The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex

Abstract

SUMMARYSuccessful brain development requires tight regulation of sequential symmetric and asymmetric cell division. Although Pax6 is known to exert multiple roles in the developing nervous system, its role in the regulation of cell division is unknown. Here, we demonstrate profound alterations in the orientation and mode of cell division in the cerebral cortex of mice deficient in Pax6 function (Pax6 Sey/Sey ) or after acute induced deletion of Pax6. Live imaging revealed an increase in non-vertical cellular cleavage planes, resulting in an increased number of progenitors with unequal inheritance of the apical membrane domain and adherens junctions in the absence of Pax6 function. This phenotype appears to be mediated by the direct Pax6 target Spag5, a microtubule-associated protein, reduced levels of which result in the replication of the Pax6 phenotype of altered cell division orientation. In addition, lack of Pax6 also results in premature delamination of progenitor cells from the apical surface due to an overall decrease in proteins mediating anchoring at the ventricular surface. Moreover, continuous long-term imaging in vitro revealed that Pax6-deficient progenitors generate daughter cells with asymmetric fates at higher frequencies. These data demonstrate a cell-autonomous role for Pax6 in regulating the mode of cell division independently of apicobasal polarity and cell-cell interactions. Taken together, our work reveals several direct effects that the transcription factor Pax6 has on the machinery that mediates the orientation and mode of cell division. KEY WORDS: Radial glia, Asymmetric cell division, Neurogenesis, Spag5The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex , 2004) or YVI mice (George et al., 2007). The day of the vaginal plug was considered as embryonic day (E) 0. For conditional deletion of Pax6 we used Pax6 flox/flox mice (Ashery-Padan et al., 2000). Immunocytochemistry and immunohistochemistryBrains isolated from E12-16 embryos were fixed in 4% (w/v) paraformaldehyde (PFA) in phosphate buffered saline (PBS), cryoprotected in 30% (w/v) sucrose in PBS, embedded in Tissue-Tek and cryosectioned (20-40 m). Cultured cells were fixed and stained as described previously (Haubst et al., 2004) After staining with fluorescently labelled secondary antibodies, nuclei were labelled by incubation in PBS containing 0.1 g/ml DAPI (4Ј,6-diamidino-2-phenylindole, Sigma), and samples were mounted in Aqua Polymount (Polyscience) and analyzed using Olympus FV1000 confocal laser scanning microscopes. Fluorescence-activated-cell-sorting (FACS) based on anti-prominin 1 staining (PE-conjugated, e-Bioscience, 1:400; supplementary material Fig. S4) was performed as previously described (Pinto et al., 2008). In utero electroporation and en face and sliced cortices live imagingPregnant mice were operated as approved by the Government of Upper Bavaria under licence number 55.2-1-54-2531-144/07 and were anaesthetized by intraperitoneal (i.p.) injec...

The Cell Biology of Neurogenesis: Toward an Understanding of the Development and Evolution of the Neocortex

“…However, the Sey/Sey (Pax6 mutant) mouse cortex shows an increased occurrence of abventricular centrosomes that is paralleled with an increase in subapical and basal mitosis (Asami et al 2011, Tamai et al 2007). Indeed, Pax6 directly regulates ciliary and centrosomal components, such as Spag5 (Asami et al 2011), suggesting a possible link between centrosome/cilium localization and fate transition.…”

“…However, the Sey/Sey (Pax6 mutant) mouse cortex shows an increased occurrence of abventricular centrosomes that is paralleled with an increase in subapical and basal mitosis (Asami et al 2011, Tamai et al 2007). Indeed, Pax6 directly regulates ciliary and centrosomal components, such as Spag5 (Asami et al 2011), suggesting a possible link between centrosome/cilium localization and fate transition. Consistent with this observation, it has been demonstrated recently that in cells undergoing delamination (e.g., newborn basal progenitors and neurons), the cilium is localized basolaterally rather than apically (Wilsch-Bräuninger et al 2012).…”

Coevolution of radial glial cells and the cerebral cortex

“…In the cerebral cortex of the mouse embryo aRGCs and bRGCs express the paired‐box transcription factor Pax6 and not the T‐box transcription factor Tbr2, whereas IPCs express Tbr2 but not Pax6 (a small percentage of mouse bRGCs have been reported to express Tbr2 by some authors, so this point remains controversial; Shitamukai et al, 2011; Wang et al, 2011). Whereas Pax6 has a critical role in the definition of telencephalic territories and multiple aspects of cortical progenitor proliferation (Asami et al, 2011; Estivill‐Torrus et al, 2002; Gotz et al, 1998; Kroll and O'Leary, 2005; Mi et al, 2013; O'Leary and Sahara, 2008; Schuurmans and Guillemot, 2002; Sur and Rubenstein, 2005), Tbr2 has an instructive role in defining the conversion of aRGCs into IPCs (Sessa et al, 2008). In agreement with these roles, Pax6 and Tbr2 expression follows a unidirectional sequence in the cortical progenitor lineage: first Pax6 (aRGCs, bRGCs), second Tbr2 (some bRGCs, IPCs and newborn neurons; Englund et al, 2005; Hevner, 2006).…”

Cell Division Modes and Cleavage Planes of Neural Progenitors during Mammalian Cortical Development

“…The expression of ZO-1-or Par3-EGFP fusion proteins (which localize to the zonula adherens) marks the cell-cell boundary at the apical junctional level (Konno et al 2008;Shitamukai et al 2011). Imaging these fluorescent markers from the apical side of brain slices allows the direct observation of the partitioning of the apical junction into daughter cells and has revealed that the segregation of the apical membrane exclusively to a single daughter cell occurs in only 10%-20% of RG divisions during the neurogenic stage of the developing murine brain (Konno et al 2008;Asami et al 2011;Shitamukai et al 2011); indeed the apical junction is partitioned between both daughter cells in the majority of RG cell divisions. The reextension of the lost apical end is rarely observed in divisions during the midneurogenic stage (E14-E16).…”