Spindle orientation in mammalian cerebral cortical development

Highlights ► Spindle orientation is a key mechanism for generating cell diversity and balancing self-renewal and differentiation of neural stem cells. ► Regulators of spindle orientation, such as mInsc and LGN, are necessary for balancing planar and oblique orientations in the developing mammalian cerebral cortex. ► Spindle orientation influences neuron production and cortical size by regulating production of intermediate progenitors and outer radial glial progenitors.


Introduction
During cell division, intracellular components must be partitioned into two daughter cells in a highly regulated manner. For some components this is done symmetrically so that both daughter cells receive an identical set. However, other components, such as cell fate regulators, may be partitioned asymmetrically to result in asymmetric cell fates following division [1]. This cell fate asymmetry contributes to developmental diversification that generates the vast array of different cell types in a fully developed metazoan. Asymmetric localization of subcellular components can be achieved by regulating the distribution of these components and the mitotic spindle relative to one another so that the two daughter cells terminate cytokinesis with distinct sets of these factors [2,3].
Regulated orientation of the spindle has been shown to be an influential factor in cell fate decisions during development of a variety of systems. For example, the Caenorhabditis elegans zygote positions the spindle along the anterior-posterior axis through interactions of spindle microtubules with polarity proteins at the cell cortex [2]. Similarly, Drosophila neuroblasts orient their spindle along the apicobasal axis through interactions at the cell cortex with apically localized factors [4].
This paradigm of positioning the spindle through interactions at the cell cortex seems to hold true in vertebrate and mammalian asymmetric divisions as well. In particular, in the developing mammalian neocortex, neurons arise from asymmetric divisions of progenitor cells, whereas symmetric divisions drive self-renewal of progenitors [5]. This process is dependent upon spindle orientation, and, like in C. elegans and Drosophila, involves polarity proteins. Orientation of the spindle has important implications in human brain evolution as well as several developmental disease states [6]. For example, disorders such as lissencephaly (smooth brain) and microcephaly (small brain) can be caused by mutations in genes with specific roles in spindle orientation in the mammalian cerebral cortex, including Lis1, Nde1, and MCPH1 to name a few [7]. In this review, we will focus on mechanisms of spindle orientation in mammals and in particular in the developing cerebral cortex.

Mechanisms of spindle orientation in mammalian cells
Astral microtubule growth and positioning. The mitotic spindle is formed during prophase when the duplicated centrosomes, or microtubule organizing centers (MTOC), nucleate spindle microtubules to position chromosomes, and astral microtubules to position the spindle relative to the cell cortex [8]. Although centrosomes are not required for spindle assembly in all cells (e.g. higher plants [9], planarians [10], and mouse oocytes [11]), there is growing evidence that centrosome function influences spindle positioning [12]. Furthermore, regulation of microtubule polymerization and stability is important not only for spindle assembly, but also for positioning [13].
Astral microtubules elongate from the MTOC and undergo microtubule-capture at the plasma membrane to position the spindle. This occurs through the concerted effort of dynein-dynactin directed microtubule transport with the help of dynein-associated proteins such as CLASP1, Lis1, and Nde1/Ndel1, thereby pulling the spindle into position [14][15][16][17]. One particularly interesting finding is that the micromechanical characteristics of the spindle allow it to move as a whole under spindle positioning forces [18 ]. Thus, microtubule anchoring at the cell cortex can position the entire spindle relative to polarity cues.
An important player in microtubule anchoring is the actin cytoskeleton. Subcortical F-actin filaments at the cell cortex interact with astral microtubules of the nascent spindle poles [19]. Through myosin transport and cortical flow of F-actin, astral microtubules can be pulled, thereby positioning centrosomes and the entire spindle [20] ( Figure 1a). However, since subcortical actin is often uniformly distributed throughout the cell, asymmetry must be conveyed by other factors as well. For example, external forces that influence cell shape can influence spindle orientation through an effect on the actin cytoskeleton [21][22][23]. Internally, polarity cues, such as apicalbasal factors, can influence spindle orientation.
The role of the apical Par complex. Epithelial cells, including the neuroepithelium, exhibit pronounced apicobasal polarity [24]. This polarity can provide an endogenous source of asymmetry to orient the spindle [25]. The apical Par complex containing Par3, Par6, and aPKC, is a master regulator of apicobasal polarity, with Par3 functioning as a scaffold for Par6 and aPKC [26]. These components, with the help of Cdc42, selectively exclude non-apical proteins from the apical domain [27].
In epithelial cells, adherens junctions help to establish and maintain this apical polarity [26]. Adherens junctions, a type of cell-cell contact, are composed of cadherins and catenins, particularly E-cadherin and a-catenin, b-catenin and p120 catenin [28]. These adherens junctions interact with Par-3 to recruit the Par-complex apically, and facilitate apical polarity maintenance by preventing mixing between apical and basolateral domains of the epithelial cell [24]. In addition, adherens junctions can influence the cytoskeleton through a-catenin interaction with actin and p120 catenin interaction with microtubules [28].
Connecting the spindle to polarity cues. Many of the factors involved in orienting the spindle relative to apicobasal 738 Neurodevelopment and disease    (Figure 1c).

Neurogenesis in mammalian cortical development
During mammalian cerebral cortical development, the neuroepithelium initially divides symmetrically and with a planar orientation to expand the progenitor pool [42] (Figure 2). At the start of neurogenesis, the progenitor pool, termed radial glial stem cells (RGs) or apical progenitors, then begin dividing asymmetrically. These asymmetric divisions can result in several outcomes leading to either direct neurogenesis to produce a neuron immediately after division, or indirect neurogenesis giving rise to an intermediate progenitor (IP) ( Figure 2). This IP can then divide again to produce two neurons, thereby expanding the neurogenic output.
The asymmetric cell fate resulting from these divisions is likely determined by many factors, such as epigenetic changes and signaling, which we are only just beginning Spindle orientation in mammalian cerebral cortical development Lancaster and Knoblich 739 to identify. In particular, there is now substantial evidence that asymmetric divisions in the cortex give rise to daughter cells with variable Notch activities resulting in daughter cells with greater Notch activity, which tend to remain as RGs, while those with lesser Notch activity become neurons or IPs [43]. Indeed, disruption of Notch signaling in the mammalian neocortex has dramatic consequences to cell fate [44]. For example, inducing Notch activation inhibits neuronal differentiation by maintaining RGs as progenitors [45].
Thus, establishment of differential Notch activities is likely a key event in fate determination following asymmetric division of RGs. Other signaling pathways, such as Shh and Wnt signaling are also vital to acquisition of specific cell fates in the neocortex [46], though their roles Increased asymmetric fates [98] Vangl2 Planar cell polarity LOF: increased planar orientation [99] Increased early-born neurons, decreased RGs [99] Not all factors affecting cortical progenitor asymmetric division are shown. This table is limited to those factors where spindle orientation was specifically examined. LOF = Loss of function, GOF = Gain of function.
in asymmetric fate determination have not been specifically addressed. Importantly, it is still largely unclear how differential signaling activities in daughter cells are established and whether this is influenced by spindle orientation. This is discussed further below.

Spindle orientation factors and their phenotypes in the mammalian neocortex
The MTOC and microtubule dynamics. Disruptions in mitotic spindle components, such as the centrosome and astral microtubules, lead to striking phenotypes in the mammalian neocortex. For example, disruption of factors involved in centrosome function leads to misorientation of the mitotic spindle in neural progenitors [47,48 ], which results in a depletion of neural progenitors [12,49]. Furthermore, disruption of microtubule dynamics as is seen with loss of Doublecortin (Dcx), a gene mutated in patients with lissencephaly and cortical band heterotopia, leads to a similar depletion of progenitor cells due to randomized spindle orientation [13]. Along these lines, disruption of dynein-dynactin function at the cell cortex, as in the case of Lis1 and Nde1/Ndel1 mutations, similarly leads to depletion of progenitor cells due to randomized spindle orientation (Table 1) (Figure 2). Our understanding of these oRGs is still in its infancy, but several studies have revealed that, like apical RGs, oRGs can divide asymmetrically to expand neuronal output. Additionally, although oRGs express identical markers to Spindle orientation in mammalian cerebral cortical development Lancaster and Knoblich 741  apical RGs, they lack an apical process and therefore the apical domain [5]. These are important characteristics as they have major implications for many aspects of asymmetric cell division in the neocortex (discussed below).
In addition, genetic studies of mInsc have now been performed in mouse neocortex and reveal that loss of mInsc leads to a decrease in oblique and vertical divisions, resulting in a decrease in IPs (Table 1) [40 ]. On the other hand, overexpression of mInsc leads to a reduction of planar divisions and drives the production of IPs as well as basal RGs [40 ,41] ( Figure 3). Interestingly, this does not seem to affect the number of apical RGs as seen in other mutants affecting spindle orientation. Furthermore, mInsc seems to specifically affect later neurogenic events, as early dynamics are not affected in mInsc mutants. Thus, mInsc seems to inhibit planar divisions and promote oblique and vertical orientations to drive production of IPs and oRGs.

Spindle orientation and cell fate
Spindle orientation seems to be a key initial factor in acquisition of asymmetric cell fates. Specifically, oblique and vertical (along the apicobasal axis) orientations tend to give rise to asymmetric fate outcomes suggesting these orientations drive asymmetric cell fate. Furthermore, the contribution of these division orientations varies temporally during neurogenesis, with early neuroepithelium dividing primarily with a planar orientation, and oblique orientation arising to a greater extent as neurogenesis increases [51,58]. Vertical orientations are somewhat rare under normal circumstances but can be induced to a greater extent upon genetic manipulation (such as with mInsc overexpression) [40 ,41].
Disruption of these orientations has been shown to have consequences for resultant cell fate. For example, early planar orientations seem to be the most sensitive to spindle orientation defects and their disruption leads to depletion of the progenitor pool [51]. Disruption of planar orientations at later stages, by either randomization or induction of vertical and oblique orientations, also leads to a gradual depletion of progenitors due to increased asymmetric neurogenic divisions at the expense of symmetric proliferative divisions. On the other hand, disruption of oblique and vertical orientations leads to a reduction in neurons due to loss of this pool of asymmetric divisions [40 ].
It is important to point out that while oblique and vertical divisions promote asymmetric cell fate, the orientation of asymmetric divisions can also be planar [2]. This suggests that while spindle orientation influences cell fate, it is not the only determining factor in generating asymmetric outcomes, leaving the possibility for other factors open for investigation.
One candidate is the apical Par complex. On the basis of the cellular architecture of the developing cortex, one model suggests that oblique or vertical spindle orientation influences the inheritance of the apical domain [59]. Recently, Par3 was shown to localize asymmetrically in dividing RGs where it promotes Notch signaling in cells inheriting greater Par3 levels, thereby promoting retention of RG fate [60]. Par3 was shown to interact with Numb, a well-known fate determinant in Drosophila neuroblasts and regulator of Notch signaling [61], and this interaction led to enhanced Notch activity. Numb is also asymmetrically localized in this context [62][63][64]. Thus, Par3 and Numb localize asymmetrically in dividing RGs to help establish Notch asymmetry post-mitosis.
There are still several open questions here, however. In particular, the link with spindle orientation is not clear since asymmetric distribution of Par3 did not correlate with spindle orientation [60]. Furthermore, Numb and its related Numb-like (Numbl) have additional roles in apicobasal polarity, which may also influence spindle orientation [64]. In addition, numerous studies have examined whether the apical and/or basal domains correlate with daughter cell fate with somewhat contradictory results [54,57 ,59,64,65]. Furthermore, the fact that oRGs lack an apical domain suggests that inheritance of the apical domain is not required for RG fate specification per say, but may be involved in oRG versus apical RG fate determination. At the moment, the evidence points to a requirement for both apical and basal domains in maintaining apical RG fate, whereas alternative fates do not correlate with inheritance of these domains [41]. Since these studies are correlative, functional studies to examine apical and basal components in cell fate determination and the role of spindle orientation are still needed.
A new model coming from data from mInsc mutant mice may help explain why oblique and vertical orientations influence asymmetric cell fates but planar divisions also give rise to asymmetric outcomes. The fact that mInsc induces production of IPs and basal RGs suggests that its effect on spindle orientation may specifically regulate indirect versus direct neurogenic asymmetric divisions [40 ]. These data point to the intriguing possibility that spindle orientation regulates the type of asymmetric division, rather than whether a division will be asymmetric (Figure 3). This model would suggest that planar asymmetric divisions primarily result in direct neurogenesis while oblique and vertical divisions result in indirect neurogenesis through IPs and basal RGs. It will be important to further test this in the future.
Furthermore, recent structural studies have shed light on a possible competition between mInsc and NuMA for binding to LGN [66 ,67 ,68 ]. This would suggest a potential model whereby spindle orientation machinery primarily directs planar orientations through the action of LGN and NuMA, but when mInsc is present, the communication between LGN and the spindle via NuMA is disrupted by competition with mInsc ( Figure 3). Thus, planar spindle orientation may be an active process of orienting the spindle, while oblique and vertical orientations may reflect a more passive result of inhibiting this orienting machinery. This is in contrast to the switch model seen in Drosophila, as mInsc does not drive strictly vertical orientations in the mammalian neocortex.

Spindle orientation in human evolution and developmental disorders
Several human brain diseases have been proposed to stem from defects in asymmetric cell division and spindle orientation [7]. In particular, microcephaly, a condition involving an abnormally small brain and head, has been linked to potential spindle defects. Notably, all the genes so far identified in primary microcephaly encode proteins with roles at the centrosome/MTOC of the spindle [12]. Three of these genes, MCPH1, ASPM, and CDK5RAP2, have been shown to be required for planar oriented divisions in the mouse neocortex [48 ,49,69], and two of these (MCPH1 and CDK5RAP2) have been shown to be required for centrosome function and centriole duplication [48 ,70]. Furthermore, several of the genes identified in the disorder lissencephaly, which is characterized by a loss of gyri and sulci (the folds and grooves of the cerebral cortex), have also been suggested to have roles in spindle orientation. As mentioned above, LIS1, NDE1, and DCX all regulate spindle orientation [13,17,51], and this has consequences to cell fate specification.
These data from developmental disorders also point to a probable role for spindle orientation in human brain evolution, as this has involved a massive size expansion as well as elaboration of foliation [6]. This may have occurred through genetic changes to factors regulating asymmetric division, and in particular spindle orientation. For example, ASPM and CDK5RAP2 have both been shown to have undergone positive selection along the primate lineage and associate with increased brain size [71][72][73][74].
We are only just beginning to understand some of the molecular factors involved in human brain evolution, but regulators of spindle orientation are likely candidates. The identification of oRGs, and the fact that they are dramatically expanded in humans and other animals with large cerebral cortexes, suggests a potential role in evolutionary expansion of neuronal production. Along these lines, it will be important to examine biological processes governing generation of oRGs. Data from LGN and mInsc mutants suggest that oblique oriented divisions give rise to oRGs [40 ,41,57 ], pointing to the intriguing possibility that oblique spindle orientation may have contributed to evolutionary expansion of the human cerebral cortex.

Concluding remarks
Studies from a diverse array of systems have provided a foundation for understanding the molecular mechanisms of spindle orientation. Many of these mechanisms hold true in the mammalian cerebral cortex as well, and can shed light on asymmetric cell division within this context. Overall, existing data suggest spindle orientation influences asymmetric cell fate though it does not strictly determine whether or not a division will be asymmetric or symmetric. Whether this is due to an inherent stochasticity [75] or an as yet unclear fate determinant, perhaps involving the apical domain, is not yet evident. However, evidence points to a more specific role for spindle orientation in determining the type of asymmetric division in the mammalian neocortex. It will be important to test this model directly as well as whether oblique and vertical divisions reflect a more passive process of disruption of planar orientation as recent interaction evidence suggests.
One of the key remaining questions is how orienting the spindle influences fate determination. Whether this is through asymmetric inheritance of Notch signaling components or other signaling cascade components is still unclear. Now that we have a clearer understanding of the role of spindle orientation, namely in generating IPs and oRGs, we can begin to examine whether factors involved in determination of these progenitor cell types are asymmetrically inherited in a spindle orientation dependent manner.
Finally, an examination of mechanisms of spindle orientation in oRGs would be very exciting, as these seem to undergo asymmetric divisions in the complete absence of an apical domain to help govern the orientation. It may be that the basal domain provides the directional cues responsible for orientation in this context. This would represent quite a divergence from the mechanism used in other systems and should be examined.

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