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• W2153292012 abstract "Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture. Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture. The neural tube at embryonic day 8–8.5 (E8–8.5) in the mouse consists of a single pseudostratified epithelial layer of neuroepithelial stem cells (NESC) that undergo rapid symmetrical proliferative divisions as the neural tube grows and the progenitor pool is expanded. Neurogenesis begins at about E12 as the NESCs first undergo asymmetric divisions to generate one radial glial progenitor cell (RGPC) and one migratory postmitotic daughter neuron. The neocortex partitions into the proliferative ventricular zone (VZ), intermediate zone (IZ), cortical plate (CP), and marginal zone (MZ). Several embryonic lamina are established between E13 and E19 in the mouse in an “inside-out” pattern as neurons generated from the RGPCs in the VZ migrate through the IZ toward the CP. Similar events occur during the development of other areas of the brain such as the midbrain and hindbrain (reviewed in Gupta et al., 2002Gupta A. Tsai L.H. Wynshaw-Boris A. Life is a journey: a genetic look at neocortical development.Nat. Rev. Genet. 2002; 3: 342-355Crossref PubMed Scopus (295) Google Scholar). The maintenance of proliferative NESCs and their transition to RGPC at the onset of neurogenesis is fundamental to the formation of the brain (reviewed in Gotz and Huttner, 2005Gotz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Crossref PubMed Scopus (1426) Google Scholar). NESCs and RGPCs span the neural tube and/or brain, attached at the apical (ventricular) and basal (pial) surfaces. Both types of progenitors display interkinetic nuclear migration (Takahashi et al., 1993Takahashi T. Nowakowski R.S. Caviness Jr., V.S. Cell cycle parameters and patterns of nuclear movement in the neocortical proliferative zone of the fetal mouse.J. Neurosci. 1993; 13: 820-833PubMed Google Scholar, reviewed in Caviness et al., 2003Caviness Jr., V.S. Goto T. Tarui T. Takahashi T. Bhide P.G. Nowakowski R.S. Cell output, cell cycle duration and neuronal specification: a model of integrated mechanisms of the neocortical proliferative process.Cereb. Cortex. 2003; 13: 592-598Crossref PubMed Scopus (156) Google Scholar). Nuclei migrate from apical to basal surfaces in NESCs, while interkinetic nuclear migration in RGPCs is restricted to the VZ. Both types of progenitors display apical-basal polarity, with apical localization of the centrosome, PAR3, PAR6/atypical PKC (aPKC), and junctional proteins (reviewed in Gotz and Huttner, 2005Gotz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Crossref PubMed Scopus (1426) Google Scholar). NESCs are generated from symmetrical proliferative divisions perpendicular to the apical-basal axis in the single layer of the neuroepithelium (Chenn and McConnell, 1995Chenn A. McConnell S.K. Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis.Cell. 1995; 82: 631-641Abstract Full Text PDF PubMed Scopus (626) Google Scholar, Haydar et al., 2003Haydar T.F. Ang Jr., E. Rakic P. Mitotic spindle rotation and mode of cell division in the developing telencephalon.Proc. Natl. Acad. Sci. USA. 2003; 100: 2890-2895Crossref PubMed Scopus (183) Google Scholar). In the RGPCs that act as neuronal progenitors during neurogenesis as well as radial guides for migration of daughter postmitotic cells (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 (1454) 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 (686) Google Scholar), both symmetric and asymmetric divisions occur (Chenn and McConnell, 1995Chenn A. McConnell S.K. Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis.Cell. 1995; 82: 631-641Abstract Full Text PDF PubMed Scopus (626) Google Scholar, Haydar et al., 2003Haydar T.F. Ang Jr., E. Rakic P. Mitotic spindle rotation and mode of cell division in the developing telencephalon.Proc. Natl. Acad. Sci. USA. 2003; 100: 2890-2895Crossref PubMed Scopus (183) Google Scholar, Kosodo et al., 2004Kosodo Y. Roper K. Haubensak W. Marzesco A.M. Corbeil D. Huttner W.B. Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.EMBO J. 2004; 23: 2314-2324Crossref PubMed Scopus (325) Google Scholar). Symmetrical divisions in NESCs likely result in the symmetrical distribution of polarized apical components, while asymmetric divisions in RGPCs distribute components asymmetrically to produce one progenitor and one neuron or glial cell (reviewed in Gotz and Huttner, 2005Gotz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Crossref PubMed Scopus (1426) Google Scholar). The importance of mitotic cleavage plane during asymmetric cell division in RGPCs was demonstrated for NDE1 (Feng and Walsh, 2004Feng Y. Walsh C.A. Mitotic spindle regulation by Nde1 controls cerebral cortical size.Neuron. 2004; 44: 279-293Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), DCLK (Shu et al., 2006Shu T. Tseng H.C. Sapir T. Stern P. Zhou Y. Sanada K. Fischer A. Coquelle F.M. Reiner O. Tsai L.H. Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression.Neuron. 2006; 49: 25-39Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), Gβγ, and AGS3 (Sanada and Tsai, 2005Sanada K. Tsai L.H. G protein betagamma subunits and AGS3 control spindle orientation and asymmetric cell fate of cerebral cortical progenitors.Cell. 2005; 122: 119-131Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), but little is known about differences in the regulation of symmetric and asymmetric divisions in NESCs and RGPCs. Importantly, no genetic mutants disrupting symmetrical divisions in NESCs or distinguishing mechanisms of symmetric from asymmetric divisions have yet been identified. Here, in the course of studying its in vivo role, we identify Lis1 (also known as Pafah1b1) as a critical component in NESCs. Heterozygous loss or mutation of LIS1 is sufficient to cause the human neuronal migration defect lissencephaly (“smooth brain”), characterized by a smooth cortical surface, abnormal cortical layering, and enlarged ventricles (reviewed in Gupta et al., 2002Gupta A. Tsai L.H. Wynshaw-Boris A. Life is a journey: a genetic look at neocortical development.Nat. Rev. Genet. 2002; 3: 342-355Crossref PubMed Scopus (295) Google Scholar). We produced an allelic series of mice with varying doses of Lis1 generated from different combinations of two mutant alleles of Lis1 in mice (see Figure S1), a null knockout (Lis1ko) and a conditional knockout/hypomorphic allele (Lis1hc), and demonstrated in vivo Lis1-dosage-dependent neuronal migration defects (Hirotsune et al., 1998Hirotsune S. Fleck M.W. Gambello M.J. Bix G.J. Chen A. Clark G.D. Ledbetter D.H. McBain C.J. Wynshaw-Boris A. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat. Genet. 1998; 19: 333-339Crossref PubMed Scopus (469) Google Scholar, Gambello et al., 2003Gambello M.J. Darling D.L. Yingling J. Tanaka T. Gleeson J.G. Wynshaw-Boris A. Multiple dose-dependent effects of Lis1 on cerebral cortical development.J. Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar, 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 (345) Google Scholar). RNAi evidence also supports the role of LIS1 in neuronal migration in vivo (Shu et al., 2004Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning.Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, Tsai et al., 2005Tsai J.W. Chen Y. Kriegstein A.R. Vallee R.B. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.J. Cell Biol. 2005; 170: 935-945Crossref PubMed Scopus (307) Google Scholar). LIS1 also has a broader role in mammalian development (reviewed in Vallee and Tsai, 2006Vallee R.B. Tsai J.W. The cellular roles of the lissencephaly gene LIS1, and what they tell us about brain development.Genes Dev. 2006; 20: 1384-1393Crossref PubMed Scopus (124) Google Scholar). Lis1 null mice are peri-implantation lethal (Hirotsune et al., 1998Hirotsune S. Fleck M.W. Gambello M.J. Bix G.J. Chen A. Clark G.D. Ledbetter D.H. McBain C.J. Wynshaw-Boris A. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.Nat. Genet. 1998; 19: 333-339Crossref PubMed Scopus (469) Google Scholar, Cahana et al., 2001Cahana A. Escamez T. Nowakowski R.S. Hayes N.L. Giacobini M. von Holst A. Shmueli O. Sapir T. McConnell S.K. Wurst W. et al.Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization.Proc. Natl. Acad. Sci. USA. 2001; 98: 6429-6434Crossref PubMed Scopus (126) Google Scholar). Lis1+/ko and Lis1hc/ko mice exhibited defects in neurogenesis and interkinetic nuclear migration at the ventricular zone of the cortex, implicating LIS1 in neurogenesis (Gambello et al., 2003Gambello M.J. Darling D.L. Yingling J. Tanaka T. Gleeson J.G. Wynshaw-Boris A. Multiple dose-dependent effects of Lis1 on cerebral cortical development.J. Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar). LIS1 overexpression or disruption by antibody injection in cell culture supports a role for LIS1 in a variety of dynein-dependent mitotic functions (Faulkner et al., 2000Faulkner N.E. Dujardin D.L. Tai C.Y. Vaughan K.T. O'Connell C.B. Wang Y. Vallee R.B. A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function.Nat. Cell Biol. 2000; 2: 784-791Crossref PubMed Scopus (364) Google Scholar, Tai et al., 2002Tai C.Y. Dujardin D.L. Faulkner N.E. Vallee R.B. Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function.J. Cell Biol. 2002; 156: 959-968Crossref PubMed Scopus (203) Google Scholar). Finally, siRNA knockdown of Lis1 in rat cortical slice cultures resulted in defects in migration, interkinetic nuclear migration, and ventricular zone/intermediate zone defects in cell division (Tsai et al., 2005Tsai J.W. Chen Y. Kriegstein A.R. Vallee R.B. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.J. Cell Biol. 2005; 170: 935-945Crossref PubMed Scopus (307) Google Scholar). These and other studies suggest an important role for LIS1 in cell division and neurogenesis, but its precise role is not well defined. We directly demonstrate an essential and surprising role for Lis1 in the neuroepithelium at times prior to the onset of radial glial neurogenesis and neuronal migration as well as in mitotic spindle orientation and symmetric division at the apical surface of cells. This effect appears to result from a requirement for LIS1 in dynein-mediated cortical microtubule capture. This novel function for LIS1 during mitosis and neurogenesis reveals a unique and critical role for the control of vertical spindle orientation and symmetric cleavage during neuroepithelial expansion of NESCs and supports the established role of spindle orientation in RGPC neurogenesis. To examine the role of Lis1 during progenitor expansion and neurogenesis, we ablated Lis1 in restricted spatial and/or temporal patterns in the developing brain using a Lis1 conditional-hypomorphic knockout allele and compared the phenotype of littermates heterozygous or homozygous for Lis1hc with various Cre transgenes (Figure S1). The Rosa26-lacZ Cre reporter (Soriano, 1999Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat. Genet. 1999; 21: 70-71Crossref PubMed Scopus (4029) Google Scholar) marked Cre-exposed cells. Pax2-Cre transgenic mice express Cre predominantly at the midbrain-hindbrain junction starting at E8.0 in the neuroepithelium prior to the onset of neuronal migration (Lewis et al., 2004Lewis P.M. Gritli-Linde A. Smeyne R. Kottmann A. McMahon A.P. Sonic hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum.Dev. Biol. 2004; 270: 393-410Crossref PubMed Scopus (232) Google Scholar). Lis1hc/hc; Pax2-Cre; Rosa26 pups did not survive past P0. Strikingly, the midbrain, midbrain-hindbrain junction, and the cerebellum were absent in nearly all (n = 10) Lis1hc/hc; Pax2-Cre; Rosa26 mice, resulting in the separation of the forebrain from the hindbrain at birth (data not shown). In whole embryos at E9.5, there was a small but noticeable reduction in X-gal staining in the branchial arches (Figures 1A, 1B, 1E, and 1F, arrows) and the midbrain-hindbrain region (Figures 1A, 1B, 1E, and 1F; arrowheads) of Lis1hc/hc; Pax2-Cre; Rosa26 compared with Lis1+/hc; Pax2-Cre; Rosa26 embryos. The majority of the X-gal positive cells in the midbrain-hindbrain but not the X-gal negative cells in the telencephalon, underwent apoptotic cell death in the Lis1hc/hc; Pax2-Cre; Rosa26 embryos (Figures 1G and 1H), whereas control embryos displayed negligible numbers of apoptotic cells (Figures 1C and 1D). Wnt1-Cre (Chai et al., 2000Chai Y. Jiang X. Ito Y. Bringas Jr., P. Han J. Rowitch D.H. Soriano P. McMahon A.P. Sucov H.M. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis.Development. 2000; 127: 1671-1679Crossref PubMed Google Scholar) is expressed at the midbrain-hindbrain junction and in the telencephalon at about E8–8.5 (see Figures 1I and 1J). The broader expression pattern of Wnt1-Cre resulted in severe neuronal loss in the telencephalon as well as in the midbrain-hindbrain junction of Lis1hc/hc; Wnt1-Cre; Rosa26 embryos and death at about E13.5. At E10.5, the telencephalon of Lis1hc/hc; Wnt1-Cre; Rosa26 embryos was significantly smaller and degenerated compared to control (compare Figures 1I and 1J with 1M and 1N), and degeneration of the telencephalon and midbrain was observed at E11.5 (compare Figures 1K and 1L with 1O and 1P). These acute phenotypes were observed in neuronal progenitor cells in the neuroepithelium prior to the onset of RGPC neurogenesis and neuronal migration, demonstrating that LIS1 is essential during neuroepithelial expansion of NESCs in the midbrain-hindbrain junction as well as the telencephalon. To determine whether LIS1 plays an important role during RGPC neurogenesis as well as NESC expansion in the neuroepithelium, we used the hGFAP-Cre transgenic mouse to inactivate Lis1 (Zhuo et al., 2001Zhuo L. Theis M. Alvarez-Maya I. Brenner M. Willecke K. Messing A. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo.Genesis. 2001; 31: 85-94Crossref PubMed Scopus (452) Google Scholar). hGFAP-Cre expression begins at E12.5–13.5 in distinct developmental stages in neuronal precursor cells of the neocortex and hippocampus (Malatesta et al., 2003Malatesta P. Hack M.A. Hartfuss E. Kettenmann H. Klinkert W. Kirchhoff F. Gotz M. Neuronal or glial progeny: regional differences in radial glia fate.Neuron. 2003; 37: 751-764Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). hGFAP-Cre is active in the RGPCs of virtually all radially migrating cells in the neocortex, but not in the neocortical neuroepithelium prior to E12.5. By contrast, hGFAP-Cre is expressed in the neuroepithelium in the hippocampal region at E12.5, a time when only immature mitotic NESCs are present. If Lis1 is critical in NESCs and RGPC, then the phenotype should be similarly severe in both the hippocampus and cortex, but if Lis1 is more important in NESCs than RGPCs, then the phenotype should be more severe in the hippocampus than the cortex. In wild-type Lis1+/+; hGFAP-Cre; Rosa26 (Figures 1Q–1T) neonates, the cortex and hippocampus were well developed. Lis1hc/hc; hGFAP-Cre; Rosa26 neonates survived to P5, but were completely missing the hippocampus (Figures 1U–1X, see arrows in Figure 1V and 1W). The cortex of these neonates was present, although thinner than control animals. Nearly all cells present in the cortex had some blue staining, indicating that Cre was present in these cells (data not shown). A few apoptotic cells were observed in the cortex of Lis1hc/hc; hGFAP-Cre; Rosa26 embryos (data not shown) in contrast to the massive and acute apoptosis observed when Lis1 was deleted during NESC expansion. These studies demonstrate an immediate and essential role for Lis1 in dividing neural progenitor cells of the neuroepithelium, while the loss of Lis1 in radial glial progenitors undergoing neurogenesis and neuronal migration have less immediate consequences for survival. To identify factors that differentiate NESCs and RGPCs (reviewed in Gotz and Huttner, 2005Gotz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Crossref PubMed Scopus (1426) Google Scholar), we determined whether LIS1 affects apical-basal polarity and/or mitotic spindle orientation during neuroepithelial expansion and/or radial glial neurogenesis. Apical determinants such as centrosomes (marked by pericentrin), β-catenin, aPKC, Numb, and the cadherin hole (Kosodo et al., 2004Kosodo Y. Roper K. Haubensak W. Marzesco A.M. Corbeil D. Huttner W.B. Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.EMBO J. 2004; 23: 2314-2324Crossref PubMed Scopus (325) Google Scholar) were apically located in wild-type or Lis1+/ko or Lis1hc/ko mutant embryos with 50% or 35% of wild-type LIS1 protein, respectively (see Gambello et al., 2003Gambello M.J. Darling D.L. Yingling J. Tanaka T. Gleeson J.G. Wynshaw-Boris A. Multiple dose-dependent effects of Lis1 on cerebral cortical development.J. Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar), in the neuroepithelium (E9.5; Figures 2A, 2B, S2, and S3) and radial glia (E14.5; Figures 2C, 2D, S2, and S3), although not as tightly localized to the apical surface of mutant embryos in a Lis1 dosage-dependent fashion. This may be due to the less-organized cellular structure from long-term deficiencies of LIS1 as indicated by nestin (Figures 2A, 2B, and S2). Of note, the cadherin hole was present at the apical surface of wild-type and all mutant embryos (Figures 2A–2D, S2, and S3) consistent with the maintenance of apical polarity. Nuclei undergoing interkinetic nuclear migration are elongated and distributed throughout the apical-basal axis, as seen in wild-types (Figures 2A and 2C), but in Lis1 mutants, the nuclei were round and accumulated near the apical surface (Figures 2B and 2D), consistent with previously described interkinetic nuclear migration defects (Gambello et al., 2003Gambello M.J. Darling D.L. Yingling J. Tanaka T. Gleeson J.G. Wynshaw-Boris A. Multiple dose-dependent effects of Lis1 on cerebral cortical development.J. Neurosci. 2003; 23: 1719-1729Crossref PubMed Google Scholar, Shu et al., 2004Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning.Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, Tsai et al., 2005Tsai J.W. Chen Y. Kriegstein A.R. Vallee R.B. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.J. Cell Biol. 2005; 170: 935-945Crossref PubMed Scopus (307) Google Scholar). Symmetrical apical mitotic cell divisions display vertically oriented cleavage planes (90° relative to the plane of the apical surface) to generate two proliferative daughter progenitor cells attached at the apical and basal surfaces, while asymmetric divisions generate one proliferative daughter cell and a postmitotic neuron or glial cell. At E9.5, all wild-type anaphase neuroepithelial cells in the neural tube displayed nearly vertical (symmetric) cleavage (Figures 2A and 2I; average angle: 80.72° ± 9.56°), while the majority of anaphase cells in wild-type cortical slices at radial glial stages (E14.5) displayed less perpendicular spindle cleavage planes with more variability (Figures 2C and 2I; average angle: 70.15° ± 18.3°, p < 0.016). In neural tubes at E9.5 and in cortex at E14.5 from Lis1+/ko or Lis1hc/hc embryos, spindle cleavage planes were more randomized and more horizontal to the ventricular surface than WT, with the degree of randomization proportional to the reduction of LIS1 (Figures 2B, 2D, and 2I). This suggests that disruption of the orientation of apical spindle cleavage planes seen with reduction in LIS1 levels results in a severe and catastrophic disruption in the neuroepithelium during NESC expansion when cleavage plane is tightly controlled. During radial glial neurogenesis, spindle orientation defects result in a change of progenitor cell fate, reducing the progenitor pool population early in cortical development and decreasing the total number of cortical neurons. Consistent with this, there are reduced numbers of Tst-1-positive layer 2 cortical neurons (Figure 1X, arrow) and neurons marked with a series of layer specific markers (data not shown) in Lis1hc/hc; hGFAP-Cre; Rosa26 neonates. To delineate the temporal sequence of events that occurred after acute loss of Lis1, we used a tamoxifen-inducible Cre (Cre-ER™, Hayashi and McMahon, 2002Hayashi S. McMahon A.P. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.Dev. Biol. 2002; 244: 305-318Crossref PubMed Scopus (946) Google Scholar) and the Tg(ACTB-bgeo/DsRed.MST) Cre reporter (Vintersten et al., 2004Vintersten K. Monetti C. Gertsenstein M. Zhang P. Laszlo L. Biechele S. Nagy A. Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.Genesis. 2004; 40: 241-246Crossref PubMed Scopus (239) Google Scholar), which switches from beta-geo to DsRed.MST expression after Cre recombination. We injected pregnant dams with tamoxifen (2 mg/40 g mouse) to activate Cre at E8.5 and examined the phenotypes of Lis1hc/hc; Cre-ER™ experimental and Lis1hc/hc control embryos without Cre from the same litter 12, 24, and 36 hr later. Within 12 hr of Cre activation, vertical spindle orientation was disrupted (Figures 2E, 2F, and 2J). This immediate disruption of spindle orientation was associated with an increase in apoptosis (Figure 2K) and metaphase arrest with mitotic cells located away from the apical surface (phospho-H3 histone, Figure 2L), without disruption of either apical polarity or epithelial integrity (Figures 2E, 2F, S2, and S4). At later time points, 24 and 36 hr after Cre activation, the spindle, apoptosis, and metaphase arrest phenotypes progressed further, with only minimal disruption of apical polarity and epithelial integrity (Figures 2G, 2H, 2J–2L S2, and S4). These findings indicate that the immediate effect of the loss of Lis1 is the disruption of the orientation of apical spindle cleavage planes and proliferation, resulting in a severe and catastrophic disruption in the neuroepithelium during NESC expansion when cleavage plane is tightly controlled. To examine cell-cycle progression and spindle organization in vitro, mouse embryonic fibroblasts (MEFs) with decreasing doses of LIS1 were generated from mice with combinations of the Lis1ko and Lis1hc hypomorphic alleles (see Figure S1), including Lis1hc/hc; Cre-ER™ MEFs to analyze the effects of complete loss of LIS1. Lis1+/ko and Lis1hc/ko MEFs displayed 50% (data not shown), 35% of wild-type LIS1, while Lis1hc/hc; Cre-ER™ MEFs 0, 24, 48, and 72 hr after tamoxifen treatment (hereafter termed Lis124h, Lis148h and Lis172h) displayed 80%, 52%, 25%, and <10%, respectively, of wild-type levels by western blot (Figure 3A). The doubling time of Lis1hc/ko, Lis1+/ko and wild-type MEFs was 38, 26, and 18 hr, respectively (Figure 3B). There was a significant decrease in growth of Lis124h cells, a nearly normal increase Lis148h cells, and a plateau thereafter (Figure 3C). No effect of tamoxifen treatment on cell growth was seen with treatment of Lis1hc/hc MEFs without the Cre-ER™ transgene (data not shown). Cell death did not significantly increase in tamoxifen-treated versus untreated cells (data not shown). Mitotic index was 4.0% ± 0.14%, 3.6% ± 0.10%, 2.2% ± 0.12% for wild-type, Lis1+/ko, and Lis1hc/ko cells, respectively, while in Lis24h and Lis48h cells it was 13.1% ± 0.09% and 1.9 ± 0.08%. Doubling time and mitotic index were significantly different among groups (p < 0.05). There was a block in mitosis from a mitotic delay in prometaphase at 24 hr (10.0% ± 0.09% versus 4.0% ± 0.11% in control cells), which disappeared at 48 and 72 hr (Figure 3D, data not shown), similar to findings from LIS1 depletion experiments (Faulkner et al., 2000Faulkner N.E. Dujardin D.L. Tai C.Y. Vaughan K.T. O'Connell C.B. Wang Y. Vallee R.B. A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function.Nat. Cell Biol. 2000; 2: 784-791Crossref PubMed Scopus (364) Google Scholar). Tamoxifen-treated Cre-ER™; Lis1hc/hc MEFs underwent one cell division and then senesced or moved more slowly through all phases of the cell cycle, since there was little increase in prometaphase cells at 48 and 72 hr. MEFs with decreased LIS1 levels displayed severely shortened and sparse astral microtubules (MTs), using an EB1 antibody to label growing MT plus ends (Figures 3E–3G), an effect on either MT growth or stability proportional to the amount of LIS1. LIS1 reduction had no affect on the formation of bipolar spindles, spindle pole maintenance, or number (Figures 3H–3J). Prometaphase spindles of LIS1 mutant MEFs were smaller and slightly disorganized with fewer MTs (Figures 3I and 3J), and a few cells in anaphase and telophase were found. Metaphase spindles of Lis1hc/ko (Figure 3M), Lis124h, and Lis148h (data not shown) MEFs treated with MG-132 (to provoke metaphase arrest) looked similar to bipolar spindles of wild-type MEFs (Figure 3K) but were smaller (Figures 3Q and 3R). Wild-type and Lis1hc/ko MEFs were treated with MG-132 for 3 hr and then placed on ice for 1 hr to depolymerize unattached MTs. Lis1hc/ko MEFs did not display a significant decrease in stabilized spindle MTs (Figure 3N) compared with wild-type (Figure 3L). Lis148h spindles had less tightly aligned chromosomes compared with wild-type and other Lis1 mutants (Figures 3O–3R), suggesting impaired chromosome congression. No lagging chromosomes or chromosomal bridges during anaphase were found in wild-type (Figures 3S and 3T), but in Lis124h and Lis148h MEFs, many lagging chromosomes at anaphase were seen (10/24, Figures 3U and 3V) with a corresponding increase in micronuclei in the general population (72 hr, 51% ± 3.3% compared to wild-type 4.5% ± 1.2%, data not shown). Similar results were found in proliferation and mitosis in undifferentiated and proliferating neural progenitor cells (NPCs) from Lis1hc/hc; Cre-ER™; Rosa26 (Lis1hc/hc NPCs) mice after tamoxifen treatment to induce inactivation of Lis1 (Figure S5), but the scant cytoplasm of NPCs precluded examination o" @default.
• W2153292012 created "2016-06-24" @default.
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• W2153292012 date "2008-02-01" @default.
• W2153292012 modified "2023-10-15" @default.
• W2153292012 title "Neuroepithelial Stem Cell Proliferation Requires LIS1 for Precise Spindle Orientation and Symmetric Division" @default.
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