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First published online August 20, 2008
doi: 10.1242/10.1242/jcs.023465

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Making bigger brains–the evolution of neural-progenitor-cell division

Jennifer L. Fish^1,*,, Colette Dehay^2,3, Henry Kennedy^2,3 and Wieland B. Huttner^1,

¹ Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany
² Inserm, U846, Stem Cell and Brain Research Institute, 69500 Bron, France
³ Université de Lyon, Université Lyon 1, 69003, Lyon, France

View larger version (19K): [in this window] [in a new window]   Fig. 1. The mode of neural-progenitor-cell division, and its effect on the direction of cortical expansion and growth (lateral expansion versus radial growth). (A) Lateral expansion (double-headed arrow), in which one AP generates two AP daughter cells, occurs as a result of the symmetric, proliferative division of APs (blue). (B,C) Radial growth (double-headed arrow) occurs as a result of either (B) asymmetric, neurogenic divisions of APs (blue), in which one AP generates a further AP as well as one neuron (red), or (C) asymmetric, differentiative divisions of APs (blue), in which one AP generates one AP and one BP (orange), which in turn generates two neurons (red) and is thereby consumed (dimmed orange).

View larger version (145K): [in this window] [in a new window]   Fig. 2. Key features of the cell biology of neural stem and progenitor cells in (A) D. melanogaster, (B) rodents and (C) primates. The apical plasma membrane and corresponding cortical domains are represented by blue lines, and the basolateral plasma membrane and corresponding cortical domains are represented by red lines. Interphase nuclei are shown in grey and representative sister chromatids are in dark blue. Black rectangles represent junctional complexes and yellow dots indicate centrosomes or mitotic-spindle poles. For clarity, only the astral microtubules of the mitotic spindle are depicted (black lines). (A) D. melanogaster neuroectodermal cells (bottom) divide with a cleavage plane that is parallel to their apicobasal axis (vertical cleavage), which results in symmetric (Sy) division. In neuroblasts (NB, top), Insc (green line) directs a 90° rotation of the mitotic spindle, aligning it along the apicobasal axis and thus generating a cleavage plane that is perpendicular to this axis (horizontal cleavage), which results in asymmetric (As) division. (Bi) In rodents, APs of the VZ exhibit apicobasal polarity (apical junctional complexes and apically located centrosomes in interphase). APs include neuroepithelial cells (not shown) and, after the onset of neurogenesis, short neural precursors (SNPs) and radial glia (RG) cells, the basal processes of which terminate at the basal side of the VZ and at the basal lamina, respectively, in interphase (two left-hand cells). APs divide at the apical surface with a vertical or nearly vertical cleavage plane that can result in either symmetric (Sy) or asymmetric (As) division (two right-hand cells; M-phase basal processes are not shown for clarity). (Bii) By contrast, BPs of the SVZ are known to have an apical process in interphase (left-hand cell), which is retracted before mitosis. Mitotic BPs (right-hand cell) are unpolarized, lack adherens junctions and divide in a basal location. BP-cell divisions are symmetric, with a random cleavage-plane orientation. (C) In primates, APs (Ci) and BPs (Cii) that are similar to those in rodents are also present, and BPs constitute the ISVZ. In addition, a novel neural progenitor that undergoes mitosis in a basal location, the OSVZ progenitor (Ciii), has evolved. Most of the cell-biological features of this progenitor are unknown. However, we hypothesize that interphase (left-hand cell) and mitotic (right-hand cell) OSVZ progenitors maintain epithelial characteristics, including radial processes and apical junctional complexes. Other features might distinguish OSVZ progenitors from APs, such as a perinuclear location of the centrosome in interphase (left-hand cell). Additionally, we hypothesize that symmetric cell divisions of polarized neural progenitors predominantly occur in APs (Ci) of the VZ (right-hand cell), whereas OSVZ progenitors (Ciii) might be restricted to asymmetric, differentiative divisions (right-hand cell).

View larger version (98K): [in this window] [in a new window]   Fig. 3. Elongation, pseudostratification and cleavage precision of APs. The apical plasma membrane and corresponding cortical domains are represented by blue lines, and the basolateral plasma membrane and corresponding cortical domains are represented by red lines. Interphase nuclei are shown in grey and representative sister chromatids are in dark blue. Black rectangles represent junctional complexes and yellow dots indicate centrosomes or mitotic-spindle poles. For clarity, only the astral microtubules of the mitotic spindle are depicted (black lines). (A) Cell elongation enables more progenitors to inhabit each unit of epithelial surface area. (Ai) An example of a cuboidal epithelial progenitor generating two similar progenitors, which require twice the area of apical surface (bars). (Aii) An example of a cuboidal epithelial progenitor generating two elongated progenitors, which can be accommodated without an increase in apical surface area. (B) Pseudo-stratification enables more apical mitoses to occur per unit of ventricular space. (Bi) In a cuboidal epithelium, progenitors in interphase (G1, S, G2) and in mitosis (M) occupy approximately the same space. Hence, as the duration of M-phase typically constitutes only a small fraction of the total length of the cell cycle, only a minor proportion of the apical surface area is used for progenitors that are engaged in mitosis. (Bii) In a pseudostratified epithelium, interphase nuclei translocate away from the apical surface, so a much greater proportion of the space at the apical surface can be filled by progenitors that are engaged in mitosis. Hence, the proliferative potential of a pseudostratified epithelium increases until the progenitor interphase nuclei that can be accommodated in the cylindrical space basal to an apical mitotic progenitor have reached a number that is equal to the length of the cell cycle divided by the duration of mitosis. According to Smart (Smart, 1972a; Smart, 1972b), in the mammalian neuroepithelium, APs in mitosis occupy three times more space beneath the ventricular surface than AP interphase nuclei. Thus, as an example, if the cell-cycle length of APs is 12 hours and mitosis takes 30 minutes, proliferation is maximized when 24 (12/0.5) AP interphase nuclei reside basal to an apical mitotic progenitor, which is equivalent to a pseudostratified neuroepithelium with eight (24/3) nuclear layers basal to the apical mitotic layer. (C) In elongated apical progenitors, the relative frequency of symmetric and asymmetric division can be controlled by the regulation of cleavage precision rather than by mitotic-spindle rotation. (Ci) In a cuboidal epithelial progenitor with apicobasal polarity, symmetric cell division (the inheritance of apical plasma membrane and junctional complexes by both daughter cells) can result from both a perfectly vertical and a slightly non-vertical cleavage plane (top and middle, respectively; broken lines). Asymmetric cell division is typically achieved by a 90° rotation of the mitotic spindle, which leads to a horizontal cleavage plane (bottom). (Cii) With increasing pseudostratification and AP elongation, the apical domain shrinks correspondingly. Consequently, asymmetric cell division can result from a slight deviation of the cleavage plane away from the vertical apicobasal axis (bottom) (Huttner and Brand, 1997), and symmetric cell division requires a cleavage-precision machinery that ensures ingression of the cleavage furrow precisely along the apicobasal axis (top) (Fish et al., 2006).

View larger version (86K): [in this window] [in a new window]   Fig. 4. In mammals, Aspm maintains the orientation of the mitotic spindle, and hence promotes the precision of cleavage, in APs after the onset of anaphase. (A,B) Knockdown of Aspm in the dorsal telencephalon of E10.5 Tis21-GFP knock-in mice (Haubensak et al., 2004) by electroporation of endoribonuclease-prepared siRNA (esiRNA) followed by 24-hour whole-embryo culture was performed as described previously (Fish et al., 2006), except that in some experiments a pCAGGS-Cherry plasmid instead of a pCAGGS-mRFP plasmid was used to identify the targeted APs. (A) Tis21-GFP-negative APs [i.e. APs that have not yet switched to neurogenic or differentiative divisions (Haubensak et al., 2004)] in metaphase (upper panels), and anaphase and telophase (lower panels) were analyzed by confocal microscopy of 16-µm cryosections (1 µm optical section shown). Control, electroporation with pCAGGS-Cherry plasmid only; Aspm RNAi, co-electroporation of Aspm esiRNAs and pCAGGS-Cherry plasmid. The left panel of each pair shows DAPI staining (blue) of the metaphase plate (upper panels) and sister chromatids (lower panels; yellow circles); the right panel of each pair shows DAPI staining, Cherry intrinsic fluorescence (red) and -tubulin immunofluorescence of centrosomes (green); this reveals the apical surface, which is towards the bottom of this image. Scale bars: 5 µm. (B) Quantification of the angle of the metaphase plate (blue), and of the predicted cleavage plane, as deduced from the position of sister chromatids in anaphase and telophase (red) relative to the apical surface of the neuroepithelium (determined by centrosome immunostaining and defined as 0°) in Tis21-GFP-negative control APs and APs subjected to Aspm RNAi [see the following references for methodological details (Fish et al., 2006; Kosodo et al., 2004)]. `Vertical' was defined as an angle of 90-75°, and `non-vertical' as an angle of 74-0°. Data are expressed as a percentage of all divisions for the control (n=10 for metaphase, n=36 for anaphase and telophase) or Aspm RNAi [n=19 for metaphase, n=41 for anaphase and telophase; anaphase and telophase data include 24 and 22 cases, respectively, that were published previously in Fig. 3B of Fish et al. (Fish et al., 2006)]. (C) Model for Aspm function after the onset of anaphase. The apical plasma membrane is represented by blue lines and the basolateral plasma membrane by red lines. Chromosomes and sister chromatids are shown in dark blue. Black rectangles represent junctional complexes, yellow dots indicate mitotic-spindle poles, black lines indicate the mitotic spindle and broken lines show the predicted cleavage plane. Aspm is not essential for correct positioning of the mitotic spindle, the axis of which in mammalian APs is typically oriented parallel to the apical surface by the end of metaphase (upper cells). Under control conditions, when Aspm is present (in particular in Tis21-GFP-negative APs), this orientation is maintained after anaphase onset and APs divide with a vertical cleavage plane, i.e. symmetrically (lower-left cell) (Fish et al., 2006; Kosodo et al., 2004). Upon Aspm knockdown, the axis of the mitotic spindle (particularly the central spindle) is increasingly likely to deviate from this orientation after anaphase onset, and hence the cleavage plane is increasingly likely to bypass the apical plasma membrane and junctional complexes (Fish et al., 2006), which results in the asymmetric division of APs (lower-right cell). This suggests an important role for Aspm in maintaining the precise alignment of the mitotic spindle perpendicular to the apicobasal AP axis after anaphase onset and throughout cytokinesis.

View larger version (77K): [in this window] [in a new window]   Fig. 5. Pax6 is present in mitotic progenitors of the primate OSVZ. Immunocytochemistry [technique modified from Kosodo et al. (Kosodo et al., 2004) to include an antigen-retrieval protocol] was performed on a 60-µm cryosection of macaque E80 cerebral cortex [area 17/18, Cynomolgus monkey foetus (Lukaszewicz et al., 2005)]. (A) Low-power overview of the cortical wall stained with DAPI (blue). IFL, inner fibre layer; OFL, outer fibre layer; SP, subplate; CP, cortical plate. The boxed area is shown at higher magnification in B. Scale bar: 250 µm. (B) Triple labelling of the area that contains the progenitor layers, as indicated by the boxed area in A. Blue, DAPI staining; red, phosphohistone H3 (PH3) immunofluorescence; green, Pax6 immunofluorescence. Note the presence of Pax6-positive mitotic cells in both the VZ and the OSVZ. Scale bar: 100 µm. (C) Detection of Pax6 (green) and PH3 (red) within the OSVZ by immunofluorescence. Note the presence of Pax6 in almost all mitotic OSVZ progenitors. The ventricular surface is below the lowest extent of the image. Scale bar: 20 µm. (D) Quantification of Pax6-positive mitotic rodent and primate APs of the VZ, rodent BPs of the SVZ and primate OSVZ progenitors. Rodent data are from 10-µm cryosections of dorsal telencephalon of E12.5 and E13.5 mice (four embryos each, three to five cryosections per embryo) that were subjected to double immunofluorescence for Pax6 and PH3 as above, and analyzed by confocal microscopy (1-µm optical sections). Primate data are from the same cryosection as is used in B and C. All mitotic rodent and primate APs were Pax6-positive (mouse 128/128, monkey 20/20). Less than 30% (9/32) of mitotic rodent BPs were Pax6-positive, and these Pax6-positive cells exhibited weak immunostaining relative to APs (indicated by the grey colour of the column). By contrast, almost 90% (94/106) of mitotic primate OSVZ progenitors were Pax6-positive and the level of immunoreactivity was equivalent to that of APs. Similar data were obtained when mitotic monkey OSVZ progenitors were analyzed for Pax6 expression without using an antigen-retrieval protocol [82% Pax6-positive (299/364), not shown].

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