms/asset/9b22519b-2aa8-44eb-8b48-e5f72af2eaff/mmc5.mp4Loading ... Download .mp4 (1.77 MB) Help with .mp4 files Movie S4. Centrosome Localization during MST and Migration" @default.
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