centrosomin
DEVELOPMENTAL BIOLOGY

Embryonic

An immunopurification method was used to clone target genes of the Antennapedia protein (ANTP). centrosomin (cnn) was one of the target genes isolated using this approach. The spatial and temporal expression of the cnn gene in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS) of wild-type and homeotic mutant embryos is consistent with the idea that cnn is a homeotic target. In the VM, Antp and abdominal-A (abd-A) negatively regulate cnn, while Ultrabithorax (Ubx) shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Characterization of a cDNA encoding CNN predicts a novel structural protein with three leucine zipper motifs and several coiled-coil domains exhibiting limited homology to the rod portion of myosin. Immunocytochemical results demonstrate that the cnn encoded protein is localized to the centrosome and the accumulation pattern is coupled to the nuclear and centrosome duplication cycles of cleavage. In addition, evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction. The centrosomal localization of CNN and the involvement of microtubules in midgut morphogenesis suggest that this protein may participate in mitotic spindle assembly and the mechanics of morphogenesis through an interaction with microtubules, either directly or indirectly (Heuer, 1995).

The detection of two transcripts by the cDNA in northern analysis indicates alternate splicing, though this was not characterized further. In situ hybridizations to whole-mount embryos using the cDNA indicates that cnn is expressed in cells of the mesoderm and developing CNS and peripheral nervous system (PNS). The cnn mRNA is detected as early as stage 5 at cellular blastoderm. High levels of expression are detected in the gastrulating embryo along the ventral and cephalic furrows and later in the mesoderm. At the time of germ band extension, at stage 10, cnn mRNA begins to accumulate in large cells presumed to be neuroblasts of the CNS and by stage 11 is seen in cells of the PNS. By stage 12, cnn expression predominates in the CNS and PNS. At a later time, expression diminishes in the PNS, and more cells in the thoracic part of the nerve cord contain cnn mRNA relative to the abdominal region. From stage 11 to stage 16, cnn mRNA is observed in two domains of the VM surrounding the midgut. Double labeling experiments for cnn mRNA and SCR, ANTP or UBX in the VM indicate that the anterior domain (domain 1) of cells expressing cnn is located just anterior to cells expressing SCR and that the posterior domain (domain 2) overlaps with cells expressing ANTP or UBX (Heuer, 1995).

The expression pattern in the VM suggests homeotic regulation. Therefore, the transcript pattern of cnn was examined in the VM of homozygous mutant embryos for homeotic proteins. In Antp mutant embryos, cnn transcripts are detected ectopically in cells that normally express Antp . Accumulation of cnn mRNA appears normal in Scr mutant embryos. Embryos that lack both Ubx and abd-A, express cnn in a pattern clearly different than wild type. The cnn expression that normally overlaps with Ubx is absent, but cnn transcripts are now found in cells of the VM where abd-A is normally expressed (Heuer, 1995).

The pattern of cnn expression in the CNS of a shortened germ band embryo also suggests homeotic regulation, so the CNS was examined for cnn expression in homeotic null mutants. Transcripts of cnn are normally visible in cells of the brain hemispheres and ventral nerve cord, with higher levels in the thoracic neuromeres compared to the abdominal neuromeres. Expression of cnn appears reduced in the thoracic neuromeres and is more similar to the abdominal neuromeres in Antp mutant embryos. In contrast, embryos deficient for both UBX and ABD-A express higher than normal levels of cnn mRNA throughout the posterior ventral nerve cord (Heuer, 1995).

CNN is localized to the centrosome at all stages of the cell cycle. The centrosomal staining pattern of the protein appears to change with different phases of mitosis. At prophase, CNN is detected in the two centrosomes located at opposite ends of the nucleus. In metaphase, CNN is associated in a compact manner with the two centrosomes located at the poles of the mitotic spindle. During anaphase, CNN distribution changes from compact to dispersed with many projections and appears to increase with the duplication of the centrosome. Between anaphase and telophase, many small staining dots are observed in addition to the larger dots. This was much more apparent with paraformaldehyde fixation than with methanol fixation (Fig. 5E). At telophase, CNN is associated with the newly duplicated centrosomes that appear at each pole (Heuer, 1995).

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).

The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis

Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. The COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. By establishing colcemid-induced microtubule depolymerization as a sensitive assay, this study examined the characteristics of COC activity and found that it functions uniquely in GSCs but not in their differentiating progeny. The COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types (Venkei, 2015).

Stereotypical orientation of the mitotic spindle is a widely utilized mechanism to achieve asymmetric cell division. Although considerable knowledge has accumulated regarding how spindle orientation is established, little is known about whether cells possess a mechanism that monitors successful spindle orientation. In the present study, using colcemid treatment as a sensitive assay, the nature of the COC, which is the only known orientation/polarity checkpoint in multicellular organisms, was was investigated. It was established that: (1) the COC specifically operates in GSCs, but not differentiating germ cells (GBs and SGs); (2) the COC operates in G2 phase of the cell cycle to prevent precocious entry into mitosis upon centrosome misorientation; and (3) as a checkpoint mechanism, the COC is distinct from the SAC (Venkei, 2015).

These results show that the COC is a GSC-specific checkpoint that monitors centrosome orientation and arrests cells in G2 phase when centrosomes are not correctly oriented. It remains unclear whether the COC-mediated G2 arrest might eventually undergo adaptation to allow mitotic entry, as is the case with mitotic slippage in the SAC. It is worth noting in this context that the GSC mitotic index never increased during 6 h of colcemid treatment, whereas SGs seem to undergo mitotic slippage by 6 h of colcemid treatment. This suggests that the COC-mediated G2 arrest is relatively strong. The findings that the mad2 mutation has no effect on G2 arrest of GSCs and that par-1 and cnn mutants have no effect on mitotic arrest in GBs/SGs upon colcemid treatment strongly support the notion that the COC and SAC constitute distinct checkpoint mechanisms. Although CySCs and SGs also orient their mitotic spindles, the present study shows that spindle orientation in these cells is not under the control of the COC. However, the lack of a COC in these cell types does not exclude the possibility that distinct polarity checkpoint mechanisms are in place to ensure correct spindle orientation. If the arrest points of such checkpoints are not prior to the arrest point of the SAC (i.e. metaphase), the assay using colcemid would not reveal their presence (Venkei, 2015).

Mutant analysis of multiple centrosomal components in this study revealed a selective requirement for centrosomal components in the function of the COC. Sas-4 and Cnn are crucial for COC function, whereas Spd-2 and Apc1 are not. This indicates that not all of the centrosomal proteins are involved in COC function. Conversely, not all COC components are localized to the centrosome. As shown in a previous study, an essential component of the COC, Par-1, is localized to the spectrosome, where it regulates the localization of Cyclin A to regulate mitotic entry. How the information on centrosomal orientation is communicated to the spectrosome, where Par-1 and Cyclin A localize, remains to be determined. A major outstanding question in understanding the COC is how it senses the location of the centrosome with respect to the hub cells. Previous studies have shown that the mother centrosome is anchored to the adherens junctions formed at the hub-GSC interface via MTs. Therefore, it is plausible that the COC senses aspect(s) of these interactions. It awaits future investigation to understand how the association of the centrosome with the hub-GSC interface is mechanistically sensed, and how such information is integrated with the activity of COC component(s) on the spectrosome (Venkei, 2015).

In summary, this present work establishes that the COC is a checkpoint mechanism that is distinct from the SAC and monitors correct centrosome orientation specifically in GSCs. It is speculated that a similar mechanism might be in place in other systems that rely on asymmetric cell division (Venkei, 2015).

Effects of Mutation or Deletion

The Centrosomin protein is required for centrosome assembly and function during cleavage

Centrosomin is a 150 kDa centrosomal protein. To study the function of Centrosomin in the centrosome, mutations were recovered that are viable but male and female sterile (cnnmfs). These alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and gamma-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions (Megraw, 1999).

The composition of the centrosome is dynamic during development and each cell cycle. The fact that centrosomes are assembled in a step-wise fashion upon fertilization has been demonstrated in partial in vitro reconstitution experiments using Xenopus sperm head and egg extracts. Numerous experiments have shown that centrosomes are modified at the onset of M phase to acquire the capability to nucleate spindle microtubules. Such modifications include the accumulation of PCM and the phosphorylation of centrosomal components. The assembly of the centrosome, either at the onset of fertilization, or at the transition from interphase to M phase, is very poorly understood. Although numerous proteins have been identified that are localized to the centrosome, the functions of most of them in the assembly of a functional centrosome are not known. Using a genetic analysis of one such protein, this study shows that Centrosomin is required for the assembly of functional centrosomes during the syncytial divisions in Drosophila. In cnn mutants, the assembly into the centrosomes of several Drosophila centrosomal components, including CP60, CP190 and gamma-tubulin, are severely affected (Megraw, 1999).

Maternal effect sterile mutations of cnn result in nuclear division and spindle organization defects during the early stages of the syncytial cleavage divisions. The effect appears to be gradual and cumulative. In the early cycles, there are more successful nuclear divisions, and the number of nuclei increases. However, by cycles 8 and 10, during cortical migration, most of the nuclei have become polyploid and remain at the interior of the embryos. Only patches of the cortex are occupied by nuclei and spindles (Megraw, 1999).

Several mechanisms could explain the nuclear fusion and nuclear fall-out phenotypes seen at these later stages. For example, protein synthesis and DNA replication defects, caused either by mutations in glutamine synthetase or by the injection of the DNA replication inhibitor aphidicolin, both lead to the recession of large numbers of nuclei into the interior of the embryo. Disruption of the cortical actin filament organization, e.g., by injection of antibody against the 95F unconventional myosin, also leads to nuclear fusions. Moreover, mutations in the nuclear-fallout (nuf) locus, which encodes a centrosomal protein, disrupt the organization of actin and other components into the metaphase and cytokinetic furrows. This results in nuclear fusions and the subsidence of these nuclei to the interior of the nuf mutant embryos. Two observations suggest that the maternal effect phenotypes in cnn mutants are also caused by centrosomal defects. First, spindle morphology, even at the very early stages, is abnormal. Many of the spindles in these embryos are anastral, and microtubule bundles fail to converge at the poles. These spindles are reminiscent of spindles built from the 'inside-out', i.e., nucleated by chromosomes and bundled by microtubule binding proteins. Second, localization of known centrosomal proteins, including CP60, CP190 and gamma-tubulin are severely affected. CP60 is never observed at the spindle poles and CP190 and gamma-tubulin are present at very low levels only in spindles with confined poles, and are apparently absent from the broad poles of the abnormal spindles (Megraw, 1999).

Furthermore, the spindle defects and nuclear fusions that are observed in the maternal effect cnn mutants are similar to those seen in parthenogenic Sciara embryos that contain no centrosomes. In the absence of live analysis however, it cannot be concluded whether the spindle and nuclear fusion defects that occur are differentially affected by the presence of astral microtubules. If Centrosomin affects the assembly of functional units into the centrosome, what centrosome functions (i.e., duplication, separation, microtubule organizing activity) are defective in embryos deficient in Centrosomin? Does a reduction of Centrosomin levels affect the assembly of all centrosomes, including the first centrosome built upon the sperm basal body, or does it only affect the maturation of the centrosomes during subsequent nuclear division cycles? The spindle defects observed indicate that all aspects of centrosomal function are affected. However, the gradual and multiphasic nature of nuclear loss suggests that none of these functions are completely blocked. Although many of the spindles do not have confined poles and therefore may not have centrosomes, there are always some spindles with astral microtubules and very low amounts of gamma-tubulin at the spindle poles, especially during the early cycles. It is suggested that the latter class of spindles have functionally defective centrosomes at their spindle poles (Megraw, 1999).

During the syncytial blastoderm divisions in Drosophila, mitotic apparati have to be separated from each other to prevent aberrant chromosomal attachments. In wild-type embryos, this is achieved by the formation of 'pseudocleavage furrows', which are transient membrane invaginations that surround each mitotic apparatus during the nuclear divisions. Formation of these furrows depends on interactions between the cortical actin filaments and the centrosomes. Defective centrosomal function could therefore lead to abnormal spindle spacing and the observed failure of somatic cellularization. Consistent with this possibility is the chaining/rosette phenotype observed in the cnn mutant animals during the syncytial cleavage divisions (Megraw, 1999).

The recovered cnn mutations do not appear to block centrosomal function completely. This is manifest in two ways. The first is seen in the fact that the observed spindle defects appear to be progressive during cleavage. That is, the earlier divisions appear to be somewhat less affected than those occurring later. A second puzzle resides in the fact that no lethal alleles of cnn have been recovered despite the fact that Centrosomin is accumulated in the centrosomes of all mitotically dividing cells. Thus, it could be assumed that spindle defects like those observed in cleavage might also be observed in dividing cells later in development. If all of the recovered mutant alleles were hypomorphs it would be possible to rationalize these observations. However, it is difficult to do so based on the fact that cnnhk21 and the other five alleles all exhibit similar phenotypes while hk21 encodes a truncated peptide of 106 amino acids and is probably a functional null (Megraw, 1999).

Consequently, it would appear that Centrosomin is dispensable for somatic cell division and is only required for the rapid syncytial cleavage divisions, male meiosis and some aspects of spermiogenesis. Therefore it is concluded that either: (1) other proteins perform Centrosomin’s centrosomal function post-cleavage, or (2) that Centrosomin is required for some specialized role in the centrosomes of the spindles of cleavage stage embryos and meiotically dividing spermatocytes. Identification of Centrosomin somatic substitutes/alternatives will be required to demonstrate the former possibility. The salient differences between the two types of cell division where Centrosomin can be shown to function makes speculation on the latter possibility difficult. Nevertheless, it appears that Centrosomin is not required in the divisions after cleavage or in the mitotic divisions leading up to meiosis in the male. Thus the centrosomes in these different cell types are qualitatively different from one another. The number and nature of these differences will of course require a further dissection of this organelle and its functional components (Megraw, 1999).

Under normal cellular environments free microtubules are extremely unstable, and most cellular microtubules are nucleated and stabilized by centrosomes. In cnn mutants, fully functional centrosomes are lacking and chromosomes seem to organize microtubules. Fragmented nuclei or chromosomal fragments can also organize a miniature spindle in the cytoplasm. The behavior of these lost chromosomes is very similar to those in experiments in which chromosomal bivalents are mechanically removed from the Drosophila spermatocyte meiotic spindle apparatus and placed in the spermatocyte cytoplasm; they are able to assemble mini-spindles and separate properly during anaphase. The ability of chromosomes to organize microtubules has also been extensively documented in many other systems. In Drosophila melanogaster, female meiosis occurs in the absence of detectable centrosomes (Megraw, 1999).

Chromosomes themselves have been proposed to nucleate microtubules, and motor proteins may be responsible for tethering the microtubules into confined poles. Morphologically, these centrosome-less spindles have microtubules connecting the poles to chromosomes and pole to pole, but lack the astral microtubules emanating from centrosomes seen in most mitotic spindles (Megraw, 1999).

In cnn mutants, chromosomes may have the ability to organize relatively normal looking spindles, but they do not achieve normal separation as in female meiosis. The chromosomes are not tightly aligned at the spindle equator, but are distributed along the length of the spindles instead. The spindles elongate as in anaphase, but the chromosome movements appear random and nonsynchronous. The lack of functional centrosomes associated with these spindles is further supported by the fact that in the anaphase configurations, the prominent astral microtubules extending from the centrosomes to the cytoplasm are not present. It is unclear why these anastral microtubule spindles do not support chromosome segregation as in female meiosis. One possibility is that proteins that participate in female meiosis are not available during the syncytial divisions (Megraw, 1999).

During female meiosis, spindle assembly and chromosome alignment and segregation all depend on motor proteins such as Nod and Ncd. It is possible that these proteins are absent, or present in insufficient quantities, during the syncytial divisions to support the large number of rapid nuclear divisions (Megraw, 1999).

In summary, using mutations that affect Centrosomin localization to the centrosome, it has been shown that Centrosomin is necessary for the assembly of functional centrosomes during Drosophila syncytial divisions. Mutational analysis of other centrosomal proteins should allow a more precise determination of the relationships between known centrosomal proteins. One interesting feature of the Centrosomin protein is the presence of three putative leucine zipper motifs. Since these motifs have been shown to mediate protein-protein interactions, isolation of interaction partners of Centrosomin will lead to the identification of more centrosomal proteins and a better understanding of the molecular construction of the centrosome (Megraw, 1999).

Mutations in centrosomin reveal requirements for centrosomal function during early Drosophila embryogenesis

Although centrosomes serve as the primary organizing centers for the microtubule-based cytoskeleton in animal cells, various studies question the requirements for these organelles during the formation of microtubule arrays and execution of microtubule-dependent processes. Using a genetic approach to interfere with centrosomal function, this study presents an assessment of this issue, in the context of early embryogenesis of Drosophila. Mutant alleles were identified of the centrosomin (cnn) locus, which encodes a core component of centrosomes in Drosophila. The cnn mutant flies are viable but sterile. The normal course of early embryonic development is arrested in all progeny of cnn mutant females. This analysis identified a failure to form functional centrosomes and spindle poles as the primary mutant phenotype of cnn embryos. Various aspects of early development that are dependent on cytoskeletal control are disrupted in cnn mutant embryos. In particular, structural rearrangements of cortical microfilaments are strongly dependent on proper centrosomal function. It is concluded that Centrosomin is an essential core component of early embryonic centrosomes in Drosophila. Microtubule-dependent events of early embryogenesis display differential requirements for centrosomal function (Vaizel-Ohayon, 1999).

These phenotypic and molecular analyses suggest that the cnn gene encodes a centrosomal protein that is essential for the proper formation of MTOCs in the syncytial-stage Drosophila embryo. The cnn gene products are supplied maternally to the developing embryo. Two of the alleles studied, cnnHK and cnnE2, represent mutations that result in drastic truncations of the Cnn protein. Embryos supplied with these mutant proteins do not appear to contain functional centrosomes, as demonstrated by the abnormal localization patterns of the established centrosomal antigens γ-tubulin and CP190. Ultrastructural studies are required to assess fully the impact of the mutation on centrosomal structure (Vaizel-Ohayon, 1999).

Immunolocalization experiments further demonstrate that Cnn is a core component of early embryonic centrosomes. The observation of a strict centrosomal localization of Cnn during early development of wild-type embryos, coupled with the functional requirements revealed by the mutant phenotype, imply that this protein performs a role essential for the structural integrity of early embryonic centrosomes. Structural attributes of Cnn, in particular the striking coiled-coil nature of the protein, suggest that the functional requirement for Cnn involves a rigid centrosomal structure, and extensive protein–protein interactions. One possibility envisaged is that Cnn is a component of a scaffold, upon which early embryonic MTOCs are established (Vaizel-Ohayon, 1999).

Expression of cnn is not restricted to early embryonic stages. Immunolocalization studies demonstrate that Cnn is a general component of mitotic spindle poles throughout fly development, whereas in non-dividing cells the protein is generally cytoplasmic. Despite these dynamic and continuous expression patterns, the mutant phenotypes indicate a clear functional requirement for cnn only in syncytial embryos and the male germline. This conclusion implies, in turn, that proper formation of only certain types of MTOCs — in particular, those present in the early embryo — are absolutely dependent on cnn function. The restricted requirement for cnn might reflect a structural diversity among MTOCs of different developmental stages. One possibility is that the functions provided by Cnn are shared with additional centrosomal elements during late stages of development. Such redundancy would compensate for the loss of any one element caused by mutation. In this context, it cannot be ruled out that the mutant alleles supply a low level of cnn function that is generally sufficient during postembryonic stages. Alternatively, it is possible that the very rapid nuclear divisions characteristic of early embryogenesis pose unique hardships on the centrosomes and spindle poles of syncytial-stage embryos that can be withstood only if a full complement of structural proteins such as Cnn are present (Vaizel-Ohayon, 1999).

The apparent absence of functional centrosomes from cnn syncytial embryos provides a unique opportunity to assess the contribution of MTOCs to early embryogenesis of the fruit fly. Complete or partial dependence of several large-scale cytoplasmic events on the presence of centrosomes is evident from the mutant phenotypes. Centrosomes appear to be absolutely required for the dynamic restructuring of cortical microfilaments during late syncytial stages, given that the cortical microfilament layer completely fails to respond to the presence of centrosome-deficient nuclei near the surface of cnnHK and cnnE2 eggs. These observations are in agreement with the results of a series of studies, in which a close correspondence between presence and position of centrosomes and the formation of microfilament caps and furrows was demonstrated, implicating centrosomes as the instructive agents driving cortical microfilament restructuring in Drosophila syncytial embryos. The elimination of functional centrosomes by genetic means, as achieved here, provides an important complementary approach to the earlier investigations, which primarily involved administration of toxic materials or exposure to extreme environmental conditions. The consistent results of all these studies strongly support the notion that order within the cortical nuclear array relies on a communication process between centrosomes and cortical microfilaments, in which centrosomes are the primary instructive agents, and provide a prominent example of interactions between the microtubule-and microfilament-based cytoskeletons (Vaizel-Ohayon, 1999).

A second aspect of syncytial cytoplasmic organization affected by cnn mutations is the stereotypic motion and spatial arrangement of the nuclei. Internal nuclei are unevenly distributed in the vast majority of cnn embryos, resulting in a non-uniform pattern when they reach the cortex. This phenotype is not unexpected, as previous studies have suggested centrosome-based mechanisms for nuclear motion in insect eggs. Thus, a mechanism for nuclear migration in early Drosophila embryos has been proposed, in which outward motion of nuclei from the embryonic interior results from ‘pushing-off’ between centrosome-based microtubule arrays of neighboring nuclei. Furthermore, a recent landmark study of parthenogenetic development in Sciara, suggested that spacing between nuclei is not properly maintained in the absense of centrosomes and astral microtubules (Vaizel-Ohayon, 1999).

Finally, it is apparent that in syncytial Drosophila embryos, execution of the mitotic divisions themselves can proceed, to varying extents, even in the absence of intact centrosomes. The microtubule structures associated with the nuclei of cnn embryos exhibit distinct morphologies during these unconventional nuclear cycles. The midpoint of mitosis is characterized by formation of an abnormally shaped spindle, reminiscent of the atypical spindle morphology that has been described in other instances of mitosis, in the apparent absence of discrete spindle poles. Additional unusual structures observed in cnn embryos are enigmatic aster-like concentrations of microtubules at the periphery of interphase nuclei. Normal microtubule midbodies appear to form in late anaphase of the division cycle. Proper organization of midbodies is possible regardless of the functional status of centrosomes. The competence of cnn embryos to sustain mitotic division cycles is in keeping with a variety of recent studies that have demonstrated formation of functional mitotic spindles despite the lack of centrosomes. Identification of cnn as a mutation with relevant phenotypes in Drosophila should enable application of a genetic approach towards elucidation of the mechanistic basis of this phenomenon (Vaizel-Ohayon, 1999).

It is concluded that progeny of female Drosophila flies that bear mutations in the centrosomin locus cease to develop during the early, syncytial stages of embryogenesis. A failure to form functional centrosomes, as judged by the abnormal distributions of key centrosomal antigens, is the underlying cause of the developmental arrest. Critical developmental processes, including patterned motion of nuclei within the egg, and structural rearrangements of cortical microfilaments, are found to be strongly dependent on centrosome function. Various microtubule-based arrays, including mitotic spindles, seem capable, however, of forming in the apparent absence of functional microtubule organizing centers. The cnn mutant phenotypes and the strict localization of the Cnn protein to MTOCs in syncytial Drosophila embryos, imply that Cnn is an essential core component of early embryonic centrosomes. It appears, however, that centrosome structure is not fully dependent on Cnn during later developmental stages (Vaizel-Ohayon, 1999).

Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome

Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. This study shows that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation (Yamashita, 2003).

Adult stem cells maintain populations of highly differentiated but short-lived cells such as skin, intestinal epithelium, or sperm through a critical balance between alternate fates: Daughter cells either maintain stem cell identity or initiate differentiation. In Drosophila testes, germline stem cells (GSCs) normally divide asymmetrically, giving rise to one stem cell and one gonialblast, which initiates differentiation starting with the spermatogonial transient amplifying divisions. The hub, a cluster of somatic cells at the testis apical tip, functions as a stem cell niche: Apical hub cells express the signaling ligand Unpaired (Upd), which activates the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway within GSCs to maintain stem cell identity (Yamashita, 2003).

Analysis of dividing male GSCs by expression of green fluorescent protein (GFP)-α-tubulin in early germ cells revealed that in 100% of the dividing stem cells observed, the mitotic spindle was oriented perpendicular to the hub-GSC interface throughout mitosis, with one spindle pole positioned within the crescent where the GSC contacted the hub. Stem cell division was rare, averaging one dividing stem cell observed per 5 to 10 testes (~2% of total stem cells) in 0- to 2-day-old adults. Spindles were not oriented toward the hub in gonialblasts (Yamashita, 2003).

Drosophila male GSCs maintained a fixed orientation toward the hub throughout the cell cycle, unlike Drosophila embryonic neuroblasts or the Caenorhabditis elegans P1 cell, in which spindle orientation is established during mitosis by a programmed rotation of the spindle. The single centrosome in early interphase GSCs was consistently located adjacent to the hub. After centrosome duplication, one centrosome remained adjacent to the hub, whereas the other migrated to the opposite side of the nucleus. The mechanisms responsible for Drosophila GSC spindle orientation may differ between sexes. In female GSCs, the spectrosome, a spherical intracellular membranous structure, remains localized next to the apical cap cells, where it may help anchor the spindle pole during mitosis. In interphase male GSCs, in contrast, the spectrosome was often located to the side, whereas at least one centrosome held the stereotyped position adjacent to the hub (Yamashita, 2003).

To investigate centrosome function in orientation of male GSCs, males were analyzed that were null mutant for the integral centrosome component centrosomin (cnn), which is required for normal astral microtubule function. In cnn mutant males, mitotic spindles were not oriented toward the hub in ~30% of the dividing GSCs examined. In an additional 10% to 20%, spindles were properly oriented, but the proximal spindle pole was no longer closely associated with the cell cortex at the hub-GSC interface and the entire spindle was displaced away from the hub. The frequency of spindle orientation defects was highest in metaphase. Loss of function of cnn also partially randomized the interphase centrosome positioning in male GSCs. In more than 35% of the cnn mutant GSCs with duplicated centrosomes that were scored, neither centrosome was positioned next to the hub (Yamashita, 2003).

The number of germ cells associated with the hub was increased 20% to 30% in cnn mutant males, from an average of 8.94 GSCs per hub in the wild type to 11.89 GSCs per hub in cnnHK21/cnnHK21 and 10.69 GSCs per hub in cnnHK21/cnnmfs3. Hub size was not significantly different in cnn compared with wild-type males. In cnn testes with many stem cells, GSCs appeared crowded around the hub and often seemed attached to the hub by only a small region of cell cortex. Finite available physical space around the hub may limit the increase in stem cell number in cnn mutant males (Yamashita, 2003).

As suggested by the increased stem cell number, there were several cases in both live and fixed samples from cnn males in which a stem cell that had recently divided with a mitotic spindle parallel to the hub-GSC interface produced two daughter cells that retained contact with the hub, a finding that was not observed in the wild type. GSCs were also observed dividing with a misoriented/detached spindle that lost attachment to the hub, probably explaining the mild increase in stem cell number relative to the frequency of misoriented spindles (Yamashita, 2003).

The normal close attachment of one spindle pole to a region of the GSC next to the hub and the effects of cnn mutants on centrosome and spindle orientation suggest that a specialized region of the GSC cell cortex touching the hub might provide a polarity cue toward which astral microtubules from the centrosome and spindle pole orient. High levels of DE-cadherin (fly epithelial cadherin) and Armadillo (Arm; fly ß-catenin) colocalized at the hub-GSC interface, as well as at the interface between adjacent hub cells, marked by high levels of Fas III. High levels of DE-cadherin and Arm were not detected around the rest of the GSC surface. Forced expression of DE-cadherin-GFP specifically in early germ cells confirmed that DE-cadherin in GSCs colocalized to the hub-GSC interface (Yamashita, 2003).

DE-cadherin and Armadillo at the hub-GSC interface may provide an anchoring platform for localized concentration of Apc2, one of two Drosophila homologs of the mammalian tumor suppressor gene Adenomatous Polyposis Coli (APC), which in turn may anchor astral microtubules to orient centrosomes and the spindle. Immunofluorescence analysis revealed Apc2 protein localized to the hub-GSC interface. In apc2 mutant males, GSCs were observed with mispositioned centrosomes, misoriented spindles, or detached spindles. Both the average number of stem cells and hub diameter increased in apc2 mutant males compared with that of the wild type. Unlike in cnn mutants, GSCs did not appear crowded around the hub in apc2 males, perhaps as a result of the enlarged hub (Yamashita, 2003).

The second Drosophila APC homolog, apc1, may also contribute to normal orientation of the interphase centrosome and mitotic spindle. Apc1 protein localized to centrosomes in GSCs and spermatogonia during late G2/prophase, after centrosomes were fully separated but before nuclear envelope breakdown. Apc1 was not detected at centrosomes from prometaphase to telophase. Spindle orientation and centrosome position were perturbed in GSCs from apc1 males, and the number of stem cells per testis and the diameter of the hub both slightly increased in apc1 mutant testes compared with those of the wild type (Yamashita, 2003).

It is proposed that the reliably asymmetric outcome of male GSC divisions is controlled by the concerted action of (1) extrinsic factor(s) from the niche that specify stem cell identity, and (2) intrinsic cellular machinery acting at the centrosome and a specialized region of the GSC cortex located at the hub-GSC interface to orient the cell division plane with respect to the signaling microenvironment. Astral microtubules emanating from the centrosome may be captured by a localized protein complex including Apc2 at the GSC cortex where it interfaces with the hub, similar to the way in which cortical Apc2 may orient mitotic spindles in the syncytial embryo or epithelial cells (Yamashita, 2003).

Mechanisms that orient the mitotic spindle by attachment of astral microtubules to specific cortical sites may be evolutionally conserved. In budding yeast, spindle orientation is controlled by capture and tracking of cytoplasmic microtubules to the bud tip, dependent on Kar9, which has weak sequence similarity to APC proteins. Kar9 has been localized to the spindle pole body and the cell cortex of the bud tip, reminiscent of the localization of Drosophila Apc1 at centrosomes and Apc2 at the cell cortex (Yamashita, 2003).

Polarization of Drosophila male GSCs toward the hub could result simply from the geometry of cell-cell adhesion. GSCs appear to be anchored to the hub in part through localized adherens junctions. Homotypic interactions between DE-cadherin on the surface of hub cells and male GSCs could concentrate and stabilize a patch of DE-cadherin. The resulting localized DE-cadherin cytoplasmic domains could then provide localized binding sites for ß-catenin and Apc2 at the GSC cortex. Although binding of E-cadherin and APC to ß-catenin is thought to be mutually exclusive, APC could be anchored at the cortical patch through the actin cytoskeleton, which in turn could interact with ß-catenin/α-catenin (Yamashita, 2003).

Orientation of stem cells toward the niche appears to play a critical role in the mechanism that ensures a reliably asymmetric out-come of Drosophila male GSC divisions, consistently placing one daughter within the reach of short-range signals from the hub and positioning the other away from the niche. Oriented stem cell division may be a general feature of other stem cell systems, helping maintain the correct balance between stem cell self-renewal and initiation of differentiation throughout adult life (Yamashita, 2003).

The meiotic spindle of the Drosophila oocyte: the role of centrosomin and the central aster

Evidence is provided that a distinct midzone is present in the Drosophila melanogaster female meiosis I spindle. This region has the ability to bind the Pavarotti kinesin-like (PAV-KLP) and Abnormal spindle (Asp) proteins, indicating a correct organization of the central spindle microtubules. The core component centrosomal protein centrosomin (CNN) has been identified at an unexpected site within the anaphase I spindle, indicating a role for CNN during the biogenesis of the female meiotic apparatus. However, there are no apparent defects in the midzone organization of cnn oocytes, whereas defects occur later when the central aster forms. The primary mutant phenotype of cnn oocytes is the failure to form a developed central microtubule organizing center (MTOC), although twin meiosis II spindles usually do form. Thus the central MTOC may not be essential for the formation of the inner poles of twin meiosis II spindles, as generally proposed, but it might be involved in maintaining their proper spacing. The proposal is discussed that, in the presence of a central MTOC, a chromatin-driven mechanism of spindle assembly like that described during meiosis I may control the morphogenesis of the twin meiosis II spindles (Riparbelli, 2005).

Eighty years ago, the pioneering studies of Huettner (Huettner, 1924) on the maturation of the Drosophila melanogaster oocyte revealed that chromosome segregation during the first meiosis is supported by a peculiar spindle apparatus that transforms during the second meiosis into twin spindles arranged in tandem and disposed perpendicularly to the longitudinal axis of the egg. These spindles ensure the reductional divisions and the formation of the haploid complements, the innermost of which is the female pronucleus. Huettner first observed that the meiotic spindles are anastral and lack centrioles at their poles, though they are tapered. Despite the potential of these observations for clarifying acentrosomal pathways of microtubule (MT) organization, the female meiotic apparatus of Drosophila has received little attention for many years. During the last decade some researches have concentrated their attention on the morphogenesis and organization of the spindle during metaphase of the first meiotic division. Immunohistochemical analysis failed to reveal centrosomal components, such as CP60, CP190 and gamma-tubulin, at the spindle poles, confirming, in its essential aspects, the general description of Huettner. The role of gamma-tubulin during meiosis is, however, controversial since mutants for this gene have abnormal first meiotic spindles, but meiosis is not terminally arrested and appears mostly normal. Several lines of evidence suggest that meiotic spindle assembly in the Drosophila female begins with a chromosome-driven mechanism of MT organization. Mutational analysis indicates that, in the absence of centrosomes, MT bundling and bipolarity of anastral meiotic spindles requires kinesin-like proteins with minus-end-directed MT motor activity. Defects in the genes nonclaret disjunctional (ncd) and subito (sub), which encode kinesin-like proteins, lead to the formation of abnormal meiotic I spindles with frayed or undefined poles. The cooperative interaction between motor proteins and the products of the genes mini spindles (msps) and transforming acidic coiled-coil protein (tacc), localized at the acentrosomal poles of the first meiotic spindle, might be crucial for the bipolarity of the meiotic spindle. The MT minus-end-associated Abnormal spindle (Asp) protein, localized at the extremities of the meiotic spindle, could be also involved in the stabilization of focused poles (Riparbelli, 2005 and references therein).

Oocytes are activated to resume meiosis by passage through the oviduct. The spindle, positioned parallel to the cortex during metaphase arrest, reorients perpendicular to the oocyte surface during completion of meiosis I. The spindle elongates during anaphase-telophase of the first meiosis and transforms during meiosis II into two tandemly arranged spindles (Huettner, 1924). Although the meiotic II spindles are anastral, an unusual structure, from which an extensive array of MTs nucleates, forms between the two internal spindle poles (Riparbelli, 1996). It has been shown that this structure, in contrast to the poles of the meiotic spindles, contains several centrosomal proteins usually found at the mitotic spindle poles, such as gamma-tubulin, CP60 and CP190 and centrosomin (CNN) (Llamazares, 1999). However, although this central MTOC contains centrosomal components and is able to nucleate an astral array of MTs, it lacks centrioles, is unable to duplicate and disappears after the completion of meiosis (Riparbelli, 2005).

Whereas previous analyses provided insight into the mechanisms of spindle assembly and chromosome segregation during metaphase of the first meiosis, they provided no clear information on the kinetics of spindle morphogenesis during transition from meiosis I to II. This aspect was investigated by time-lapse analysis of ncd-gfp oocytes (Endow, 1998). The main event of the transforming of meiotic I spindle in twin tandem arranged meiotic II spindles is the formation in the elongated meiotic I spindle of lateral puckers, which are correlated with the organization of the central MTOC (Endow, 1998). However, despite the usefulness of this elegant in vivo approach, some aspects, such as the individualization of the twin spindles and the origin and function of the central MTOC, remain unclear. The findings presented in this report both expand and clarify earlier studies on the structural organization and dynamics of the female meiotic spindle, suggesting an alternative model of meiosis II spindle assembly (Riparbelli, 2005).

After oocyte activation, meiosis resumes and the metaphase-arrested spindle elongates and undergoes a pivoting movement to reorient perpendicularly to the surface (Endow, 1997). Two distinct populations of interpolar MTs, interior and peripheral, form a top-like anaphase spindle. The interior MTs run parallel to the main axis of the spindle to form longitudinal thick bundles, whereas the peripheral ones spread out from the poles to form opposite cone-shaped half spindles. The peripheral MTs, which form a cage around the interior bundles, intersect at the equatorial plane of the spindle and extend both deeply into and above this plane until they reach the cell cortex. Thus, the spindle appears to be anchored to the oocyte surface through the peripheral MT set that forms the inner half spindle. Some clusters of MTs appear through the equatorial region of the spindle where opposite MTs overlapped and/or interacted tangentially. As anaphase progresses the clustering of MTs is more evident through the spindle equator. Most of the interior MT bundles terminate in a small non-fluorescing area at the opposite poles. Co-labeling with propidium iodide indicated that these regions of lower MT density correspond to the chromosomes and that the bundles might therefore be kinetochore fibers. The kinetochore MTs of different chromosomes form separated bundles and do not focus on a common pole. Thus the spindle poles appear broad and frayed. Although the density of the MTs made it difficult to resolve the structure of the central region of the spindle, suitable optical sections showed that some MT bundles overlap in this region. During telophase a diffuse tubulin accumulation was observed at the middle of the spindle at the sites where the interior MTs overlap (Riparbelli, 2005).

Transition from meiosis I to meiosis II is marked by the expansion of the cage of MTs that surround the interior interpolar MTs to form an extensive network. Some of the peripheral MTs also contact the oocyte cortex. Astral arrays of MTs of various size become apparent at this time within the equator of the spindle and outside its boundaries. Intermediate to the formation of these structures might be the clusters of overlapping MTs observed during the first meiosis. These clusters become increasingly compact at the onset of second meiosis and could become centers from which MTs spread. A distinct tubulin aggregate was seen to bulge out at the middle of the interior MT bundles. Kinetochore MTs run almost parallel in each half spindle both outward and inwards to each chromosome set they hold. Thus, kinetochore fibers form distinct parallel 'minispindles' and the outer poles appear frayed. The inner poles cannot be distinguished at this time. Optical sections midway along the spindle show an irregular belt-shaped equatorial tubulin aggregate that could be derived from the tubulin accumulation observed during telophase of the first meiosis. Several MTs that span from this aggregate end at the cytoplasmic asters or mingle within the peripheral network (Riparbelli, 2005).

As meiosis progresses the peripheral MT array gradually disassembles and the MT asters become free in the cytoplasm or remain connected to the remnant of the peripheral MT framework or to the spindle by thin threads and the equatorial tubulin cluster is more evident and protruding. Free asters are transient structures that disappear over time and are no longer visible from anaphase II on. The outward extremities of the minispindles formed by the kinetochore fibers focus on common outer poles, whereas the inner kinetochore fibers still run almost parallel. Thus, the individualization of the meiosis II twin spindles occurs gradually and requires the formation of the internal poles that start to be organized during late prophase/early prometaphase when the inwards extremities of the minispindles have partially coalesced midway along the spindle. Short MTs form a loose cloud at the spindle equator. During prometaphase/early metaphase, the twin spindles begin to be distinguishable, though their inner poles are formed by three to four distinct bundles and appear broad. The MTs at the spindle equator are more prominent and form an extensive astral array, the 'central aster', which increases in size during subsequent metaphase and anaphase. At telophase the MTs of the central aster reach their maximum length, although they have decreased in density (Riparbelli, 2005).

Although female meiosis in Drosophila differs from male meiosis and mitosis in that spindle poles lack centrosomes and cytokinesis does not occur, this study demonstrates that the central region of the spindle has a common organization in these systems and that a structured midzone exists in the female meiotic spindle. At least two main events are involved in the formation of the spindle midzone in Drosophila cells (Inoue, 2004): the overlay of antiparallel MTs and the release of a subpopulation of MTs from the spindle poles. The central region of the female meiosis spindle during late anaphase I has the ability to bind PAV-KLP, the Drosophila ortholog of MKLP. This protein has been found in association with the putative MT plus ends at the midzone of anaphase spindles of mitotic cells and spermatocytes where it might function in regulating the dynamics and organization of the overlapping MTs. Thus the female meiotic spindle could have a structured midzone formed by overlapping antiparallel MTs. This is consistent with the finding, from sagittal optical sections, that opposite MT bundles overlap at the middle of the central spindle. The increased density of MTs in the central region of the spindle at telophase of the first meiosis might be consistent with the release of a subset of MTs from the opposite spindle poles. When this process occurs in mitotic cells and spermatocytes (Riparbelli, 2002), the Asp protein accumulates at the minus ends of the central spindle MTs. The Asp staining observed in the central region of the spindle during telophase of the first meiosis could be such a protein localization, at the predicted MT minus ends, suggesting that MTs of the central spindle undergo similar dynamics during female meiosis and in both mitosis and male meiosis (Riparbelli, 2005).

Morphological evidence indicates that several differences exist between the meiosis I and II spindles: the meiosis I spindle is anastral, whereas the twin meiosis II spindles have outer anastral poles and an unusual central aster between the inner poles (Riparbelli, 1996; Endow, 1997). These observations suggest that spindle assembly could require different mechanisms during meiosis I and II. Whereas the organization of the meiosis I spindle involves a chromosomal-dependent pathway of MT organization, the individualization of the twin spindles during meiosis II requires the formation of new poles in the center of the prophase II spindle. However, the mechanism by which the inner poles form is unclear. This issue has been difficult to address because the formation of the inner spindle poles occurs in a region of very high MT density, and thus any stage of this process is obscured from view. It has been proposed that the formation of the inner poles might require the reorganization of the central spindle in which the polarity of MT ends reverse (Endow, 1998). This hypothesis is supported by the presence of an unusual MTOC that has been postulated to play a major role in the process of MT nucleation for the formation of the inner half spindles during meiosis II (reviewed by Megraw, 2000). However, normal-looking twin bipolar spindles can form during meiosis II, both when the central aster is defective as in polo mutants (Riparbelli, 2000) and when it is very reduced as in wispy, KLP3A and cnn mutants (Riparbelli, 2005).

A new model of spindle assembly during meiosis II is suggested. Spindle elongation during anaphase/telophase of the first meiosis moves the homologous chromosomes to opposite poles that appear broad and frayed because the kinetochore fibers of different chromosomes do not focus on a common pole during later meiosis I stages. Starting from late prophase II, two opposite half spindles become evident, with outer focused poles where Asp accumulates. By contrast, inner poles are not evident at this point. However, the accumulation of Asp midway along the spindle suggests that the minus ends of opposite interpolar MTs are facing into the central region of the spindle. These observations are consistent with the finding, in each prophase II half spindle, of two opposite MT populations, with their plus ends near the chromatin and their minus ends outwards. The outward minus ends are focused on distinct poles, whereas the minus ends facing the spindle equator remain unbound. Control of spindle assembly could be achieved during meiosis II by the same chromosome-driven mechanisms of MT organization working in meiosis I. MTs in the vicinity of the condensed chromatin could be sorted into bundles and focused in stable poles by the cooperative interaction of molecular motors and cross-linking proteins. Loss of ncd function results, indeed, in the destabilization and fragmentation of both inner and outer spindle poles (Endow, 1998). Accordingly, the inner half spindles are formed during late prophase by parallel MTs that are first bundled at the spindle equator in distinct foci, and that then coalesce into a single pole. This model of meiotic spindle assembly is consistent with the formation of twin spindles in oocytes lacking normal-shaped central asters. It is proposed, therefore, that the acentrosomal pathway makes an essential contribution to spindle formation during meiosis II, even when a functionally active MTOC is present in the oocyte. An alternative mechanism might be that the inner half meiosis II spindles could be formed by a mixture of two MT populations: MTs nucleated in the vicinity of the chromosomes and MTs of the central aster. The gradual tethering together of these two MT populations could give rise to the tapered inner spindle poles, as proposed during the assembly of the mitotic spindle in cultured vertebrate cells. To what extent inner half spindles might be formed by two MT populations is difficult to determine, since individual MTs and their origin cannot be resolved within the assembling spindle. However, the Asp staining observed during transition from prophase to metaphase of the second meiosis suggests that the MTs radiating from the central aster contribute minimally, if at all, to the formation of the internal half spindles. The Asp staining is strong in the central region of the spindle, whereas it is weaker in the central aster, pointing to a lower MT density. This suggests that the contribution of the central MTOC is not essential for the higher MT density within the region where the inner poles will organize. Consistently, optical sections at the level of the spindle equator during prophase of the second meiosis reveal a continuity between the peripheral MT network and the ring-like tubulin aggregate, but no link is observed between this structure and the MTs of the central spindle. Finally, staining of Asp indicates that the minus ends of the inner half meiosis II spindle MTs start to coalesce into a single pole and, therefore, are completed before a distinct central aster is apparent. On the basis of these considerations the first hypothesis is favored in which the function of the central aster is redundant for the meiosis spindle organization (Riparbelli, 2005).

However, if the meiosis II spindles are assembled by an acentrosomal pathway, what is the role of the central aster? In the absence of a developed central aster, the twin spindles can be improperly spaced or oriented with respect to the long axis of the oocyte, resulting in the failure to correctly position the female pronucleus. The central aster could, therefore, be needed to keep the correct spacing between neighboring twin spindles. Accordingly, twin spindles are misaligned and at variable distances from each other when the central aster is defective as in polo mutants (Riparbelli, 2000). Mutation in cnn has been shown (Vaizel-Ohayan, 1999) to impair the spatial organization of mitotic spindles in the early Drosophila embryo (Riparbelli, 2005).

In vivo observations revealed that the precursor of the central spindle could be identified by a tubulin pucker midway along the central spindle at the end of meiosis I (Endow, 1998). This structure first appears as a tubulin aggregate to the spindle midzone, where antiparallel MTs overlap and recruit the PAV-KLP motor and CNN proteins. There are two possible mechanisms in the formation of the central aster. One possibility is that the MTs are nucleated by a discrete MTOC. The other possibility is that motor proteins may cross-link and organize randomly nucleated MTs into aster-like structures. The finding of several centrosomal proteins at the focus of the central aster during meiosis II (Megraw, 2000), strongly points to the first possibility. Since gamma-tubulin at the centrosome seems to be dependent on cnn function (Terada, 2003), the finding of CNN at the spindle midzone during anaphase I could indicate the prerequisite for the recruitment of gamma-tubulin at the central spindle and, therefore, for the nucleation of the central aster MTs. However, in cnn mutants that lack this protein at the spindle midzone in anaphase I and fail to accumulate gamma-tubulin, a faint astral array of MTs forms at the middle of the spindle during prometaphase/early metaphase II. In the absence of gamma-tubulin the central aster becomes poorly organized during subsequent meiotic stages and then disappears. It is therefore proposed (see Fig. 8 of Riparbelli, 2005 ) that the assembly of this structure might first rely on the cooperative interaction of motor proteins and acentrosomal MTs to form a polarized array of MTs that spread out from the equatorial region of the spindle. This process does not require ncd function, since mutants for the Ncd motor protein assemble an astral array of MTs at the beginning of meiosis II (Endow, 1998). Recruitment of material along these MTs might then contribute to the accumulation of centrosomal proteins, thus leading to the formation of a true MTOC that in turn could lead to further growth of the central aster. According to this model the presence of centrosomal material and other components, such as PAV-KLP, at its focus might be due to a fortuitous recruitment along its MTs. This is consistent with the finding that when the integrity of the central aster is affected in cnn mutant oocytes, there is little or no accumulation of PAV-KLP between twin spindles. By contrast, the motor protein is always found at the midzone of mutant spindles during both anaphase I and anaphase II, where it is needed for spindle dynamics. This is consistent with the observation that the accumulation of pericentrin and gamma-tubulin at the vertebrate centrosome is inhibited in the absence of tubulin or by microinjection of antibodies against cytoplasmic dynein. This two step mechanism could explain why gammaTub37CD mutants show an astral array of MTs at the beginning of metaphase II in the absence of detectable gamma-tubulin. The sperm aster is usually retained to be assembled by a true centrosome derived from both paternal and maternal sources. In particular the male gamete provides the centriole around which maternal components accumulate to form a mature centrosome able to nucleate MTs and to reproduce. Although rigorous conclusions are difficult to draw from negative results, the observation that cnn mutant oocytes have a developed sperm aster lacking CNN and gamma-tubulin, points to alternative mechanisms of sperm aster assembly. However, the possibility cannot be excluded that the amount of these proteins could be so much lower in mutant oocytes that they have escaped immunofluorescence analysis (Riparbelli, 2005).

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells, yet mitotic spindles can still form after laser ablation or disruption of centrosome function. Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. In this study live-cell microscopy of GFP-tubulin to examine spindle formation was performed in Drosophila S2 cells after RNAi depletion of either γ-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits γ-tubulin to the centrosome. In these RNAi-treated cells, it was show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving γ-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, it was also demonstrated that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. It is proposed that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes (Mahoney, 2006).

Drosophila S2 cells depleted of centrosomin (Cnn) formed anastral spindles and do not recruit γ-tubulin to spindle poles. γ-Tubulin, however, was present on spindle microtubules after Cnn depletion, as was also observed for wild-type cells (Mahoney, 2006).

To probe the dynamics of spindle formation after Cnn depletion, time-lapse microscopy was performed on GFP-tubulin-expressing cells. In wild-type cells, the majority of the early prophase microtubules originate from the centrosomes immediately after nuclear-envelope breakdown (NEB). However, microtubules also clearly form around the chromosomes. In Cnn-RNAi-treated cells, time-lapse microscopy revealed a very different pathway of spindle formation. Centrosomal microtubule asters did not form at prophase, but robust microtubule nucleation still occurred at chromosomes. The interphase microtubule array, which only gradually destabilized after NEB, also incorporated into the spindle, and in some cases attachment and capture of pre-existing microtubules by chromosomes was observed. After initially collecting around chromatin, microtubules then elongated and became focused to create a bipolar spindle with broad, dynamic poles, as described for meiotic spindle formation. Time-lapse microscopy also revealed that Cnn RNAi cells proceeded into anaphase without any significant delay, a result that is consistent with their normal mitotic index. Thus, even when the dominant pathway of microtubule-based search-and-capture of chromosomes by centrosomal microtubules is completely disrupted in Cnn RNAi cells, live-cell imaging reveals that chromosome-mediated nucleation and incorporation of existing microtubules generate a functional bipolar spindle in a time period comparable to that in wild-type cells (Mahoney, 2006).

Next, S2 cells were depleted of ~90% γ-tubulin by RNAi, although residual γ-tubulin may create a hypomorphic situation rather than a true null. (Note that γ-tubulin refers to the ubiquitously expressed 23C isotype; RNAi of an ovary-specific (37C) isotype did not produce a phenotype in S2 cells. Mitotic S2 cells depleted of γ-tubulin by RNAi still contained microtubules, although the mitotic spindles were virtually all abnormal. The most common morphologies were monopolar spindles (~40%) and anastral bipolar spindles with poorly focused poles (~60%). The mitotic index was elevated 3.3-fold, but anaphase cells were observed in the population. These results are consistent with prior studies showing that interfering with γ-tubulin function results in severe spindle defects, although microtubules can still form occur and chromosome alignment can occur (Mahoney, 2006).

To better understand the mechanism of spindle formation after γ-tubulin depletion, live-cell imaging was performed of GFP-tubulin-expressing cells. In cases where NEB was observed, microtubule formation from the chromosomal region was dramatically reduced or significantly delayed in comparison with wild-type and Cnn-RNAi-treated cells. This result, combined with observation of spindle localization of γ-tubulin in Cnn-depleted cells, suggests that γ-tubulin functions as a microtubule nucleator in the chromosome-mediated microtubule assembly pathway. This finding indicates that γ-tubulin need not be anchored at the centrosome to stimulate microtubule nucleation. While this paper was in review, a similar conclusion was reached in mammalian cells (Luders, 2006) on the basis of RNAi of a γ-tubulin-associated subunit (Mahoney, 2006).

The mechanism of assembly of spindle microtubules in γ-tubulin RNAi cells was difficult to decipher from time-lapse movies, and it is possible that the microtubules originate from several sources. One such source appears to be pre-existing interphase microtubules, which coalesce into bundles after NEB and can engage chromosomes as described above for Cnn-depleted cells. Fragments of former 'interphase' microtubules might also act as nucleating seeds for new microtubule growth. In addition, 'focal points' of microtubule growth were also observed, that could represent nucleation from centrioles, or from sites on the fragmenting nuclear envelope. After γ-tubulin RNAi, time-lapse imaging showed that cells usually formed a monopolar spindle initially, as seen in fixed cell images, that often converted to a bipolar spindle through the formation of a second pole. Such bipolar spindles, however, are very unstable and exhibit much more splaying and disorganization than Cnn RNAi cells. Bipolar metaphase spindles in γ-tubulin RNAi cells stall for at least twice as long as in wild-type cells, explaining the increased mitotic index, but eventually can complete anaphase and cytokinesis (Mahoney, 2006).

In conclusion, live-cell imaging reveals several redundant mechanisms for creating mitotic spindles via (1) centrosome-based nucleation, (2) chromosome-based assembly, and (3) recruitment of microtubules created at other sites. Centrosome nucleation of microtubules constitutes the dominant pathway of spindle formation in wild-type cells, but the other processes can generate spindles in the absence of centrosome function (i.e., after Cnn or γ-tubulin depletion) (Mahoney, 2006).

Time-lapse microscopy of GFP-tubulin was effective for examining the initial events in bipolar spindle formation. However, because of the high density of microtubules in the spindle, it was difficult to visualize sites of microtubule nucleation and growth during metaphase. To gain information on these issues, live-cell imaging was performed of a stable S2 cell line expressing low levels of EB1-GFP, a microtubule-plus-end-tracking protein that localizes to the terminal ~0.5 μm tip of growing microtubules and that has been used to investigate cell-cycle-dependent microtubule nucleation (Mahoney, 2006).

In wild-type metaphase cells, EB1-GFP punctae emerged in a radial pattern from the centrosome. In addition, individual EB1-GFP punctae were visible in the microtubule-dense regions of the spindle. A semiautomated program was employed to identify and track EB1-GFP over time and create vectors plots for the growth of individual microtubules. The vector maps of EB1-GFP revealed an overall image that corresponded to the shape of the wild-type mitotic spindle, and computer-generated vectors were in good agreement with manual tracking and visual inspection of the movies. However, limitations exist in establishing the precise origin of all EB1-GFP puncta, given that some may have originated out of the plane of focus (most punctae, however, appeared suddenly as expected for nucleation in the focal plane) and that the automated program often terminated tracking when punctae crossed and overlapped in dense regions of the spindle (Mahoney, 2006).

For Cnn-RNAi-treated cells in metaphase, the radial distribution seen of vectors at the poles for wild-type cells was not observed. Surprisingly, however, EB1-GFP tracking revealed vectors originating throughout the spindle, including many vectors arising from the acentrosomal poles and traveling toward the chromosomes. This result was not anticipated, given that imaging of GFP-tubulin showed initial microtubule nucleation around chromosomes after NEB and not from a peripheral nucleating site. Thus, whereas microtubule formation initially relies upon the chromosomes, the spindle itself acquires a mechanism for forming new EB1 punctae distal from the chromosomes. However, microtubules also probably continue to form around the metaphase chromosomes in Cnn-RNAi-treated cells, because there are many EB1-GFP vectors in the chromosomal region that do not extrapolate back to the spindle poles. In γ-tubulin RNAi cells that formed anastral bipolar-like spindles, the EB1-GFP vector diagrams looked similar to those described for Cnn-RNAi-treated cells, revealing microtubule growth originating throughout the spindle and from the broad polar regions. In contrast to the selective growth of microtubules from acentrosomal poles to chromosomes in the Cnn RNAi cells, however, the orientation of the vectors in the γ-tubulin RNAi spindles tended to be more random (Mahoney, 2006).

It was of interest to determine whether EB1-GFP punctae form within wild-type spindles or whether this phenomenon is a consequence of γ-tubulin depletion/mislocalization. In some wild-type cells, the centrosome and its astral array became transiently disconnected and displaced from the kinetochore fibers, producing a clear spatial separation of the two microtubule networks in the spindle. In such cells, EB1 vectors were still observed originating from the focused minus-end region of kinetochore fibers; these acentrosomal vectors again were preferentially directed toward the chromosomes. RNAi of abnormal spindle protein (Asp) was also performed, and this treatment resulted in centrosome detachment from the main body of the spindle and splaying of kinetochore fibers. In this situation, movies of EB1-GFP also revealed fluorescent punctae originating from acentrosomal regions of splayed kinetochore fibers and moving toward the chromosomes. Thus, a process of spindle-based microtubule formation and growth occurs from acentrosomal foci of kinetochore fibers and within spindles, even in cells that possess functional centrosomes (Mahoney, 2006).

In summary, this analysis of both γ-tubulin and Cnn RNAi cells, as well as untreated cells, shows EB1-GFP punctae forming within the spindle and from acentrosomal poles and traveling toward the chromosomes. Four mechanisms are presented by which EB1 punctae might be generated in the spindle: (1) Catastrophe of stable microtubules followed by rescue can generate new growth and plus-end labeling by EB1 within the spindle, although observations of astral-microtubule dynamics show that rescue is rare and thus perhaps unlikely to be the sole source by which EB1-GFP punctae are generated within the spindle; (2) Microtubule severing of existing microtubules generates new microtubule plus ends that grow and recruit EB1; (3) γ-tubulin-mediated growth of new microtubules, potentially nucleating from the sides of pre-existing microtubules; (4) An unidentified protein nucleates new microtubules (Mahoney, 2006).

Although discussed separately, it is emphasized that these models are not mutually exclusive, and indeed, it is plausible that multiple mechanisms might be contributing to this phenomenon. One possibility for generating new EB1 punctae is through the rescue of a kinetochore microtubule that undergoes catastrophe. Observation of individual microtubule catastrophe and rescue events in the spindle is not technically possible because of the high microtubule density. Microtubule depolymerization has been induced by severing Xenopus spindles with a microneedle and it has been concluded that rescue and regrowth is rare, especially near the poles. It was not possible to image single astral microtubules in GFP-tubulin-expressing S2 cells, and observations also reveal a very low rescue frequency of depolymerizing astral microtubules (only 5% of the shrinking microtubules underwent a clear rescue event. Nevertheless, the possibility of a novel mechanism that selectively stimulates rescue in spindle versus the astral microtubule population in S2 cells cannot be excluded (Mahoney, 2006).

EB1 punctae in the spindle could also reflect the generation of additional microtubules, by either microtubule fragmentation or nucleation. An important clue in considering such models is that the EB1-GFP vectors tend to be constrained within the cone angle of the spindle and grow toward the chromosomes, in contrast to the radial nucleation/growth that occurs from centrosomes. Microtubule severing followed by regrowth of the newly created plus end could produce such results. Alternatively, a de novo templating reaction from existing spindle microtubules could occur. For example, a nucleator could bind to the side of existing microtubules and template new microtubules at a shallow angle to the mother filament, followed by crosslinking/bundling to pre-existing kinetochore microtubules. Such a mechanism is analogous to the binding and nucleation of new actin filaments by Arp2/3 bound to a pre-existing actin filament. Precedence for this idea comes from recent reports showing that γ-tubulin can nucleate microtubules from pre-existing interphase microtubules in S. pombe and in plants. γ-tubulin is present throughout the spindle in S2 cells, which might favor such a possibility in mitosis as well. The fact that γ-tubulin RNAi cells still form EB1-GFP punctae at the broad polar regions cannot necessarily be taken as evidence against γ-tubulin involvement in such a de novo microtubule nucleation mechanism, because residual γ-tubulin remains after RNAi. Moreover, EB1-GFP vectors in γ-tubulin RNAi spindles differ from those in wild-type and Cnn RNAi cells in being less dense and more random in orientation (less selective growth toward the midzone compared with γ-tubulin-containing spindles). Alternatively, a novel microtubule nucleator may be involved or could contribute in addition to γ-tubulin (Mahoney, 2006).

Clearly, further studies will be required to identify the molecule(s) responsible for generating microtubule growth at acentrosomal poles and within spindles. However, the present study illustrates that, in addition to the well-described pathways of centrosomal and chromosomal microtubule nucleation, the metaphase spindle possesses a mechanism (or mechanisms) for propagating its own architecture by promoting microtubule assembly (Mahoney, 2006).

Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila

Precise regulation of stem cell self-renewal/differentiation is essential for embryogenesis and tumor suppression. Drosophila neural progenitors (neuroblasts) align their spindle along an apical/basal polarity axis to generate a self-renewed apical neuroblast and a differentiating basal cell. This study genetically disrupted spindle orientation without altering cell polarity to test the role of spindle orientation in self-renewal/differentiation. Correlative live imaging of polarity markers and spindle orientation over multiple divisions were performed within intact brains, followed by molecular marker analysis of cell fate. It was found that spindle alignment orthogonal to apical/basal polarity always segregates apical determinants into both siblings, which invariably assume a neuroblast identity. Basal determinants can all be localized into one sibling without inducing neuronal differentiation, but overexpression of the basal determinant Prospero can deplete neuroblasts. It is concluded that the ratio of apical/basal determinants specifies neuroblast/GMC identity, and that apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation (Cabernard, 2009).

It is critical for these studies to use mutants that affect spindle orientation but not cortical polarity, so it was first confirmed that the spindle orientation mutants mud and cnn had no detectable effect on apical/basal cortical polarity. It was found that 12-13% of the metaphase neuroblasts in these mutants showed aberrant spindle orientation orthogonal to the apical/basal cortical polarity axis, and that both had an increased number of brain neuroblasts. Thus, these two mutants are appropriate tools for studying the role of spindle orientation in regulating neuroblast self-renewal versus differentiation (Cabernard, 2009).

To determine if mud and cnn mutants act autonomously within neuroblast lineages to increase neuroblast number, as predicted if neuroblast spindle orientation defects lead to increased brain neuroblast numbers, GFP-marked mutant clones were generated within single neuroblasts using the MARCM technique. Clones were induced in first instar larvae and analyzed in third instar larvae. Mutant clones were identified by GFP expression and scored for the neuroblast markers Deadpan (Dpn) or Mira and the GMC/neuron marker nuclear Pros (nPros). Wild-type single neuroblast clones always contained a single large Dpn+ nPros neuroblast and several smaller nPros+ GMC/neurons. In contrast, mud and cnn mutant single neuroblast clones often contained two or more Dpn+ nPros neuroblasts; the multiple neuroblasts in a clone were of similar size and were always tightly adjacent. Importantly, mutant clones containing zero neuroblasts were never observed, which would be expected if defects in spindle orientation resulted in some divisions producing GMC/GMC siblings, and live imaging confirms that GMC/GMC siblings are never generated. These data are consistent with a model in which mud and cnn mutant neuroblasts generate neuroblast/neuroblast sibling cells, but not GMC/GMC sibling cells (Cabernard, 2009).

To determine whether ectopic neuroblasts in mud and cnn mutants arise occasionally or invariably from neuroblast orthogonal divisions, live imaging of neuroblast cell lineages was performed within intact larval brains. This method allowed tracking of individual neuroblasts from mitotic spindle orientation through to subsequent sibling cell fates. Spindle orientation was monitored with a microtubule-associated Cherry::Jupiter fusion protein, cortical polarity was monitored using the basal marker GFP::Mira, and neuroblast/GMC cell fates were determined by multiple cell biological criteria (subsequent cell division profile, cell lineage, cell cycle length, and cell size. Wild-type neuroblasts always showed apical/basal spindle orientation, production of unequally sized daughter cells, and partitioning of the basal cortical marker GFP::Mira into the smaller daughter cell. As expected, cnn and mud mutant neuroblasts also frequently showed normal apical/basal spindle orientation, divided asymmetrically, and generated neuroblast/GMC siblings (Cabernard, 2009).

Importantly, a subset of cnn and mud mutant neuroblast divisions showed spindle orientation orthogonal to the apical/basal polarity axis, allowing determination of the role of spindle orientation in neuroblast self-renewal versus differentiation. Live imaging showed that neuroblasts undergoing orthogonal divisions always generated equally sized siblings that both invariably assumed a neuroblast identity based on their ability to maintain a neuroblast-like short cell cycle and ability to subsequently undergo asymmetric cell division. To provide an independent molecular assay of sibling cell identity, correlative microscopy was performed in which live imaging was used to identify orthogonal neuroblast cell divisions and then subsequently the identical neuroblast lineage were fixed and stained for molecular marker expression. It was found that neuroblast orthogonal divisions always generated two siblings that expressed the neuroblast marker Deadpan (Dpn) and lacked the differentiation marker nPros. It is concluded that neuroblast orthogonal divisions always generate two equally sized cells that assume a neuroblast identity: they have a short cell cycle, can divide asymmetrically, express the neuroblast marker Dpn, and lack the GMC/neuronal marker nPros. Thus, altering neuroblast spindle orientation from apical/basal to orthogonal results in the invariant production of two sibling neuroblasts, based on both cell biological and molecular criteria (Cabernard, 2009).

Neuroblasts dividing orthogonally to the apical/basal polarity axis invariably generate two sibling neuroblasts. To determine how apical/basal cortical determinants correlate with cell fate specification -- if they correlate at all, the partitioning of apical or basal cortical domains was quantitated in cnn or mud mutant orthogonal neuroblast divisions. As expected, wild-type or mutant neuroblasts with apical/basal spindle orientation always segregated the majority of the apical marker Baz::GFP into the neuroblast, and the majority of the basal marker Cherry::Mira into the GMC. In contrast, mud mutant neuroblasts with orthogonal spindle orientation always segregated the apical marker Baz::GFP equally into both sibling cells. The apical protein aPKC is also symmetrically partitioned during orthogonal divisions. The basal marker Cherry::Mira could also be segregated equally to both siblings, but surprisingly was more frequently partitioned unequally to only one sibling. Similar results were obtained with cnn mutant neuroblasts. Clearly the segregation of all basal determinants into just one sibling was insufficient to induce neuronal differentiation, as all orthogonal divisions generated two sibling neuroblasts. It is concluded that the apical cortical domain is perfectly correlated with acquisition of neuroblast identity, whereas the basal cortical domain is insufficient to specify GMC identity (Cabernard, 2009).

Orthogonal neuroblast divisions always partition apical proteins into both siblings and always generate two neuroblasts; basal proteins can all be localized into one sibling without inducing differentiation. It is thus tempting to conclude that only apical proteins are used to specify cell fate. However, an alternative model is that cell fate is determined by the ratio of apical:basal proteins and that a sibling containing half the apical proteins and all of the basal proteins still has an apical:basal ratio high enough to promote neuroblast identity (Cabernard, 2009).

It is possible to distinguish between these two models by increasing the amount of the basal cell fate determinant Prospero: the 'apical dominant' model predicts no effect on neuroblast identity, whereas the 'apical:basal ratio' model predicts at least some loss of neuroblast identity. It was found that overexpressing Prospero in neuroblasts results in coexpression of nuclear Prospero and the neuroblast marker Deadpan and a striking depletion of larval neuroblasts. Importantly, no change was observed in the localization or function of apical cortical proteins: Baz::GFP formed an apical crescent, and aPKC was able to exclude Miranda from the apical cortex. Thus, increasing the amount of the cell fate determinant Prospero, without altering apical cortical proteins, is sufficient to block neuroblast specification or maintenance, resulting in a decrease in neuroblast numbers. It is concluded that the ratio of apical:basal cortical polarity markers is important for determining neuroblast/GMC identity and that apical/basal spindle orientation maintains neuroblast homeostasis and promotes neuronal differentiation by allowing the production of a basal cell with a high basal:apical ratio of cell fate determinants (Cabernard, 2009).

This study used a combination of genetic mutants that specifically disrupt spindle orientation without affecting cell polarity, live imaging of apical/basal spindle orientation for multiple neuroblast divisions within intact larval brains, and correlative microscopy to determine the molecular profile of terminal progeny of the imaged lineages. The results show that apical/basal spindle orientation is essential for maintaining neuroblast pool size and promoting neuronal differentiation: direct observation shows that all mutant neuroblasts with orthogonal spindle orientation generate two neuroblast siblings, whereas all mutant and wild-type neuroblasts with apical/basal spindle orientation generate neuroblast/GMC siblings. This provides strong evidence that spindle orientation defects in these mutants lead to the observed increase in neuroblast numbers, rather than other possible defects including brain patterning, nonautonomous effects in glia or GMCs, or altered cell polarity (Cabernard, 2009).

Analysis of orthogonal divisions reveals that only apical proteins are correlated with cell fate (being 100% correlated with neuroblast identity), whereas inheritance of all the basal proteins by one sibling is insufficient to induce neuronal differentiation. This is strikingly similar to mammalian embryonic neural stem cells, where only the apical cortical domain is correlated with self-renewal, while the basolateral and adherens junctional domains distribute independently of cell fate. Nevertheless, this study shows that the apical cortical domain is not the sole determinant of cell fate, but rather it is the ratio of apical:basal proteins that specifies neuroblast/GMC identity. This model is supported by the observation that increasing levels of the apical determinant aPKC can switch GMCs into neuroblasts, and that decreasing the levels of basal determinants can turn GMCs into neuroblasts. A high apical:basal ratio may promote neuroblast identity by inactivating basal proteins, increasing cell size, promoting cell proliferation, or altering centrosome composition/function. Conversely, a high basal:apical ratio may promote differentiation via Prospero repression of genes promoting cell proliferation or neuroblast identity, by Brain tumor suppression of Myc-dependent cell growth, and/or by Numb inhibition of Notch-dependent neuroblast self-renewal (Cabernard, 2009).

In wild-type Drosophila neuroblasts, the mitotic spindle is always aligned with the apical/basal axis, which maintains neuroblast pool size and allows neuronal differentiation. In other insects and mammals, regulated spindle orientation may allow switching between neural progenitor expansion and homeostasis. In the honeybee Apis, mushroom body neuroblasts expand via symmetric divisions prior to switching to an asymmetric division mode to generate neurons. Neuroblast expansion may be due to an increased apical:basal determinant ratio or a phase of orthogonal spindle orientation. Similarly, mammalian neural progenitors switch between phases of progenitor expansion, homeostasis, and depletion. Clues that spindle orientation plays an important role come from the analysis of mammalian mutants CDK5RAP2 and lis1, which cause microcephaly in mammals; the orthologous Drosophila mutants cnn and lis1 both disrupt spindle orientation, but not cortical polarity, and lead to an increase in neuroblast numbers. However, the respective contribution of apical/basal determinant ratio and spindle orientation remains to be determined in mammals, primarily due to the lack of candidate cell fate determinants and the difficulty of performing correlative microscopy within intact brain tissue. These results suggest that concurrent live imaging of cell polarity, spindle orientation, and sibling cell fate will be necessary to determine the role of spindle orientation in regulating mammalian neural stem cell self-renewal versus differentiation (Cabernard, 2009).

Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts

Centrosomes comprise a pair of centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division. In some cases, centrosome asymmetry is linked to centriole age because the older, mother centriole always organizes more PCM than the daughter centriole, thus ensuring that the mother centriole is always retained in the stem cell after cell division. This has raised the possibility that an 'immortal' mother centriole may help maintain stem cell fate. It is unclear, however, how centrosome size asymmetry is generated in stem cells. This study provides compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter centrioles. Shortly after centriole separation, mother and daughter centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother centriole, while it is maintained at the daughter centriole. This ensures that the daughter centriole maintains its PCM and so its position at the apical cortex. Thus, the daughter centriole, rather than an 'immortal' mother centriole, is ultimately retained in these stem cells (Conduit, 2010).

The conclusion that the daughter centriole is specifically retained in neuroblasts is unexpected and depends on the ability to distinguish mother and daughter centrioles. Drosophila mother and daughter centrioles are morphologically indistinguishable at the electron microscopy level, and so GFP-PACT (Pericentrin/AKAP450 centrosomal targeting domain) fluorescence intensity was used to infer centriole age. In Drosophila embryos, centriolar GFP-PACT fluorescence intensity increases as centrioles age. This also appears to occur in neuroblasts, because the separating centrioles display different levels of GFP-PACT fluorescence and, as in embryos, the bright centriole initially organizes more Cnn than the dim centriole. GFP-PACT is known to irreversibly incorporate into centrioles, and so for the daughter centriole to be brighter than the mother centriole in neuroblasts, either the GFP-PACT molecules would have to become less fluorescent as they age or GFP-PACT would have to be partially removed from the mother centriole before it separates from its daughter. Moreover, given the observations of neuroblasts and their GMC progeny, if daughter centrioles were brighter than their mothers, not only would mother centrioles have to exhibit the same level of GFP-PACT fluorescence irrespective of their age, daughter centrioles of the same age would have to exhibit widely varying levels of GFP-PACT fluorescence. Thus, it seems very likely that GFP-PACT preferentially labels the mother centrioles in neuroblasts and that the daughter centriole is specifically retained in these stem cells (Conduit, 2010).

It can only be speculated as to why it might be an advantage for neuroblasts to specifically retain their daughter centriole. It seems to be of benefit to neuroblasts to retain one centrosome at the apical cortex throughout interphase, because this helps to establish correct spindle orientation for the subsequent asymmetric division. It is also easy to envisage the benefits of retaining only one centrosome at the cortex, because retaining both might hinder efficient centrosome separation prior to mitosis. Because several centriolar proteins are irreversibly incorporated into centrioles during their assembly, continuously selecting the mother centriole might lead to the retention in the neuroblast of a centriole that accumulates damage. Male GSCs, which appear to specifically retain their mother centriole, exist in a niche environment, and so defective stem cells can be replaced by cells that dedifferentiate; this option is probably not available to neuroblasts (Conduit, 2010).

It is difficult to test whether perturbing the preferential retention of the daughter centriole would be detrimental to neuroblasts. The functional significance of randomizing centrosome inheritance (for example, by removing Cnn) is hard to assess, because cnn mutant brains appear largely morphologically normal, but this is also true in flies that completely lack centrioles and centrosomes -- even though ~30% of asymmetric neuroblast divisions fail in these cells. Thus, centrosome defects in flies can lead to a high frequency of defective asymmetric neuroblast divisions without producing any obvious defects in brain morphology. Mutations in several human centrosomal proteins (including CDK5RAP2, the human homolog of Cnn) are linked to microcephaly, a condition where patients have perturbed brain development. Thus, human brains may not be able to compensate for defects in asymmetric neural stem cell divisions in the same way that flies apparently can. It seems to be an emerging principle that neural progenitor/stem cell divisions are particularly sensitive to defects in centrosome function, and so potentially in asymmetric centrosome inheritance (Conduit, 2010).


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centrosomin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 October 2014

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