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Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism

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Developmental Biology 302 (2007) Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous
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Developmental Biology 302 (2007) Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism Jane C. Quinn a, Michael Molinek a, Ben S. Martynoga a, Paulette A. Zaki a, Andrea Faedo b,1, Alessandro Bulfone b, Robert F. Hevner c, John D. West d, David J. Price a, a Genes and Development Group, Department of Biomedical Sciences, Centres for Integrative Physiology and Neuroscience Research, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK b Stem Cell Research Institute, Dibit, H. S. Raffaele, Via Olgettina 58, Milan, Italy c Department of Pathology, University of Washington, Seattle, Washington, WA 98104, USA d Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK Received for publication 7 June 2006; revised 11 August 2006; accepted 15 August 2006 Available online 22 August 2006 Abstract Many cerebral cortical neurons and glia are produced by apical progenitors dividing at the ventricular surface of the embryonic dorsal telencephalon. Other neurons are produced by basal progenitor cells, which are derived from apical progenitors, dividing away from the ventricular surface. The transcription factor Pax6 is expressed in apical progenitors and is downregulated in basal progenitors, which upregulate the transcription factor Tbr2. Here we show that Pax6 / cells are under-represented in the cortex of Pax6 +/+ Pax6 / chimeras early in corticogenesis, indicating that Pax6 is required for the production of normal numbers of cortical cells. We provide evidence that this underproduction is attributable to an early depletion of the progenitor pool caused by greater than normal proportions of newly divided cells exiting the cell cycle. We show that most progenitor cells dividing away from the ventricular surface in Pax6 / embryos fail to express the transcription factor Tbr2 and that Pax6 is required cell autonomously for Tbr2 expression in the developing cortex of Pax6 +/+ Pax6 / chimeras. Transcription factors normally expressed ventrally in the telencephalic ganglionic eminences (Mash1, Dlx2 and Gsh2) are upregulated cell autonomously in mutant cells in the developing cortex of Pax6 +/+ Pax6 / chimeras; Nkx2.1, which is expressed only in the medial ganglionic eminence, is not. These data indicate that early functions of Pax6 in developing cortical cells are to repress expression of transcription factors normally found in the lateral ganglionic eminence, to prevent precocious differentiation and depletion of the progenitor pool, and to induce normal development of cortical basal progenitor cells Elsevier Inc. Open access under CC BY license. Keywords: Chimera; Pax6; Proliferation; Telencephalon; Mouse; Tbr2; Mash1; Nkx2.1; Gsh2; Dlx2; Apical progenitor cell; Basal progenitor cell Introduction Correct development requires regulation of the number of cells and the types of cell produced in each region. Regulating Corresponding author. Fax: address: (D.J. Price). 1 Current address: Nina Ireland Laboratory of Developmental Neurobiology, Center for Neurobiology and Psychiatry Genetics, Development and Behavioral Sciences Building, th Street, University of California at San Francisco, San Francisco, CA , USA. the numbers of postmitotic cells generated in the cortex requires control of two key aspects of proliferation: (i) the length of the cell cycle and (ii) the proportion of newly generated cells that re-enter the cell cycle as opposed to leaving it to differentiate. A number of cell cycle regulators and transcription factors, including Pax6, have been implicated in the control of these processes (Calegari and Huttner, 2003; Estivill-Torrus et al., 2002; Heins et al., 2002; Iacopetti et al., 1999; Roy et al., 2004). Two types of progenitor cell exist in the developing neocortex. Radial glia, also known as apical progenitor cells (APCs), divide at the ventricular Elsevier Inc. Open access under CC BY license. doi: /j.ydbio J.C. Quinn et al. / Developmental Biology 302 (2007) surface either symmetrically, giving rise to two mitotic offspring, or asymmetrically to produce one mitotic and one postmitotic daughter [for review see Fishell and Kriegstein, 2003]. A second proliferative population, derived from the APCs, forms in the subventricular zone: the majority of these cells divide symmetrically to produce two postmitotic neurons, and they have been designated non-surface dividing cells or basal progenitor cells (BPCs) (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004; Smart, 1976; Takahashi et al., 1995b). BPCs are thought to generate many of the neurons in the upper cortical layers (Altman and Bayer, 1990; Tarabykin et al., 2001). Pax6 is expressed in APCs and is downregulated in BPCs (Englund et al., 2005) and, in the present study, we addressed unanswered questions about the functions of Pax6 in the development of these cell types so as to generate a coherent model of the early functions of Pax6 in cortical development. First, we examined how the overall production of Pax6 / cortical cells compared to that of Pax6 +/+ cells early in corticogenesis. The fact that the dorsal telencephalon of Pax6 / embryos is smaller than that of wild types is not sufficient evidence for underproduction since it does not exclude the possibility that cells are more densely packed in the mutants, which is certainly the case in the later stages of corticogenesis (Caric et al., 1997; Kroll and O'Leary, 2005; Schmahl et al., 1993). We examined the production of Pax6 / cells in the cortex of Pax6 +/+ Pax6 / chimeras, allowing us to compare the numbers of cells with the two genotypes in the same animals and to test whether abnormalities persist even in the presence of wild-type cells, i.e., whether they likely reflect a cell autonomous requirement for Pax6. The results showed reduced production of mutant cells in our chimeras. We then investigated whether Pax6 is required to prevent excessive cell death, to regulate the length of the cortical progenitor cell cycle or to control the proportion of newly generated cells that re-enter the cell cycle as opposed to leaving it to differentiate. We found that the last of these parameters was altered in the Pax6 / cortex, indicating that Pax6 expression is required to maintain the size of the cortical progenitor pool. Next, we examined the BPCs in Pax6 / embryos. A recent study (Englund et al., 2005) showed that BPCs express the transcription factor Tbr2. The number of progenitors dividing away from the ventricular zone (or abventricularly) is increased in Pax6 / mutants (Estivill-Torrus et al., 2002; Haubst et al., 2004).We tested whether these cells resemble normal BPCs in expressing Tbr2 and found that the majority of abventricular mitoses in the Pax6 / mutant cortex did not express Tbr2. Since Pax6 is normally expressed in APCs and downregulated in BPCs, we determined whether Pax6 is required cell autonomously for Tbr2 expression using Pax6 +/+ Pax6 / chimeras. The dorsal telencephalon of Pax6 / mutants becomes progressively ventralized throughout corticogenesis and this is due to a change in the fate of dorsal telencephalic progenitors (Kroll and O'Leary, 2005). What remains unclear is whether this fate change is a direct cell autonomous consequence of the loss of Pax6 in cortical progenitors or whether it results indirectly from a loss of Pax6 in interacting cells. We addressed this issue by examining the expression of ventral genes in mutant cells in the cortex of Pax6 +/+ Pax6 / chimeras. Methods Production of Pax6 +/+ Pax6 / chimeras Chimeras used to estimate the numbers of mutant cells contributing to the cortex were produced as described in Quinn et al. (1996). In brief, eight-cell embryos were obtained from the parental cross Pax6 Sey-Neu/+, Gpi1 b/b female Pax6 +/Sey, Gpi1 b/b, Tg/Tg male, where Tg denotes the presence of the reiterated β-globin transgene TgN(Hbb-b1)83Clo (Keighren and West, 1993; Lo et al., 1987). Embryos of the following four genotypes were obtained from this parental cross: Pax6 +/+, Pax6 Sey-Neu/+, Pax6 +/Sey and Pax6 Sey-Neu/Sey, all of which were Gpi1 b/b and contained a single copy of the β-globin transgene (Tg+). Donor embryos for aggregation were obtained from (BALB/c x A/J) F 2 intercrosses, producing embryos that were Pax6 +/+, Gpi1 a/a and negative for the β-globin transgene (Tg ). Embryos were collected from superovulated females at 2.5 days post coitum and aggregated according to West and Flockhart (1994). Aggregated embryos were cultured overnight, transferred to recipient pseudopregnant F1 females (Pax6 +/+, Gpi1 c/c,tg ). To increase the efficiency of return of Pax6 +/+ Pax6 / chimeras for subsequent studies of the identities of Pax6 / cells, we derived Pax6 / mutant embryonic stem (ES) cells from mice on an inbred 129/Sv background that were hemizygous for the β-globin transgene TgN(Hbb-b1)83Clo (Keighren and West,1993;Loet al.,1987) (designated 129SeyD). 129SeyD1 ES cells (Pax6 /, Gpi1 a/a, Tg+) were injected into (C57Bl/6 x CBA/Ca) F 2 intercross blastocysts (Pax6 +/+, GpiI b/b, Tg ), which were transferred to the uterus of a pseudopregnant recipient and allowed to develop to the appropriate embryonic stage. Fetuses were dissected into cold PBS and staged according to forelimb development (Palmer and Burgoyne, 1991; Wanek et al., 1989). Tail and forelimb samples were taken for analysis of glucose phosphate isomerase (GPI1) isotype contribution to give a proportion of global chimerism for each embryo (West and Flockhart, 1994). A mean percentage GPI1 was determined to give the proportion of cells in the chimera derived from the Pax6 Sey-Neu/+ Pax6 +/Sey 8-cell embryo or 129SeyD1 ES cells. The genotype of each chimera was determined by PCR and restriction digest analysis of genomic DNA as described previously (Quinn et al., 1996). The use of two predicted null mutant Pax6 alleles, Pax6 Sey and Pax6 Sey-Neu (Hill et al., 1991), allowed distinction between aggregation chimeras containing Pax6 Sey-Neu/Sey compound heterozygous (described here as Pax6 / ) cells and those containing Pax6 Sey-Neu/+ or Pax6 +/Sey heterozygous (described as Pax6 +/- ) cells. Chimeric embryos obtained by ES cell injection were genotyped for the presence of the Pax6 Sey allele alone. Histological visualization of cells derived from the Pax6 Sey-Neu/+ Pax6 +/Sey embryos or from 129SeyD1 ES cells was achieved by DNA DNA in situ hybridization using a digoxigeninlabeled probe to the reiterated β-globin transgene (Keighren and West, 1993; Quinn et al., 1996). Detection of in situ signal was achieved either with peroxidase-labeled antibody visualized with diaminobenzidine (DAB) (Keighren and West, 1993) or by reaction with an anti-digoxigenin rhodamine antibody (Roche). We observed no phenotypic differences between Pax6 +/+ Pax6 / chimeras obtained by aggregation or ES cell injection. Quantitative analysis of contribution of Pax6 / cells to Pax6 +/+ Pax6 / chimeras Percentages of Tg+ cells in various regions of the forebrain and other tissues of E12.5 chimeras were measured in a minimum of three non-consecutive sections, with cells counted per area per section, depending on tissue. As in previous studies (Quinn et al., 1996), observed percentages for each tissue (O) were corrected to allow for failure to identify Tg+ signals in all Tg+ cells due to sectioning artefact. To generate tissue-specific correction factors (c), percentages of Tg+ nuclei in Pax6 +/+, Tg+ embryos (i.e., non-chimeric embryos in which all cells should be Tg+) were counted at E12.5. Corrected observed 52 J.C. Quinn et al. / Developmental Biology 302 (2007) percentages of Tg+ cell contribution (Oc) for each chimeric tissue were then divided by the percentage of cells expected in that tissue (E) if the percentage was to equal the global percentage chimerism estimated by GPI1B analysis (Oc/E). Values were compared by Student's t-test. Production of Pax6 / mice All non-chimeric mouse embryos designated Pax6 / were derived from Pax6 Sey heterozygote crosses maintained on an inbred Swiss background. Wildtype siblings were used as controls. The day of the vaginal plug following mating was designated E0.5. Pregnant females were killed by cervical dislocation. Fetuses were dissected from the uterus at the required gestational age before fixing and processing to either wax or plastic sections. Wax sections were cut at 10 μm and plastic sections at 5 μm. Quantitative analysis of cell densities and cortical depth In all cases, images were captured using Leica NTS confocal microscopy. Areas for cell density analysis were defined using Image-Tool. Cells were counted on a minimum of 5 non-consecutive sections at E12.5. Cell counts/ densities were compared using Sigmastat. Cortical depths were measured in the center of the neocortex using Image Tool and data compared using Sigmastat. Studying cell cycle with iododeoxyuridine (IdU) and bromodeoxyuridine (BrdU) On embryonic day E10.5 or E12.5, IdU was injected i.p. into pregnant dams followed by BrdU injection (both at 70 μg/g body weight) 1.5 h later (Fig. 2A). Dams were killed at 2.0 h after the first injection; embryos were fixed, sectioned and processed to reveal IdU/BrdU using mouse monoclonal anti-brdu (Becton Dickson Ltd., UK), which recognized both IdU and BrdU, in conjunction with rat monoclonal anti-brdu (Abcam Ltd., UK) which recognized BrdU alone. Directly conjugated AlexaFluor secondary antibodies were anti-mouse AlexaFluor 488 and anti-rat AlexaFluor 568. Images were captured using Leica NTS confocal microscope, viewed using LCS Lite (Leica) and imported into Adobe Photoshop for counting. Proportions of IdU/BrdU-labeled cells in the ventricular zone of telencephalon were counted in a minimum of three (E10.5) or five (E12.5) nonadjacent sections from each embryo. Cell cycle lengths were calculated using the following paradigm (Martynoga et al., 2005): cells in the initial IdUlabeled cohort that leave S-phase during the interval between IdU and BrdU (T i =1.5 h), designated the leaving fraction (L cells ), will be labeled with IdU but not BrdU. The proportion of cells labeled with BrdU is designated S cells. The length of S-phase (Ts) can be calculated using the formula: Ts 1:5 ¼ S cells L cells and the length of the cell cycle (Tc) estimated from the formula: Tc Ts ¼ P cells S cells where P cells is the total number of proliferating cells in the ventricular zone (VZ) (Martynoga et al., 2005). Since previous studies have shown that a prolonged pulse of BrdU will label virtually all VZ cells at E12.5, in both wild-type and Pax6 / embryos, P cells was estimated by counting all VZ cells (Estivill-Torrus et al., 2002). Cumulative BrdU analyses BrdU was given (70 μg/g body weight, i.p.) to E12.5 pregnant dams either once or every 2 h over a 12-h period. Dams were killed 0.5 and 12.5 h after the first injection; embryos were fixed, sectioned and processed to reveal BrdU as described previously (Gillies et al., 1990). The relative intensity of BrdU label in the nucleus of each BrdU-labeled cell in three 200-μm-wide strips through the cortex, in three non-adjacent sections from each embryo, was measured using a Leica digital camera and QWin (Leica) software. Immunocytochemistry on cortical cells Cells from E12.5 neocortex of Pax6 +/+ and Pax6 / embryos were dissociated using papain as per manufacturer's instructions (Papain Dissociation System, Worthington Biochemicals, UK) and stained for β-tubulin isotype III (mouse monoclonal IgG2b, 1:100, Sigma, UK). Visualization was achieved using directly conjugated AlexaFluor 488 (goat anti-mouse IgG, 1:200) or AlexaFluor 546 (goat anti-mouse IgG, 1:200) (Molecular Probes, Inc.). For cell counts, viable cells per culture were assessed in six randomly selected microscope fields. Flow cytometric analysis of cortical cells Cortices were dissociated as above and fixed in ice cold 70% ethanol. Cortical tissue was collected from E12.5 and E14.5 embryos from 4 separate litters and a minimum of 3 individuals of each genotype pooled at dissection. Dissociated cells were stained for β-tubulin isotype III (1:800); primary antibody binding was revealed using directly conjugated AlexaFluor 488 (1:800) as above. Cells were then stained with propidium iodide (PI) to allow discrimination of single cells and analysis of DNA content. Staining reactions were carried out in duplicate. Cells were analyzed on a Beckman-Coulter XL flow cytometer with Expo32 software (Beckman-Coulter, Inc.) ,000 cells were analyzed per sample. Cell death analysis Cells from E12.5 neocortex of Pax6 +/+ and Pax6 / fetuses were dissociated and cultured for 24 h as described previously (Estivill-Torrus et al., 2002). Cultures contained cells of either genotype alone or both genotypes mixed, with one set of cells stained using PKH26 fluorescent cell linker (Sigma) according to the manufacturer's instructions. Cells were fixed with 4% paraformaldehyde and visualized for localization of activated caspase-3 using rabbit polyclonal anticaspase-3 antibody (Chemicon International; 1:100). Secondary amplification was with anti-rabbit biotin-conjugated antibody (DAKO; 1:200) and visualization was with streptavidin-conjugated AlexaFluor 488 (Molecular Probes; 1:200). Cells were counterstained with bisbenzimide. Apoptotic cells were identified either by immunoreactivity for activated caspase-3 activity (Lesuisse and Martin, 2002; Srinivasan et al., 1998) and/or nuclear chromatin condensation, as described previously (Kerr et al., 1972). Cell counts were done in six randomly selected microscope fields per culture. Microarray hybridization Total RNA from E14.5 neocortex was isolated using a method based on guanidinium lysis and phenol chloroform extraction (ToTALLY RNA, Ambion). Labeling of total RNA was performed using the dendrimer technology (3DNA Submicro Expression Array Detection Kit, Genisphere). The cdna was then hybridized to a cdna chip representing 1026 different genes of the TESS subtractive cdna library (Faedo et al., 2004) which had been generated by subtracting genes expressed in adult telencephalon from those expressed in E14.5 cortex (Porteus et al., 1992). Differential gene expression was assessed by scanning the hybridized arrays as described previously (Faedo et al., 2004). Changes in Tbr2 gene expression were confirmed at a protein level by Western blotting for Tbr2 protein expression using standard protocols. Equal loading of lanes was confirmed using β-actin immunostaining. Immunohistochemistry Embryos were fixed overnight in 4% paraformaldehyde/pbs and processed to wax. All embryos were sectioned in the coronal plane. Slides were microwaved in 10 mm sodium citrate to achieve maximal antigen retrieval before addition of primary antibody. Antibodies used were mouse monoclonal anti-β-tubulin isotype III (Sigma, UK), mouse anti-phosphohistone 3 (Abcam Ltd., UK), rabbit polyclonal Tbr2 (Englund et al., 2005), mouse anti-mash1 (BD Biosciences), rabbit anti-dlx2 antibody (Abcam Ltd., UK), anti-gsh2 (Toresson et al., 2000), anti-nkx2.1 (Biopat) and directly conjugated AlexaFluor secondary antibodies (Molecular Probes). Nuclei were stained with TOPRO3 J.C. Quinn et al. / Developmental Biology 302 (2007) (Molecular Probes). Where necessary, signal amplification was achieved using either Dako ABC or Dako Envision Kit prior to staining with DAB. Results Cortical thickness is reduced at E12.5 in the Pax6 / mutant embryo A reduction in cortical thickness is a reported feature of the homozygous Pax6 / telencephalic phenotype during mid-late corticogenesis (Caric et al., 1997; Fukuda et al., 2000; Haubst et al., 2004; Schmahl et al., 1993; Warren et al., 1999). To determine whether reduction had occurred by E12.5, we measured thickness in the cent
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