By interfering with β-catenin signaling using focal electroporation techniques, we observe that that precursor proliferation in the cerebral cortical ventricular zone requires cell-autonomous β-catenin signaling. Focal elimination of β-catenin by cre-mediated gene excision or inhibition of β-catenin signaling with ICAT and dominant-negative TCF-4 leads to premature cell cycle withdrawal and neuronal differentiation.
It has been proposed that the number of neural precursor cells generated during development controls the size of the cerebral cortex (Rakic, 1995
). Although our previous work showed that overexpression of β-catenin led to increased numbers of cortical precursors and a subsequent expansion of cortical surface area (Chenn and Walsh, 2002
), the cell-autonomous requirement for β-catenin in the normal regulation of cortical precursor number remained unexplored. Conditional knock-out studies suggested a role for β-catenin in cell proliferation in the cortex, but interpretation of β-catenin function was complicated by the severe architectural abnormalities of the tissue (Machon et al., 2003
). Although recent studies using overexpression of Wnts or dominant-negative Wnts have suggested a similar function for Wnt-β-catenin signaling in hippocampal progenitors (Lie et al., 2005
), the methodology did not permit the examination of cell-autonomous function. The electroporation approach allows us to investigate cell-autonomous function of β-catenin in the absence of architectural disruption, and demonstrates that β-catenin signaling function is required to maintain progenitor proliferation. Together, the gain- and loss-of-function studies suggest a model in which high levels of β-catenin signaling expand the neural precursor population, whereas inhibition of signaling causes withdrawal from the cell cycle and depletion of the precursor pool.
During cortical development, cell cycle exit and neuronal differentiation are closely linked (Caviness et al., 1995
), and cells that continue to cycle while initiating neuronal development exhibit increased cell death (Lee et al., 1992
). Recent studies of the cyclin-dependent kinase inhibitor p27Kip1 suggest that neuronal differentiation and cell cycle exit may be independently regulated, because p27Kip1 can promote neuronal differentiation and radial migration of cortical projection neurons separable from its role in cell cycle regulation (Nguyen et al., 2006
). Although β-catenin can regulate cell cycle through transcriptional regulation of cyclin D1 and c-myc (Peifer and Polakis, 2000
) as well as promote neuronal differentiation by regulating Neurogenin1 (Israsena et al., 2004
), our methods do not provide sufficient resolution to distinguish whether β-catenin independently regulates cell cycle and neuronal differentiation. Although inhibition of β-catenin signaling leads both to increased cell cycle exit and neuronal differentiation (at the expense of precursor number), we did not observe evidence that neuronal differentiation occurred at rates different from would be predicted by cell cycle exit. Regardless of whether cell cycle exit and differentiation are independently regulated by β-catenin, our findings suggest that inhibition of β-catenin signaling results in depletion of the precursor pool.
From the perspective of proliferation and differentiation, the size of the precursor pool can be regulated by the types of cell division that occur. During development, neural progenitors can undergo three types of cell divisions with regard to cell cycle reentry and exit (for review, see Caviness et al., 1995
): (1) symmetric divisions that generate two additional progenitors serve to expand the progenitor pool. (2) Asymmetric divisions generate one daughter that exits the cell cycle and one daughter that reenters the cell cycle to divide again. These asymmetric divisions maintain the progenitor population while generating postmitotic neuronal progeny during cortical development. (3) Symmetric terminal divisions generate two postmitotic daughters, and these divisions will ultimately deplete the progenitor population.
The existence and relative contributions of these types of divisions have been suggested by population studies of proliferating cells (Takahashi et al., 1996
; Miyama et al., 1997
) and time-lapse microscopy (Chenn and McConnell, 1995
; Adams, 1996
; Noctor et al., 2001
; Haydar et al., 2003
). Recent studies modeling ret-roviral lineage tracing suggest that different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexist during cortical development period (Cai et al., 2002
), supporting the notion that changes in the relative proportions of these three types of divisions would influence the production of neurons from the progenitor population (Takahashi et al., 1996
). Our experiments showing that inhibition of β-catenin signaling increases cell cycle exit cannot distinguish between the relative contributions of these different types of divisions, and multiple possibilities can generate similar outcomes. For example, increasing the fraction of symmetric terminal divisions at the expense of asymmetric divisions is difficult to distinguish on a population level from increasing the relative fraction of asymmetric divisions at the expense of symmetric proliferative divisions, and time-lapse imaging would provide definitive evidence of the relative contributions of cell divisions with varying symmetry.
Although we did not perform direct observation of changes in division symmetry, the localization of β-catenin at adherens junctions suggests a testable hypothesis: asymmetric localization of β-catenin may serve a role in regulation of signaling after asymmetric division. Although the factors that inhibit β-catenin signaling in the ventricular zone remain unknown, the observation that the bulk of β-catenin is asymmetrically localized at the adherens junctions of dividing precursors (Chenn et al., 1998
) suggests that one way of regulating signaling is through unequal inheritance of β-catenin after a vertically oriented cell division. Recent studies suggest that asymmetric inheritance of the apical membrane appears sufficient to confer differences to two daughters after mitosis (Kosodo et al., 2004
). Mitotic cleavage planes only slightly oblique from vertical were capable of distributing the apical membrane preferentially to one daughter (Kosodo et al., 2004
). We noted that such divisions could lead to the asymmetric segregation of the bulk of the adherens complex to only one daughter as well. It has been proposed that the cadherin-bound pool of β-catenin might serve as a reservoir for signaling competent β-catenin (Cox et al., 1996
; Barth et al., 1997
), and recent studies provide support for cross talk between the cadherin bound and signaling pools of β-catenin (Klingelhofer et al., 2003
). Therefore, after an asymmetric cleavage (as defined by asymmetric inheritance of apical membrane) (Kosodo et al., 2004
), the cell adjacent to the ventricular lumen (the apical cell) will inherit the adherens complex and β-catenin, and thus may be influenced to remain in the cell cycle because of maintained β-catenin signaling. In contrast, the basal daughter after an asymmetric cleavage may be predisposed to exit the cell cycle as a consequence of receiving less β-catenin. Our model would suggest that inhibition of β-catenin signaling could potentially influence previously asymmetric divisions to become symmetric and terminal divisions, and symmetric proliferative divisions might also be influenced to become symmetric terminal divisions.
However, additional complexity is introduced by recent experiments that indicate that secondary proliferative populations identified in the subventricular zone (SVZ) (Takahashi et al., 1995
) contribute significantly to neuronal production during cortical development (Noctor et al., 2004
). Whereas progenitor divisions in the ventricular zone can be asymmetric, generating one daughter that exits the cell cycle and one daughter that reenters the cell cycle, asymmetric VZ divisions can also generate one daughter that moves to the SVZ (to divide again), and one daughter that remains in the ventricular zone. Although this mode of division is symmetric in the sense that it generates two daughters that reenter the cell cycle, these resultant daughters are distinct, in both physical location and behavior. Furthermore, symmetric divisions in the VZ could also generate two daughters that move to the SVZ, which then divide again. In the SVZ, the majority of cell divisions (~90%) that subsequently occur in the SVZ are symmetrically terminal, generating two postmitotic neurons that migrate to the cortical plate (Noctor et al., 2004
). Our findings that inhibition of β-catenin signaling increase cell cycle exit and neuronal differentiation cannot distinguish between effects on cell cycle exit in the ventricular zone versus the subventricular zone; although β-catenin signaling could conceivably regulate overall cell cycle exit by altering the frequency of asymmetric ventricular divisions that generate SVZ precursors, it could also influence the patterns of subsequent SVZ divisions as well. Our observation that β-catenin signaling is reduced in the SVZ suggests that such changes in SVZ behavior may be a later consequence of β-catenin inhibition in the VZ rather than a direct role for β-catenin in SVZ divisions.
Our studies target a period approximating the midpoint of cortical neurogenesis in the mouse (Takahashi et al., 1999
). Although we have not yet determined whether β-catenin signaling occurs in precursors before the onset of neurogenesis, we suspect based on gain-of-function studies (Backman et al., 2005
) that β-catenin functions similarly early during development. Nevertheless, our current studies do not provide information on whether other aspects of cell fate choice are affected by β-catenin (for example, choice of cortical lamina, or decision to generate glia). Although these choices normally appear to be regulated by when a cell exits the cell cycle (McConnell, 1989
), we do not know whether forcing a precursor to exit the cell cycle early will cause it to adopt a relatively earlier phenotype. For example, although we observe β-catenin signaling later in cortical development (G. J. Woodhead and A. Chenn, in preparation), it remains unexplored whether loss of β-catenin signaling will lead to increased differentiation into non-neuronal cells appropriate for that period of development.
Recent studies provide support for the notion that mitotic spindle orientation plays a crucial role in neural precursor proliferation and brain size (for review, see Woods et al., 2005
). The most common cause of primary autosomal recessive microcephaly is mutation in the abnormal spindle-like microcephaly associated (ASPM) gene. ASPM
is highly expressed in neural precursors and the protein is localized to mitotic spindle poles (Kouprina et al., 2005
), and its Drosophila
counterpart, Asp, is an asymmetrically localized centrosomal protein required for mitotic spindle integrity (do Carmo Avides and Glover, 1999
). Similarly, knock-down of Aspm in telencephalic neuroepithelial cells causes alterations in cleavage plane orientation and leads to an increased frequency of asymmetric division (Fish et al., 2006
). Furthermore, recent studies in mouse have also shown the importance of Doublecortin-like kinase and G-proteins in the regulation of the mitotic spindle and the symmetric/asymmetric mode of division (Sanada and Tsai, 2005
; Shu et al., 2006
The model proposed above places β-catenin signaling downstream of mitotic spindle orientation, as an apical factor that can be distributed symmetrically or asymmetrically to daughter cells depending on the mitotic cleavage angle. However, our data cannot rule out a role for β-catenin signaling in the organization of mitotic spindle orientation, and recent studies suggest that multiple Wnt/β-catenin signaling pathways serve to orient the mitotic spindle in early Caenorhabditis elegans
embryos (Walston et al., 2004
β-Catenin is likely to function at multiple times during the lifetime of a developing neuron, and the function of β-catenin in promoting neuronal differentiation has been suggested by previous studies. Gain-of-function studies in neurosphere cultures suggest that developmental context and presence of other growth factors (FGF2) influence whether β-catenin signaling promotes proliferation or differentiation (Israsena et al., 2004
), and β-catenin overexpression increases neuronal production from embryonic stem cells differentiated in vitro
(Otero et al., 2004
). Although our studies suggest that one key function of β-catenin is to maintain precursor proliferation in vivo
, they do not exclude subsequent roles for β-catenin in promoting neuronal differentiation. Indeed, our observations that β-catenin signaling reappears in newly arriving neurons of the cortical plate support later functions for β-catenin signaling in neuronal differentiation. Although additional studies are necessary to determine what function this later signaling plays in neuronal development, early aspects of cortical neuronal differentiation (such as development of long-distance callosal projections) appear grossly normal in the absence of β-catenin (data not shown).
What is the role of β-catenin signaling versus cell adhesion in the developing cortex? Here, we have shown by electroporation of a reporter construct responsive to β-catenin signaling that β-catenin signaling exists in cortical precursors during cortical neurogenesis. Focal deletion of β-catenin leads to premature ventricular zone exit, and furthermore, two different specific inhibitors of β-catenin signaling (that do not affect adhesion) lead to premature cell cycle exit and neuronal differentiation. Although these approaches provide multiple lines of evidence that β-catenin signaling function is required for cortical precursor proliferation, the role of β-catenin cell adhesion in neural precursors is more difficult to examine directly.
Although β-catenin has distinct functions in signaling and cell adhesion, the protein domain of β-catenin required for signaling through TCF/LEF binding domains overlaps significantly with the cadherin binding domains (Huber et al., 1997
). This substantial overlap may underlie alterations in cell proliferation that result when epithelial architecture is disrupted by mutation or deletion of other adherens junction proteins (Lien et al., 2006
). Loss of adherens junction integrity may allow for the redistribution and accumulation of previously membrane-localized β-catenin leading to increased signaling and proliferation. Although the role of β-catenin-mediated cell adhesion in neuronal differentiation remains unclear, our studies suggest that β-catenin signaling is essential to maintain neural precursor proliferation during cortical development.
Our observations that robust β-catenin signaling is found primarily in the ventricular zone support a role for localized signaling in cortical precursor maintenance. It is likely that neuronal differentiation and exit from the ventricular zone are linked, because in vivo
, the early cortical ventricular zone is notably devoid of differentiated neurons (Menezes and Luskin, 1994
). Our findings that inhibiting β-catenin signaling in vivo
causes exit from the ventricular zone suggest (1) inhibition of signaling is sufficient to lead to cell cycle withdrawal and VZ exit, and (2) exit from the ventricular zone may ensure continued inhibition of signaling. A role for cell adhesion to physically maintain cortical precursors in the ventricular zone remains an attractive, but unexplored possibility.