Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex

The size of brain regions depends on the balance between proliferation and differentiation. During development of the mouse cerebral cortex, ventricular zone (VZ) progenitors, neuroepithelial and radial glial cells, enlarge the progenitor pool by proliferative divisions, while basal progenitors located in the subventricular zone (SVZ) mostly divide in a differentiative mode generating two neurons. These differences correlate to the existence of an apico-basal polarity in VZ, but not SVZ, progenitors. Only VZ progenitors possess an apical membrane domain at which proteins of the Par complex are strongly enriched. We describe a prominent decrease in the amount of Par-complex proteins at the apical surface during cortical development and examine the role of these proteins by gain- and loss-of-function experiments. Par3 (Pard3) loss-of-function led to premature cell cycle exit, reflected in reduced clone size in vitro and the restriction of the progeny to the lower cortical layers in vivo. By contrast, Par3 or Par6 (Pard6alpha) overexpression promoted the generation of Pax6+ self-renewing progenitors in vitro and in vivo and increased the clonal progeny of single progenitors in vitro. Time-lapse video microscopy revealed that a change in the mode of cell division, rather than an alteration of the cell cycle length, causes the Par-complex-mediated increase in progenitors. Taken together, our data demonstrate a key role for the apically located Par-complex proteins in promoting self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex.     

Live imaging at the onset of cortical neurogenesis reveals differential appearance of the neuronal phenotype in apical versus basal progenitor progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.     

Role of Pax6 in development of the cerebellar system.

Post-mitotic neurons generated at the rhombic lip undertake long distance migration to widely dispersed destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Here we show that Pax6, a key regulator in CNS and eye development, is strongly expressed in rhombic lip and in cells migrating away from it. Development of some structures derived from these cells is severely affected in Pax6-null Small eye (Pax6(Sey)/Pax6(Sey)) embryos. Cell proliferation and initial differentiation seem unaffected, but cell migration and neurite extension are disrupted in mutant embryos. Three of the five precerebellar nuclei fail to form correctly. In the cerebellum the pre-migratory granule cell sub-layer and fissures are absent. Some granule cells are found in ectopic positions in the inferior colliculus which may result from the complete absence of Unc5h3 expression in Pax6(Sey)/Pax6(Sey) granule cells. Our results suggest that Pax6 plays a strong role during hindbrain migration processes and at least part of its activity is mediated through regulation of the netrin receptor Unc5h3.     

Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates

Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3' untranslated region (3'UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved.     

Experimental Sey mouse chimeras reveal the developmental deficiencies of Pax6-null granule cells in the postnatal cerebellum

Pax6 has been implicated in cerebellar granule cell development, however the neonatal lethality of the Sey/Sey mutant has precluded a more detailed study of this late developing neuronal type. In this study we use experimental mouse chimeras made from wildtype and Pax6-null embryos to circumvent early lethality and assess the developmental potential of mutant cells in the construction of the cerebellum. We have identified the granule cell as a direct target of mutant gene action, with glia and Purkinje cells being affected in what is largely a non-cell autonomous manner.Most dramatically, in postnatal day 21 (P21) chimeras, mutant cells are largely absent in the anterior and posterior cerebellum while present in central lobules, but amidst disorganized cerebellar architecture. Analysis of P0/1 and P10 chimeras demonstrates a profound temporally based defect where mutant cells colonize the anterior and posterior EGL but fail to migrate to the IGL. Mutant granule cells in the central lobules can reach the IGL in an abnormal manner, with large streams of cells forming raphes through the molecular layer.These studies provide new insights into the role of Pax6 in postnatal cerebellar development that pinpoint the granule cell as an intrinsic target of the mutant gene and key events in the life of the developing granule cell that depend upon normal Pax6 expression.     

Mitotic spindle orientation can direct cell fate and bias Notch activity in chick neural tube

Inheritance of apical membrane is proposed to maintain vertebrate neural stem cell proliferation. However, evidence for this is contradictory. Using direct clonal analysis and live imaging in chick neural tube, we show that divisions that separate apical and basal components generate an apical daughter, which becomes a neuron, and a basal daughter, which rapidly re-establishes apico-basal polarity and divides again. Using a recently described real-time reporter of Notch activity, we confirm progenitor status and demonstrate that division orientation can influence Notch signalling. In addition, we reveal loss of apical complex proteins on neuronal differentiation onset, suggesting that removal of this inherited complex is part of the neuronal differentiation mechanism. These findings reconcile contradictory data, link asymmetric division to Notch signalling dynamics and identify apical complex loss as a new step towards neuronal differentiation.     

BMP7 acts in murine lens placode development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.     

Pax6 is misexpressed in Sox1 null lens fiber cells

Sox1 null lens fiber cells fail to elongate and have disrupted expression of gamma-crystallin. We have evaluated the expression of Sox1 and Pax6 proteins during critical stages of lens morphogenesis, with particular focus on fiber cell differentiation. While Pax6 and Sox1 are co-expressed during early stages of fiber cell differentiation, Sox1 up-regulation coincides temporally with the down-regulation of Pax6, and these proteins therefore display a striking inverse expression pattern in the lens fiber cell compartment. Furthermore, Pax6 is inappropriately expressed in the fiber cells of Sox1 null mice and the Pax6 target, alpha5 integrin, is simultaneously misexpressed. Finally, we demonstrate a genetic interaction between Sox1 and Pax6, as Sox1 heterozygosity partially rescues the diameter of Pax6(Sey) lenses by increasing the number of cells in the fiber cell compartment.     

Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord

Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neuron-generating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.     

The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial glia that form the subcommissural organ.

During brain development, Pax6 is expressed in specific regions of the diencephalon including secretory cells of the subcommissural organ (SCO), a circumventricular organ at the forebrain-midbrain boundary that originates from the pretectal dorsal midline neuroepithelial cells beneath the posterior commissure (PC). Homozygous small eye (Sey/Sey) mice lack functional Pax6 protein and fail to develop the SCO, a normal PC and the pineal gland. Small eye heterozygotes (Sey/+) show defective development of the SCO's basal processes which normally penetrate the PC, indicating that normal development of the gland requires normal Pax6 gene-dosage. A correlation between the defects of SCO formation and altered R- and OB-cadherin expression patterns in the SCO is observed in mutants suggesting a role for cadherins in SCO development.     

Asymmetric self-renewal and commitment of satellite stem cells in muscle

Satellite cells play a central role in mediating the growth and regeneration of skeletal muscle. However, whether satellite cells are stem cells, committed progenitors, or dedifferentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre-reporter alleles, we observed that in vivo 10% of sublaminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells gave rise to Pax7(+)/Myf5(+) satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7(+)/Myf5(-) and an apical Pax7(+)/Myf5(+) cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7(+)/Myf5(+) cells exhibited precocious differentiation, Pax7(+)/Myf5(-) cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of neuromuscular diseases.     

Niche regulation of muscle satellite cell self-renewal and differentiation

Muscle satellite cells have been shown to be a heterogeneous population of committed myogenic progenitors and noncommitted stem cells. This hierarchical composition of differentiating progenitors and self-renewable stem cells assures the extraordinary regenerative capacity of skeletal muscles. Recent studies have revealed a role for asymmetric division in satellite cell maintenance and offer novel insights into the regulation of satellite cell function by the niche. A thorough understanding of the molecular regulation and cell fate determination of satellite cells and other potential stem cells resident in muscle is essential for successful stem cell-based therapies to treat muscular diseases.     

Strabismus regulates asymmetric cell divisions and cell fate determination in the mouse brain

The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn–enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.