Asymmetric inheritance of mother versus daughter centrosome in stem cell division

Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. Developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation (Yamashita, 2007).

Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell. The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation (Yamashita, 2007).

Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Unpaired, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity. Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (Yamashita, 2007).

The stereotyped orientation of the mitotic spindle is set up by the positioning of centrosomes during interphase. GSCs remain oriented toward the niche throughout the cell cycle. In G₁ phase, the single centrosome is located near the interface with the hub. When the duplicated centrosomes separate in G₂ phase, one stays next to the hub, whereas the other migrates to the opposite side of the cell. Centrosomes in the GSCs separate unusually early in interphase, rather than at the G₂-prophase transition, so it is common to see GSCs with fully separated centrosomes without a spindle (Yamashita, 2007).

Differences between the mother and daughter centrosomes underlie the stereotyped behavior of the centrosomes in Drosophila male GSCs. The mother centrosome normally remains anchored to the hub-GSC interface and is inherited by the GSC, whereas the daughter centrosome moves away from the hub and is inherited by the cell that commits to differentiation. Mother and daughter centrosomes were differentially labeled by transient expression of green fluorescent protein-pericentrin/AKAP450 C-terminus (GFP-PACT) from the Drosophila pericentrin-like protein under heat shock-Gal4 control. The PACT domain, which is necessary and sufficient for centriolar localization, is incorporated into centrioles only during centrosome duplication and does not exchange with the cytoplasmic pool. Both the mother and daughter centrosomes are labeled by GFP-PACT in the first cell cycle after heat shock. In the second cell cycle, the daughter centrosome retains GFP-PACT, whereas the mother centrosome is not labeled, thus distinguishing the mother and daughter centrosomes. After a short burst of GFP-PACT expression induced by a 2.5-hour heat shock, 20% to 30% of the GSCs had GFP-labeled centrosomes, indicating the duplication of centrosomes during the window of GFP-PACT expression. By 12 hours after heat shock, >90% of the labeled GSCs had two GFP-positive centrosomes, indicating that they had progressed to the G₂ phase of the first cell cycle after GFP-PACT incorporation (Yamashita, 2007).

By 18 to 24 hours after heat shock, the number of GSCs with two GFP-positive centrosomes had decreased, whereas the number of GSCs with one GFP-positive and one GFP-negative centrosome had increased, suggesting progression into the second cell cycle. Generally, the centrosome distal to the hub was labeled, whereas the centrosome proximal to the hub was GFP-negative, indicating that the daughter centrosomes migrate away from the hub-GSC interface during asymmetric GSC divisions (Yamashita, 2007).

Labeling the mother rather than the daughter centrosomes confirmed that the male GSCs in the niche preferentially retain mother centrosomes over time. Centrioles assembled during early embryogenesis were labeled using the NGT40 Gal4 driver to drive the expression of GFP-PACT in blastoderm-stage embryos, shutting off after germband extension. In the first cell cycle after the depletion of the cytoplasmic pool of GFP-PACT in the GSCs, both the mother and daughter centrosomes should be labeled. In subsequent cell cycles, only the mother centrosomes should be labeled (Yamashita, 2007).

In most GSCs in the second or later cell cycle after the depletion of cytoplasmic GFP-PACT, the labeled centrosome was positioned next to the hub-GSC interface, and the unlabeled centrosome had moved away from the hub. The frequency of GSCs that had the proximal, but not distal, centrosome labeled remained constant over time for 10 days (L3 larvae to day-3 adults), suggesting that the mother centrosomes are reliably retained by the GSCs, even through multiple rounds of GSC divisions. Some GSCs maintained cytoplasmic GFP-PACT, especially in L3 larvae, suggesting that the GFP-PACT had not yet been diluted out. Some GSCs were also observed with two labeled centrosomes, suggesting that they are in the first cell cycle after the depletion of cytoplasmic GFP-PACT (Yamashita, 2007).

The mother centrosomes in GSCs appeared to maintain robust interphase microtubule arrays. Ultrastructural analysis of the GSCs revealed that the centrosome proximal to the hub was commonly associated with many microtubules throughout the cell cycle. Nineteen centrosomes in GSCs were scored in serial sections of the apical tips of five wild-type testes. Eleven centrosomes were localized close to the adherens junctions between the hub and the GSCs. Nine of these proximal centrosomes appeared to be in interphase cells, based on nuclear morphology and microtubule arrangement. Typically, these interphase centrosomes proximal to the hub were associated with numerous microtubules. In some samples, microtubules appeared to extend from the centrosome toward the adherens junctions. The other two proximal centrosomes appeared to be in cells in mitotic prophase, based on their robust microtubule arrays containing bundled microtubules running parallel to or piercing the nuclear surface (Yamashita, 2007).

In contrast, of the five distal centrosomes in the apparently interphase cells that were scored, four had few associated microtubules. The remaining three distal centrosomes appeared to be in cells in mitotic prophase, based on microtubule arrays containing bundled microtubules. Thus, the mother centrosomes may maintain interphase microtubule arrays that anchor them to the hub-GSC interface, whereas the daughter centrosomes may initially have few associated microtubules and be free to move, establishing a robust microtubule array only later in the cell cycle (Yamashita, 2007).

Consistent with the idea that astral microtubules anchor the mother centrosomes to the hub-GSC interface, mother versus daughter-centrosome positioning was randomized in GSCs that were homozygous mutant for centrosomin (cnn), an integral centrosomal protein required to anchor astral microtubules to centrosomes. Analysis of mother and daughter centrosomes after transient expression of GFP-PACT revealed that, for cnn homozygous mutant GSCs where one of the two centrosomes was positioned next to the hub, it was essentially random whether the mother or the daughter centrosome stayed next to the hub. In addition, in >25% of total labeled GSCs, neither of the two centrosomes was next to the hub (Yamashita, 2007).

These results indicate that the two centrosomes in Drosophila male GSCs have different characters and fates. The mother centrosome stays next to the junction with the niche and is inherited by the cell that self-renews stem cell fate. Thus, GSCs can maintain an old centriole assembled many cell generations earlier. In contrast, the daughter centrosome migrates away from the niche and is inherited by the cell that will initiate differentiation. It is postulated that the mother centrosomes in male GSCs may remain anchored to the GSC-niche interface throughout the cell cycle by attachment to astral microtubules connected to the adherens junction, whereas the daughter centrosomes may initially have few associated microtubules and thus can move away from the niche. Microtubule-dependent differential segregation of mother and daughter spindle-pole bodies (equivalent to centrosomes in higher organisms) is observed in budding yeast (Pereira, 2001). In cultured vertebrate cells, the centrioles mature slowly over the cell cycle, and the mother centrosomes (containing a mature centriole) attach astral microtubules more effectively and are more stationary than daughter centrosomes in interphase (Piel, 2000). The unusually early separation of centrosomes in interphase male GSCs may provide a way to move the daughter centrosome out of range of the stabilizing influence of the adherens junction complex before it becomes competent to hold a robust microtubule array (Yamashita, 2007).

Developmentally programmed anchoring of the mother centrosome may provide a key mechanism to ensure the stereotyped orientation of the mitotic spindle and thus the reliably asymmetric outcome of the male GSC divisions. Although it is tempting to speculate that determinants associated with the mother or daughter centrosome may play a role in specifying stem cell or differentiating-cell fates, such determinants are yet to be identified. Rather, the asymmetric inheritance of mother and daughter centrosomes in male GSCs may be a consequence of the cytoskeletal mechanisms that are imposed as part of the stem cell program to anchor one centrosome next to the niche throughout the interphase, ensuring a properly oriented spindle (Yamashita, 2007).

Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. It has been shown that their mitotic spindle orientation is established before spindle formation. This study investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a 'dominant centrosome.' This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. A two-step mechanism is proposed to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment (Rusan, 2007).

By prophase, NBs contain two MTOCs that are almost fully separated and aligned along the NB/GMC axis, but analysis of fixed NBs revealed a single MTOC positioned opposite the GMCs before mitotic entry. Thus MTOC behavior was examined throughout the cell cycle as an initial approach to test the hypothesis that fixing the position of one MTOC through successive divisions helps ensure persistent spindle orientation. Live NBs expressing GFP-G147, an MT-associated protein were examined, revealing a striking temporal difference in MTOC activity. During interphase, a single detectable MTOC persists opposite the previous division site; this is referred to as the dominant MTOC. As NBs approach mitosis, this MTOC increases activity (matures; empirically judged by size and MT fluorescence intensity), forming an MT basket around the nucleus. This is referred to as preprophase; this stage is also seen in fixed samples stained for tubulin. Soon after, sometime before the dominant MTOC fully matures, something striking happens: a second MTOC appears distant from the first. This is the second MTOC and this stage is referred to as prophase onset. The second MTOC increases activity, maturing ~10 min before nuclear envelope breakdown (NEB). Using 4D imaging, the possibility was excluded that the second MTOC was present earlier in another focal plane. To further assess this, forming spindles were imaged end on. It is clear that the second MTOC did not emerge from the dominant MTOC or travel around the nucleus. Instead, the second MTOC appeared roughly opposite the dominant MTOC. MTOC separation began immediately, and by NEB, they were 146 ± 20° apart. Thus, NBs form two distinct MTOCs: an MTOC persisting from the previous division and another only activated at mitotic entry (Rusan, 2007).

This distant activation of the second MTOC raised questions about the centrosome cycle. One possibility is that NBs have two MT nucleating centrosomes, but only one can retain MTs and act as an MTOC during interphase, whereas the second acquires MT retention ability during mitotic entry, explaining the second MTOC's sudden appearance. There is precedent for this: mouse L929 cells have two γtub-bearing centrosomes that can nucleate MTs, but only one contains Ninein and can retain MTs to form an MTOC (Rusan, 2007).

To test this hypothesis in NBs, EB1-GFP was used. This binds growing MT plus ends and reliably identifies MT nucleation sites. Only one nucleation site was present in interphase and preprophase, and a new nucleation site appeared distant from the first, consistent with spatially and temporally distinct second MTOC activation. Thus, NBs regulate MT nucleation and not just MT retention (Rusan, 2007).

To examine how the new nucleation center forms, centrosomes were imaged using a PCM protein, GFP-Cnn. NBs contain a single detectable centrosome during interphase. When NBs reenter mitosis, a second Cnn-positive centrosome appears distant from the first, mimicking activation of the second MTOC. To verify that these occur simultaneously, NBs expressing mCherry-Tubulin (chTub) and GFP-Cnn were imaged. This revealed perfect temporal and spatial correlation between the appearance of the second centrosome and activation of the second MTOC. Physical separation of two centrosomes/MTOCs was never seen. This is the first example of asynchronous and physically distant centrosome maturation, suggesting that NBs use a novel centrosome cycle (Rusan, 2007).

Higher temporal/spatial resolution imaging revealed that two GFP-Cnn spots separate during mitotic exit. One GFP-Cnn spot persists as the NB interphase centrosome, forming the dominant MTOC, whereas the other spot disappears. The persistent Cnn spot (centrosome) remains relatively stationary in interphase, consistent with the hypothesis that coarse spindle alignment begins in interphase by anchoring the dominant centrosome (Rusan, 2007).

Centrosome fate was examined in the two daughters (new NB and GMC). They differ dramatically in PCM retention, in contrast to mammalian cells, where both daughters' centrosomes retain PCM. The GMC centrosome sheds all PCM. The new NB centrosome (that becomes the dominant MTOC) retains PCM throughout interphase and further accumulates PCM during the next mitosis. The complete shedding of PCM in GMCs appears to be the normal behavior of interphase centrosomes in most fly cells, whereas in syncytial early embryos, both daughters retain PCM foci through the cell cycle. In contrast to both cell types, the NB daughters exhibit differential PCM retention (Rusan, 2007).

These data suggest that NBs have a novel centrosome cycle in which the second centrosome matures distant from the dominant centrosome/MTOC. One hypothesis to explain this would be the distal positioning of a differentially regulated centriole that is blocked from recruiting PCM in interphase and thus cannot form an MTOC until 'activated' during mitosis. If this centriole is always inherited by the GMC, it might also explain complete PCM loss as GMCs exit mitosis (Rusan, 2007).

Centrioles were examined live to test this hypothesis, using the centriole marker GFP-PACT and Histone-GFP Mother/daughter centrioles disengaged in late telophase, as in mammalian cells and fly embryos. Thus, two NB centrioles are present throughout interphase despite the presence of only one MTOC (Rusan, 2007).

The two centrioles then exhibit different behaviors. One remains fairly stationary, whereas the second moves to roughly the other side of the nucleus. Disengagement perfectly correlates with separation of Cnn spots, suggesting that the stationary centriole retains PCM to form the dominant MTOC and the mobile centriole completely sheds PCM. To test this, NBs were imaged expressing chTub and GFP-PACT. The stationary centriole retained MTs, whereas the mobile centriole did not. Upon reentering mitosis, the mobile centriole regained nucleation activity, forming the second MTOC (Rusan, 2007).

This suggests that full separation of the MTOCs that organize the spindle is biphasic. It begins in interphase, when one centriole retains PCM, remains stationary, and forms the dominant MTOC, whereas the second centriole sheds PCM and becomes mobile. Movement of the second centriole away from the dominant MTOC in interphase accounts for ~70% of the separation needed to form a spindle. Mechanisms of transporting the mobile centriole remain to be identified, but it is nonrandom, as in 26/30 NBs, the second MTOC emerged more than 90° from the dominant MTOC. After the second MTOC is activated, the two separate the last 30%, most likely via MT sliding forces. This might explain defects in lissencephaly1 mutants, where MTOCs are only separated by 124° at NEB. Perhaps interphase centriole movement is normal, but MT-based MTOC separation is defective (Rusan, 2007).

These data suggest that NBs differentially regulate the activity of their two centrioles within the same cytoplasm. Interestingly, a similar observation was made in clam eggs, which have three centrosomes just after fertilization. The sperm centrosome is functionally inactivated, whereas female centrosomes organize the meiotic spindle (Rusan, 2007).

Next NB centrosome regulation was examined. In preprophase, one centriole (marked by anti-DPLP) formed the dominant MTOC, whereas the second centriole had no associated MTs and was randomly positioned, confirming the live-cell data. Thus whether γtub is recruited asymmetrically was examined. Fixed preprophase NBs had two centrioles; only that opposite the GMCs accumulated γtub. Further, both γtub and Cnn are absent from the NB centriole nearest the GMCs in interphase/preprophase (Rusan, 2007).

Polo kinase promotes centrosome maturation by promoting γtub recruitment during mitotic entry. Differences in Polo localization/activity might underlie differences in timing of NB centrosome maturation. NBs expressing Polo-GFP and the centriole marker mCherry-DSAS-6 were examined. Only the centriole pair that forms the dominant centrosome was Polo-GFP positive during preprophase. Polo-GFP accumulated on the mobile centriole pair as the NB entered mitosis, increased on both centriole pairs through prophase, and moved on to kinetochores. When Polo-GFP was examined in cells exiting mitosis, it was retained at low levels on the dominant centrosome. In the future, it will be interesting to examine the localization of Aurora A, another centrosome regulator (Rusan, 2007).

Unlike the distal appendages of mammalian mother centrioles, fly mother and daughter centrioles have been thought to have no known ultrastructural or molecular differences. These data suggest that differences exist. It is unlikely that this differential regulation is a result of location, as both centrioles are initially adjacent after disengagement. The differences may be due to centriole age or procentriole maturation state (Rusan, 2007).

The NB spindle is largely aligned by NEB. Based on the current data, the hypothesis was tested that the dominant centrosome helps define one spindle pole before prophase. The angle between the dominant centrosome/MTOC axis and the anaphase axis was calculated, using the nuclear centroid as a fixed reference. This revealed two phases in defining the future spindle axis. Through prophase onset, the dominant centrosome remains fairly stationary roughly opposite the GMCs, agreeing with fixed images, whereas the second centriole moves to a distal position (to within 46 ± 33° of the anaphase axis). This is consistent with the hypothesis. The dominant centrosome may be immobilized by aster–cortex interactions or by absence of an active displacement mechanism. In the second phase, alignment is refined in prophase and prometaphase (the angle between the NB centrosome and anaphase axes decreases from 31 ± 29° to 15 ± 12° (Rusan, 2007).

Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. This study shows that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. GSCs containing misoriented centrosomes accumulate with age, and these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, it is proposed that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. It was also shown that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia (Cheng, 2008).

GSCs with misoriented centrosomes accumulate as flies age. Since such misoriented GSCs divide less frequently as compared to oriented GSCs, accumulation of misoriented GSCs contributes to the decline in spermatogenesis that occurs with age. Although misoriented GSCs rarely divide, they are not permanently arrested (or senescent) and are correctly oriented when they divide. Whether correction of GSC orientation is an active process that is part of the acquisition of stem cell identity remains to be determined. The low cell cycle activity of misoriented GSCs may also suggest that mechanisms are in place to detect misorientation and induce cell cycle arrest in response to this change, although the underlying mechanisms remain to be identified (Cheng, 2008).

It was also demonstrated that misoriented GSCs originate, at least in part, from dedifferentiation of spermatogonia. Although dedifferentiated GSCs have high frequency (>40%) of centrosome misorientation, they can function as stem cells by resuming the cell cycle, with correctly oriented mitotic spindles just like as constitutive GSCs. GSC numbers do not decrease as quickly as expected from the calculated GSC half-life, suggesting that a mechanism to compensate for the loss of GSCs exists. Since misoriented spindles, or symmetric stem cell division, was rarely observed, it is speculated that dedifferentiation is the major mechanism to replace stem cells over time in the Drosophila male germ line (Cheng, 2008).

A decline in GSC number in older males (day 50) was reported recently (Boyle, 2007) This decrease in stem cell number is likely due to failure of the niche function (via decreased signal from the niche as well as decreased E-cadherin-based attachment between the niche and GSCs. However, the decrease in the production of spermatogonia and testis involution precede the loss of GSCs such that decreasing GSC numbers cannot explain the testis involution that is observed at younger ages (Cheng, 2008).

The present results provide a novel mechanistic link between the control of stem cell polarity and the age-related decline in tissue regenerative capacity. Mechanisms responsible for monitoring stem cell orientation with respect to the niche not only prevent overproliferation of stem cells by ensuring the asymmetric outcome of the stem cell division, but they contribute to the decline in tissue regenerative capacity during aging. Many of the misoriented GSCs originate from the dedifferentiation of spermatogonia, a mechanism thought to be responsible for maintaining the stem cell population over extended periods of time. Therefore, although GSCs produce less progeny over time, the system appears to maximize the number of progeny produced throughout life, while maintaining asymmetric stem cell division (Cheng, 2008).

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. This study shows that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex (Wang, 2009).

Radial glia cells constitute a major population of neural progenitor cells that occupy the proliferative VZ in the developing mammalian neocortex. In addition to their well-characterized function as a scaffold in supporting neuronal migration, radial glia cells display interkinetic nuclear oscillation and proliferate extensively at the luminal surface of the VZ (that is, the VZ surface). During the peak phase of neurogenesis [around embryonic day 13-18 (E13-E18) in mice] they predominantly undergo asymmetric division to self-renew while simultaneously giving rise either directly to a neuron, or to an intermediate progenitor cell which subsequently divides symmetrically to produce neurons. Whereas differentiating progeny progressively migrate away from the VZ to form the cortical plate (CP) (the future neocortex) renewing radial glia progenitors remain in the VZ for subsequent divisions. The distinct migratory behaviour of radial glia progenitors and their differentiating progeny is fundamental to the proper development of the mammalian neocortex; however, little is known about the basis of these behavioural differences (Wang, 2009).

Centrosomes, the main microtubule-organizing centres in animal cells, have an important role in many cell processes, particularly during cell division. All normal animal cells initially inherit one centrosome, consisting of a pair of centrioles surrounded by an amorphous pericentriolar material. The two centrioles differ in their structure and function. The older 'mother' centriole, which is formed at least one-and-a-half generations earlier, possesses appendages/satellites that bear specific proteins, such as cenexin (also known as Odf2), and anchor microtubules and support ciliogenesis. In contrast, the younger 'daughter' centriole, which is formed during the preceding S phase, lacks these structures. Full acquisition of appendages/satellites by the daughter centriole is not achieved until at least one-and-a-half cell cycles later. During each cell cycle, the centrosome replicates once in a semi-conservative manner, resulting in the formation of two centrosomes: one of which retains the original old mother centriole (that is, the mother centrosome) while the other receives the new mother centriole (that is, the daughter centrosome). This intrinsic asymmetry in the centrosome has recently been demonstrated to be important for proper spindle orientation during the division of male germline stem cells and neuroblasts in Drosophila, although female germline stem cells appear to divide normally in the absence of centrioles/centrosomes. These studies indicate a critical role for the differential behaviour of centrosomes with differently aged mother centrioles in asymmetric division of the progenitor/stem cells, although it remains unclear whether proper behaviour and development of the progenitor/stem cells and their differentiating daughter cells depend on centrosome asymmetry. Asymmetric division of radial glia progenitors accounts for nearly all neurogenesis in the developing mammalian neocortex. Three out of four autosomal recessive primary microcephaly (MCPH) genes identified so far encode centrosomal components, suggesting that proper neocortical neurogenesis and development entail a tight regulation of the centrosome, which is so far poorly understood. To address these issues, centrosome regulation during the peak phase of mammalian neocortical neurogenesis was investigated (Wang, 2009).

To examine centrosome behaviour, a plasmid encoding centrin 1, a central component of the centriole, fused with enhanced green fluorescent protein (EGFP-CETN1) was introduced into the developing neocortex of E13.5 mouse embryos by in utero electroporation. As expected, EGFP-CETN1 formed pairs of dots that co-localized with γ-tubulin, a centrosomal marker, suggesting that transient expression of EGFP-CETN1 reliably labels the two centrioles of individual centrosomes in the developing neocortex in vivo. Moreover, it was observed that at the onset of peak neurogenesis (E13-E14), EGFP-CETN1-labelled centrosomes were predominantly located at the VZ surface with a small subset located in the subventricular zone (SVZ) and the intermediate zone (IZ) (Wang, 2009).

To identify the cell types harbouring EGFP-CETN1-labelled centrosomes, a plasmid encoding DsRedexpress (DsRedex), a red fluorescent protein that diffuses throughout cells and thereby reveals their morphology, was co-electroporated . In bipolar radial glia progenitors in the VZ the centrosome was located in their ventricular endfeet at the VZ surface, whereas in multipolar cells in the IZ and the SVZ the centrosome was harboured in their cell bodies. Moreover, dividing radial glia progenitors were observed that possess a pair of centrosomes together with condensed chromosomes at the VZ surface. Consistent with this differential centrosome localization between radial glia progenitors and their differentiating daughter cells, a progressive increase was observed in the appearance of EGFP-CETN1-labelled centrosomes in the IZ and the CP as development proceeded, in addition to some that remained at the VZ surface. This gradual increase in centrosome localization in the IZ and the CP coincided with the production and migration of differentiating cells such as neurons to these regions during this period (Wang, 2009).

The distinct positioning of the centrosome in radial glia progenitors versus their differentiating progeny prompted asking whether the centrosomes in these two cell populations/types are different. To explore this, a plasmid encoding ninein, a mature centriole-specific protein that localizes to appendages/satellites, fused with EGFP (EGFP-Nin) together with a plasmid encoding CETN1 fused to DsRedex (DsRedex-CETN1) were electroporated into the developing mouse neocortex at E13.5. As expected, both EGFP-Nin and DsRedex-CETN1 formed dot-like structures and co-localized to the centrosomes, especially those at the VZ surface, as identified by an antibody to the integral centrosomal protein pericentrin 1. Notably, EGFP-Nin was preferentially concentrated at one of the two centrioles marked by DsRedex-CETN1 in individual centrosomes, suggesting that the two centrioles in interphase radial glia progenitors are not identical. Given that Nin specifically associates with mature centrioles, these results indicate that the centriole with abundant EGFP-Nin is the more mature mother centriole, whereas the one with little EGFP-Nin is the less mature daughter centriole. A similar inequity in the recruitment of EGFP-Nin by the duplicated centrosomes was also observed in dividing radial glia progenitors at the VZ surface, indicating that the duplicated centrosomes are not identical during late mitosis. The centrosome with abundant EGFP-Nin is probably the centrosome that retains the mature old mother centriole and the centrosome with little EGFP-Nin is probably the centrosome that bears the relatively immature new mother centriole (Wang, 2009).

Having found that the centrosomes in dividing radial glia progenitors exhibit asymmetry in their maturity, it was next asked whether this centrosome asymmetry is related to the distinct behaviour of radial glia progenitors and their differentiating progeny in the developing neocortex during neurogenesis. To address this, the relative distribution of centrosomes labelled by DsRedex-CETN1 versus those labelled by EGFP-Nin in the developing neocortex were compared as development proceeded. Interestingly, whereas DsRedex-CETN1-labelled centrosomes progressively occupied the IZ and the CP, where differentiating cells are situated, EGFP-Nin-labelled centrosomes were mostly found in the VZ, where radial glia progenitors are located. Given that DsRedex-CETN1 labels all centrosomes whereas EGFP-Nin selectively labels mature centrosomes, these results point to an intriguing possibility that the duplicated centrosomes in dividing radial glia cells are differentially inherited depending on their age and maturity during neocortical neurogenesis. It is known that during each cell division one centrosome retains the old mature mother centriole and the other bears the new less mature mother centriole. Thus, these results suggest that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors (Wang, 2009).

To test this, an assay was developed to distinguish explicitly between the centrosome containing the old mother centriole and the centrosome containing the new mother centriole in the developing neocortex in vivo. The assay takes advantage of the photoconvertible fluorescent protein Kaede, which changes from green to red fluorescence on exposure to violet light. Centrioles in the developing neocortex were labelled by transient expression of CETN1 fused with Kaede (Kaede-CETN1). Photoconversion was then performed to switch labelled centrioles from green to red fluorescence. This green-to-red fluorescence conversion of Kaede proteins is irreversible and the red protein is very stable, thus allowing long-term tracking of the existing photoconverted proteins and the structures with which they are associated. Moreover, all newly synthesized Kaede proteins are green fluorescent. It is known that centriole duplication requires new protein synthesis of centrin. As a result, newly duplicated centrioles that contain newly synthesized Kaede-CETN1 are green fluorescent, whereas previously existing centrioles are red fluorescent. Hence, in the first cell cycle after photoconversion, both centrosomes contain a red fluorescent mother centriole and a green fluorescent daughter centriole. However, in the second and subsequent cell cycles, centrosomes with the new mother centriole contain only green fluorescent centrioles, whereas centrosomes retaining the original old mother centriole harbour both red and green fluorescent centrioles, thus distinguishing between centrosomes with differently aged mother centrioles (Wang, 2009).

To carry out this assay in the developing neocortex in vivo, an in utero photoconversion procedure was developed and and combined with in utero electroporation. Kaede-CETN1, which localized specifically to the centrosomes, was introduced into the developing mouse neocortex at E13.5. One day later, that is, E14.5, the forebrain of electroporated embryos was treated with a short exposure of violet light while still in the uterus, which effectively converted nearly all Kaede-CETN1 proteins and their labelled centrosomes from green to red fluorescence [E13.5-E14.5(PC)]. The uterus was replaced and the embryos continued to develop in vivo. The localization and inheritance of centrosomes were analysed at different developmental stages thereafter (Wang, 2009).

It was found that one day after photoconversion [E13.5-E14.5(PC)-E15.5], around 95% of centrosomes contained both red and green fluorescent centrioles (indicated by yellow colour in the merged image), consistent with the notion that the labelled cells have undergone one round of division and have duplicated their centrioles during the 24-h period after photoconversion. This was shown directly by imaging centrosomes at high magnification, revealing that each centriole was mostly only red or green fluorescent. This also demonstrates that there is little diffusion of centrin proteins between duplicated centrioles, or between the centrioles and a cytoplasmic pool which was confirmed by fluorescence recovery after photobleaching (FRAP) experiments. Moreover, it was found that more than 30% of centrosomes possessed only green fluorescent centrioles 2 days after photoconversion [E13.5-E14.5(PC)-E16.5]. The appearance of the solely green fluorescent centrosomes 48 h after photoconversion indicates that the initially labelled radial glia progenitors have undergone two rounds of division during this period; this is consistent with the previous observation that the duration of the neocortical progenitor cell cycle is about 12 to 20 h around this developmental stage. The ongoing division of labelled radial glia cells at a normal rate suggests that expression of Kaede-CETN1 and the photoconversion procedure had no effect on their cell cycle. In addition, no obvious DNA damage or cell death was induced by the photoconversion treatment. Besides the green and yellow fluorescent centrosomes, about 4% of solely red fluorescent centrosomes were observed, indicating that a few labelled cells do not undergo cell division during this period (Wang, 2009).

Having successfully distinguished the centrosomes with differently aged mother centrioles in the developing neocortex in vivo, their distribution was examined to determine whether they are asymmetrically segregated. Remarkably, it was found that more than 76% of centrosomes with the new mother centriole (that is, only green fluorescent) were located in the IZ and the CP, whereas around 78% of centrosomes with the old mother centriole (that is, both green and red fluorescent) were located in the VZ in addition to the SVZ. These results demonstrate that the centrosomes with differently aged mother centrioles are asymmetrically segregated in the developing neocortex during the peak phase of neurogenesis. It is worth noting that a small fraction of both green and red (that is, yellow) fluorescent centrosomes was found in the IZ and the CP and that these centrosomes probably originated from the first cell cycle after photoconversion (Wang, 2009).

The asymmetric segregation of centrosomes suggests differential regulation of the duplicated centrosomes in dividing radial glia progenitors. To gather further evidence for this, time-lapse imaging experiments were carried out to monitor the behaviour of centrosomes with differently aged mother centrioles in dividing radial glia progenitors at the VZ surface in situ. Kaede-CETN1 was introduced into radial glia cells together with mPlum, a far-red fluorescent protein, to label cell morphology. Around 24 h later, cortical slices were prepared. Photoconversion of the existing Kaede-CETN1 proteins was then performed in individual slices, which were then cultured for another 24 h before being subjected to time-lapse imaging. Labelled dividing radial glia cells with enlarged and rounded cell bodies possessing a pair of centrosomes at the VZ surface were monitored at 10-min intervals over a period of 5 to 8 h. In six out of seven dividing radial glia cells that proceeded through mitosis at the VZ surface and reached the two-cell stage, the centrosome retaining the old mother centriole in both red and green fluorescence stayed at the VZ surface, whereas the centrosome containing the new mother centriole in solely green fluorescence migrated away from the VZ surface. These results demonstrate that the centrosomes with differently aged mother centrioles in dividing radial glia progenitors exhibit distinct behaviour during the peak phase of neurogenesis (Wang, 2009).

The distinct behaviour of the centrosomes suggests that they are differentially inherited by the two daughter cells embarking on different routes of fate specification and development. On the basis of their behaviour, it is postulated that the centrosome with the new mother centriole is largely inherited by differentiating cells, such as neurons, whereas the centrosome with the old mother centriole that remains located at the VZ is mostly inherited by radial glia progenitors. Indeed, it was found that 2 days after photoconversion [E13.5-E14.5(PC)-E16.5] the centrosomes with the new mother centriole, marked by green fluorescence alone, were mostly associated with cells expressing TUJ1, a differentiating neuronal marker, in the CP and the IZ. In contrast, the centrosomes that retained the old mother centriole in yellow (that is, both green and red) fluorescence at the VZ were largely associated with cells expressing Pax6, a radial glia progenitor marker. These results show that the centrosomes with differently aged mother centrioles in dividing radial glia cells are asymmetrically inherited by the two daughter cells: whereas the renewing radial glia progenitor inherits the centrosome with the old mother centriole, the differentiating daughter cell inherits the centrosome with the new mother centriole (Wang, 2009).

These data thus far show that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors in the developing neocortex. Next, whether the selective inheritance of the centrosome with the old mature mother centriole by radial glia progenitors is necessary for their maintenance at the VZ was tested. Should this be the case, given that Nin is an essential component of the appendage/satellite structures specific to the mature centriole, it was predicted that removal of Nin, which prevents centriole maturation, would disrupt asymmetric segregation of centrosomes with differently aged mother centrioles and impair the maintenance of radial glia progenitors in the developing neocortex (Wang, 2009).

To test this, short hairpin RNA (shRNA) sequences were developed against Nin, that effectively suppressed its expression (Nin shRNAs). Consistent with the prediction, expression of Nin shRNA, but not control shRNA, disrupted asymmetric segregation of centrosomes with differently aged mother centrioles labelled with Kaede-CETN1 in the developing neocortex, suggesting that Nin is necessary for centriole maturation, thereby generating asymmetry between duplicated centrosomes. The presence of solely green fluorescent centrosomes in Nin shRNA-expressing cortices indicates that centrosome duplication and segregation and cell division are not severely affected by removal of Nin. More importantly, it was found that removal of Nin caused a premature depletion of cells from the VZ, where radial glia progenitors reside. This effect of Nin shRNAs correlated with their efficacy in suppressing Nin protein expression and was rescued by a shRNA-insensitive Nin plasmid, suggesting that the effect of the Nin shRNA is due to a specific depletion of the endogenous Nin protein. A similar reduction in cells in the VZ was observed when Nin expression was suppressed using small interfering RNA (siRNA) (Wang, 2009).

To characterize further the extent to which removal of Nin leads to a depletion of radial glia progenitors, the fate specification was examined of cells expressing either control or Nin shRNA. When compared with the control, expression of Nin shRNA led to a marked reduction in the percentage of cells positive for Pax6 and glutamate transporter (GLAST), two radial glia progenitor markers, and a significant increase in the percentage of cells positive for TUJ1, a differentiating neuronal marker. These results suggest that removal of Nin leads to a depletion of radial glia progenitors and a concomitant increase in differentiating neurons. Consistent with this, a significant reduction was observed in phospho-histone 3 (P-H3)-labelled mitotic cells at the VZ surface and a marked increase in cell cycle exit. No obvious change in the cleavage plane orientation of late stage mitotic cells at the VZ surface was observed (Wang, 2009).

Previous studies showed that the carboxy-terminus of Nin is responsible for its localization to the centriole and expression of this region displaces endogenous protein at the centriole. Interestingly, it was found that, similar to removal of Nin, expression of the carboxy-terminus of Nin (Nin-Cter) led to a premature depletion of radial glia progenitor cells from the VZ, suggesting that centriolar Nin is critical for maintaining radial glia progenitor cells in the VZ. Taken together, these results indicate that preferential inheritance of a centrosome containing the mature mother centriole is required for the maintenance of radial glia progenitors in the proliferative VZ of the developing neocortex (Wang, 2009).

The results presented in this study suggest that the centrosomes with differently aged centrioles in asymmetrically dividing radial glia progenitors exhibit different behaviour and are differentially inherited by the two daughter cells during the peak phase of mammalian neocortical neurogenesis. Whereas the centrosome with the less mature new mother centriole migrates away from the VZ surface and is largely inherited by differentiating cells, the centrosome with the more mature old mother centriole stays at the VZ surface and is predominantly inherited by renewing radial glia progenitors. Recently, asymmetric behaviour of centrosomes has been observed during asymmetric division of Drosophila male germline stem cells and neuroblasts. The findings of this study suggest that this type of asymmetric centrosome regulation may be a general feature of asymmetric cell division across species. Furthermore, the findings provide new insight into centrosome regulation in the developing mammalian neocortex, which has been linked to the pathogenesis of human microcephaly (Wang, 2009).

Centrosomes with differently aged mother centrioles differ in their protein composition and thereby in their biophysical properties, such as microtubule anchorage activity. In this study, it was found that Nin, an appendage/satellite-specific protein required for centriole maturation, localized differently to the duplicated centrosomes in radial glia progenitors in late mitosis. Notably, another appendage/satellite-specific protein cenexin was recently found to be asymmetrically localized to centrosomes in sister cells after mitosis; moreover, the cell receiving the more mature old mother centriole usually grew a primary cilium first (Anderson, 2009). The asymmetric inheritance of centrosomes with distinct biophysical properties may thereby differentially regulate the behaviour and development of the daughter cells that receive them. For example, given that primary cilia have essential roles in a number of signal transduction pathways, including Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signalling, the asynchrony in cilium formation could differentially influence the ability of the two daughter cells to respond to environmental signals and thereby their behaviour and fate specification. Furthermore, the strong microtubule anchorage activity associated with the centrosome retaining the older mother centriole would facilitate its anchorage to a specific site (for example, the VZ surface), thereby tethering the cell that inherits it. Indeed, it was found that disruption of centriole maturation by removing Nin not only impairs asymmetric segregation of centrosomes, but also depletes radial glia progenitors from the VZ, a proliferative niche in the developing mammalian neocortex. Aside from their participation in microtubule organization and ciliogenesis, centrosomes associate with messenger RNAs (mRNAs) and membrane-bound organelles such as the Golgi and recycling endosomes and regulate protein degradation, thereby raising the possibility that asymmetric centrosome inheritance might contribute to proper segregation of cell fate determinants to the two daughter cells of asymmetrically dividing progenitor/stem cells (Wang, 2009).