To analyze Mud localization in asymmetric cell division, Mud in embryonic neuroblasts were stained by using an anti-Mud antibody (Yu, 2006). At neuroblast delamination, Mud colocalizes with Pins on the apical cell cortex. This cortical localization is maintained through interphase, when alternative methods of fixation also reveal a pool of Mud on the nuclear rim (see also Yu, 2006). At metaphase, when the spindle aligns with the apical crescents of Mud and Pins, Mud can also be observed on spindle poles. At telophase, Mud preferentially segregates into the neuroblast. This localization is consistent with recent work showing that Mud decorates mitotic and meiotic spindle poles and is required for positioning spindles in meiosis II (Yu, 2006). Although Mud is expressed in larval brains, fixation conditions could not be found for analyzing Mud localization in larval tissue. It is concluded that Mud colocalizes with Pins on the cortex of asymmetrically dividing neuroblasts (Bowman, 2006).
To test if the localization of Mud depends on its binding partner Pins, pins Δ50 maternal and zygotic mutant embryos were analyzed. The apical enrichment of Mud is lost in pins mutant neuroblasts in metaphase, and the cortical association is weaker, but Mud remains associated with spindle poles. To test whether Pins is sufficient for directing apical localization of Mud, transgenic inscuteable under the control of the hsp70 promoter was used to express Inscuteable in epithelial cells. Epithelial cells normally divide parallel to the plane of the epithelium. Introduction of ectopic Inscuteable recruits Pins and Gαi from the basolateral to the apical cortex, inducing a spindle reorientation. Mud is also recruited apically and colocalizes with Pins. This suggests that Pins recruits Mud to the apical cortex of epithelial cells in the presence of Inscuteable, and, by extension, that Pins recruits Mud apically in neuroblasts. It is concluded from these experiments that Pins is required and sufficient for the apical recruitment of Mud, but that the spindle pole localization of Mud is independent of Pins. Both the apical localization of Mud and its association with microtubules are consistent with a role in spindle orientation (Bowman, 2006).
In the developing central nervous system of Drosophila, proliferation follows a reproducible and well-described spatial and temporal pattern. This pattern involves a defined number and distribution of neural stem cells (neuroblasts), as well as a precisely regulated time course of division of these neuroblasts. Mutations in the mushroom body defect (mud) gene interfere with the regulation of this pattern in a rather specific manner. In the abdominal neuromeres a subset of neuroblasts prolongs the period of proliferation. Additional daughter cells persist into the imago. Similar defects are expressed in the anterior ventral nerve cord and in the lateral central brain region. In the mushroom body cortex, however, mutations in mud affect the proliferation pattern by increasing the number of neuroblasts. These additional neuroblasts behave like normal mushroom body neuroblasts according to their time course of proliferation and the specification of their progeny (Prokop, 1994).
A Pins-Gαi interacting protein that also binds microtubules is a good candidate for a regulator of spindle orientation in asymmetric cell division. To find out if Mud controls spindle orientation, larval neuroblast divisions weRE analyzed in animals homozygous for mud4, a presumptive null allele affecting all Mud isoforms (Guan, 2000). For this, third instar larval brains were immunostained for Miranda and Centrosomin. Neuroblasts were defined as Miranda-expressing cells greater than 10 μm in diameter. In wild-type neuroblasts, Miranda forms a crescent in metaphase, and it segregates into a single daughter cell at telophase. In mud zygotic mutants, the Miranda crescent can be bisected by the cleavage plane and inherited by both daughter cells. Missegregation of Miranda could be due to defective spindle orientation, or it could be a secondary consequence of a general loss of polarity. Alternatively, Mud could regulate mitotic spindle morphology or formation. All mud mutant neuroblasts form crescents of aPKC and opposing crescents of Insc and Miranda. Furthermore, spindles in mud mutant neuroblasts appear bipolar with no gross morphological differences from wild-type (see also Yu, 2006). From these data, it is concluded that mud mutants form functional spindles and the neuroblasts are correctly polarized. Consistent with this, Brat and Numb form crescents in mud mutant neuroblasts, but the spindle is not aligned with them. Therefore, the spindle orientation defect is a direct consequence of Mud loss of function. To quantify this defect, the angle between a line connecting the two centrosomes and a line bisecting the crescent of Miranda was measured in metaphase neuroblasts. A small angle indicates tight coupling of the mitotic spindle with the polarity axis. In wild-type, the measured angle is almost always less than 10°. In mud mutants, the majority of spindles show more oblique orientations, and only a minority of spindles have measured angles of 10° or less. It is concluded that Mud is required for coordinating the mitotic spindle with the axis of polarity. Together, these observations demonstrate that mud mutant neuroblasts polarize correctly, but, in the absence of Mud, the polarized cortical domains cannot direct the orientation of the mitotic spindle. As a result, cell fate determinants can fail to segregate asymmetrically (Bowman, 2006).
Mud gets its name from defective formation of the mushroom body, an adult brain structure required for olfactory learning and memory. The neurons forming the mushroom body, called Kenyon cells, are generated by four mushroom body neuroblasts that divide repeatedly throughout embryonic, larval, and pupal development. Mushroom body neuroblasts, like all neuroblasts in Drosophila, divide asymmetrically to yield a GMC that produces two neurons and a self-renewing neuroblast. Notably, wild-type mushroom body neuroblasts form crescents of Miranda, which segregate into a single small cell. This shows that neuroblasts of this lineage segregate cell fate determinants asymmetrically. In mud mutants, Miranda also forms a crescent, but spindle misorientation leads to missegregation of Miranda in ~4% of mushroom body neuroblasts. It is proposed that the remaining neuroblasts divide asymmetrically by repositioning either the spindle or the cell polarity axis during telophase. A similar rescue of defects during asymmetric cell division at late stages of mitosis has been described for other mutants, where it is called telophase rescue. Although the vast majority of mud mutant neuroblasts still divide asymmetrically, in those cells that inherit equal amounts of Miranda, and presumably equal amounts of the apical complex members known to regulate cell size asymmetry, the daughter cell size is equal. It is concluded that mushroom body neuroblasts segregate Miranda and therefore its binding partners Brat and Prospero asymmetrically, but faulty spindle orientation leads to occasional missegregation in mud mutants (Bowman, 2006).
Missegregation of cell fate determinants can result in the transformation of GMCs to neuroblasts. Therefore whether symmetric segregation of Miranda resulted in increased numbers of mushroom body neuroblasts in mud mutants was examined. For this, the mushroom body-specific GAL4 line OK107 and UAS CD8-GFP were used to label mushroom body neuroblasts and their progeny, and Miranda, which is present in neuroblasts but rapidly degraded in GMCs, was immunostained. While the number of mushroom body neuroblasts in wild-type late third instar brains is always 4, the average number of mushroom body neuroblasts in mud mutants is nearly 14. Neuroblast number increases over time; an average of around eight mushroom body neuroblasts were observed in the early third instar brains of mud mutants. From these experiments, it is concluded that mud mutants generate excess mushroom body neuroblasts, a conclusion consistent with an earlier study (Prokop, 1994). Increased numbers of neuroblasts were observed in the posterior half of the larval brain hemisphere, ventral nerve cord, and the anterior brain regions of mud mutants. Ectopic neuroblasts in mud mutants express the neuroblast marker Deadpan and incorporate BrdU, showing that they are correctly specified and mitotically active (Bowman, 2006).
In brat mutants, transformation of GMCs to neuroblasts leads to a decrease in the number of neurons. To see if this is true for mud mutants, Kenyon cells were analyzed in late third instar larval brains by using OK107-GAL4-driven CD8-GFP to mark the progeny of the mushroom body neuroblasts. Wild-type larval brains contain around 500 Kenyon cells, but, in mud mutants, the average number of Kenyon cells increases. This suggests that unlike in brat mutants, the ectopic neuroblasts in mud mutants produce ectopic progeny cells. It is proposed that the penetrant symmetric division phenotype in brat mutants makes neuroblasts nearly incapable of producing GMCs, while the frequent asymmetric divisions in mud mutants can still give rise to differentiated progeny (Bowman, 2006).
To test whether the ectopic neuroblasts and progeny cells develop normally, the morphology of the mushroom body was investigated in mud mutant adults. During development, repeated divisions of mushroom body neuroblasts sequentially generate three types of morphologically distinct Kenyon cells. These neurons project axons that form the characteristic lobed structure of the adult mushroom body. Kenyon cells born from late embryogenesis to the early third instar project their axons into the γ lobe, Kenyon cells born between early third instar and puparium formation project branched axons into the α′ and β′ lobes, and, finally, Kenyon cells born after puparium formation project their branched axons into the α and β lobes. Like larval brains, mud mutant adult brains contain an increased number of Kenyon cells. The number of wild-type Kenyon cells is lower than expected, but OK107 may not detectably label every Kenyon cell. Interestingly, while a small γ lobe is present in mud mutants, the ectopic Kenyon cells are unable to project axons into the α′β′ or αβ lobes. The absence of these lobes could indicate a role for Mud in axon guidance. Alternatively, the presence of the γ lobe suggests that Kenyon cells born early in development, when the maternal supply of Mud is sufficient, are correctly specified. Consistent with this, the mud mutant γ lobe expresses low levels of Fasciclin-II, as in wild-type γ lobes. At later stages, the reduced levels of Mud may result in misspecification of the α′β′ and αβ neurons, resulting in an absence of projections. Taken together, these observations suggest that in mud mutants, the occasional transformation of a GMC into a neuroblast causes overproliferation and cell fate misspecification (Bowman, 2006).
In addition to their well-known effects on the development of the mushroom body, mud mutants are also female sterile. This study shows that, although the early steps of ovary development are grossly normal, a defect becomes apparent in meiosis II when the two component spindles fail to cohere and align properly. The products of meiosis are consequently mispositioned within the egg and, with or without fertilization, soon undergo asynchronous and spatially disorganized replication. In wild-type eggs, Mud is found associated with the central spindle pole body that lies between the two spindles of meiosis II. The mutant defect thus implies that Mud should be added to the short list of components that are required for the formation and/or stability of this structure. Mud protein is also normally found in association with other structures during egg development: at the spindle poles of meiosis I, at the spindle poles of early cleavage and syncytial embryos, in the rosettes formed from the unfertilized products of meiosis, with the fusomes and spectrosomes that anchor the spindles of dividing cystoblasts, and at the nuclear rim of the developing oocyte. In contrast to its important role at the central spindle pole body, in none of these cases is it clear that Mud plays an essential role. But the commonalities in its location suggest potential roles for the protein in development of other tissues (Yu, 2006).
This work describes a fully penetrant phenotype that is associated with strong mutations in the mud gene. Specifically, mud mutants have a striking maternal-effect lethal phenotype: although eggs produced by mutant females look normal and accept sperm, they fail to undergo normal cleavage divisions. Instead of forming a well-ordered syncytial blastoderm, these eggs accumulate disordered arrays of replicated material and then necrose. A number of mutants in other genes have been reported to have abnormal early embryonic mitoses. One class of these, comprising strong alleles of the genes pan gu (png), plutonium (plu), and giant nuclei (gnu), reflects defects in a complex with protein kinase activity. It seems unlikely that mud is a member of this class. While png, plu, and gnu mutants typically form one to five giant nuclei, strong alleles of mud characteristically generate dozens of smaller chromatin masses that are scattered throughout the egg. Moreover, in contrast to mutations in genes of the giant nuclei class, mud mutations do not interfere with degradation of maternal mRNA. A closer resemblance to the mud phenotype is shown by mutants that have defects in spindle-associated proteins. In such mutants, as in mud, the meiotic products undergo inappropriate and disorganized mitoses that produce eggs with scattered chromatin masses. The possibility is considered that Mud and the products of one or more of these genes (ncd, alphaTub67C, or gammaTub37C) serve mutually interdependent roles. If so, double heterozygotes might display a maternal-effect phenotype lacking in any of the single heterozygotes. However, when either the mud3 or the mud4 mutation was combined with the ncdD, alphaTub67C1, or gammaTub37C3 mutation, no decrease in fertility was seen over the robust levels seen in the single heterozygotes. Although this outcome is not decisive, it argues against possibilities such as Mud being involved in the localization of these spindle components. In any case, it should be emphasized that the observation that unfertilized mutant eggs undergo the same rounds of disorganized mitoses as do early embryos strongly implies that the mud maternal lethal phenotype is due to a defect in meiosis and not in a subsequent stage of development (Yu, 2006).
What is the basis for the meiotic defect? A central spindle pole body that incorporates several centrosomal proteins together with a diffuse aster of alpha-tubulin is normally found between the two spindles that make up the meiosis II apparatus. As judged by alpha-tubulin staining, this structure is defective or absent in mud mutant eggs. In wild-type eggs, Mud is found associated with the spindle pole body, implying that the protein is needed for the formation or stability of this structure. Because Mud staining appears to surround the alpha-tubulin framework, an attractive possibility is that the former is needed for the latter to be recruited to or be maintained at the central spindle pole body. In any case, just as with weak alleles of polo, which also show defects in the formation of the central spindle pole body, disjoined meiosis II spindles, and disorganized early mitoses, the observations of this study provide a plausible scenario for the female sterility of mud mutants. To wit, without a functional spindle pole body the two meiosis II spindles are not properly held together and the products of meiosis do not get correctly positioned with respect to the egg cortex. It is presumed that positioning is important for condensation of the meiotic products into inactive polar bodies. These structures may normally serve to shield the dead-end meiotic products from the replication machinery, which according to this scenario is competent to operate in unfertilized Drosophila eggs. If so, when proper condensation fails, inappropriate replication ensues. Regardless of the correctness of this model, the similar phenotypes of mud and polo mutations suggest that corresponding proteins might serve interdependent roles in activated eggs. But no decrease in fertility was observed in double heterozygotes of polo1 and either mud3 or mud4. It remains to be seen whether Polo and Mud are similarly distributed in activated oocytes (Yu, 2006).
In addition to its association with the central spindle pole body, Mud protein is readily detected at other spindle poles. However, in those cases where it can be tested, it appears that Mud is not essential for the formation or function of these structures. In this regard, Mud is reminiscent of the fly orthologs of CP190 and pericentrin, which, despite being consistently found at the centrosome, are dispensable for mitosis. It may be that the structures built at the spindle poles are designed to withstand the undersupply of a few ingredients. If so, it might be useful to look for synthetic phenotypes when mud mutations are combined with those of other genes whose products are concentrated at spindle poles. Of course, because eggs that lack a maternal contribution of functional Mud develop so anomalously, it is undecided whether it plays an essential role at the spindle poles of embryonic mitoses or in polar bodies. Insight into these cases will have to await the availability of a conditional mutant that can be shifted to nonpermissive conditions after meiosis II is completed. Despite the doubts about its role at places other than the central spindle pole body, the frequent associations of Mud with microtubular structures in the oocyte and egg suggest that the protein might be regarded as a MAP; direct tests of this hypothesis are underway. Another attractive, albeit speculative, idea is that Mud is functionally related to the vertebrate NuMA protein. Like Mud, NuMA is a large coiled-coil protein that is found at spindle poles. The two proteins are apparently not orthologous in that they cannot be globally aligned outside of their coiled-coil regions; they also differ in subcellular localization during interphase. However, the carboxy terminus of one particular isoform of Mud shares a short region of similarity with NuMA, hinting at a conserved interaction (Yu, 2006).
Two other sites of Mud localization that suggest a connection with microtubules are fusomes and spectrosomes. These are membrane-rich structures that form during the earliest stages of ovary development and are surrounded by microtubules. Mud is present within these structures at a time when they serve to anchor the mitotic spindles of the dividing cystoblasts but not later, when they serve to focus the microtubule network in postmitotic cells. Although this distribution hints at a role for Mud in spindle anchoring, inactivation of the gene causes no obvious defect in fusomes or spectrosomes. These structures not only form normally in mud mutants but the processes they govern during early oogenesis proceed without defect. It is concluded that, if Mud plays a role in building fusomes and spectrosomes or connecting them to spindles, it is a role for which there is adequate redundancy (Yu, 2006).
In cells that are not in metaphase or anaphase, Mud can also be found at the nuclear envelope. It is not clear how Mud gets from the nuclear rim of the oocyte and the early embryo, respectively, to the meiotic and mitotic spindle apparatus. In contrast, Mud might lose contact with the nuclear envelope, where it could have been held by its transmembrane domain, and be directed to the spindle by a distinct targeting signal. But, since the spindles of the Drosophila germ line and pre-syncytial egg are typically enclosed by membranous structures, the movement of Mud might be part of a concerted redistribution of elements of the nuclear envelope. Although the mechanism is thus unclear, it should be pointed out that shuffling between the nuclear envelope and the spindle is not unique to Mud but has been reported for several other proteins. Another open question is whether Mud plays a microtubule-related role at the nuclear rim. However, even though the oocyte nucleus is surrounded by a cage of microtubules and the dynactin complex is concentrated at its rim, the structure and positioning of the oocyte nucleus are not grossly affected by loss of Mud function. Accordingly, the idea is favored that Mud is simply stored at the nuclear rim to ensure a local supply for subsequent delivery to the spindle apparatus but the possibility that a parallel system renders obscure a more active function for Mud at the nuclear envelope cannot be ruled out (Yu, 2006).
To what extent do the observations on the female sterility shed light on the other phenotypes of mud mutants, particularly the role of Mud in the development of the adult nervous system? It is easy to imagine that a protein that can associate with the nuclear envelope might be part of the mechanism that prepares the way for exit from the cell cycle. And a protein that is commonly found around spindle poles might be involved in the regulation of spindle orientation that governs the transition from symmetric to asymmetric division of neuroblasts and thus the switch from neuroblast proliferation to stem cell behavior. Similarly, a protein that associates with microtubules might play a role in the precision of growth cone movement that is needed for proper axon pathfinding. Thus, although speculative, concrete suggestions for places to look for Mud action in the nervous system can be gleaned from the insights gained from a description of this protein in the oocyte and early embryo (Yu, 2006).
Asymmetric cell division generates cell diversity during development1, 2 and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-Galphai crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. This study shows that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. It is proposed that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division (Siller, 2006).
The Mud-Pins interaction was confirmed by showing that a short C-terminal portion of Mud containing the NLM domain and 142 amino acids of the amino-terminal sequence (amino acids 1825-1997) directly interacts with Pins in vitro. Further analysis revealed that Mud binds the amino-terminal Pins tetratricopeptide (TPR)1-7 domain, but not the C-terminal GoLoco domain. Although Insc binds TPR1-4, no Mud binding was observed to any region of Pins that was smaller than TPR1-7, indicating that all seven TPRs are required for proper presentation of the Mud-binding epitope. Consistent with the Mud-Pins direct interaction, Mud and Pins can be coimmunoprecipitated from embryonic lysates. The Mud-Pins interaction is likely to be evolutionarily conserved, since homologous domains in NuMA and Mud mediate their interaction with LGN and Pins, respectively. It is concluded that the Mud C-terminus can bind the Pins TPRs, and both proteins are part of a common protein complex in vivo (Siller, 2006).
In embryonic neuroblasts, Mud and Pins were both enriched at the cortex over the apical centrosome/spindle pole from late interphase and up to the end of metaphase. By late anaphase-telophase, Mud showed bipolar apical and basal cortical crescents over both spindle poles; this can be seen most clearly in neuroblasts that are cultured in vitro, where there are fewer surrounding cells. In addition, Mud shows strong spindle-pole/centrosome localization and weaker spindle and astral microtubule localization in all neuroblasts. In larval neuroblasts, Mud is always present at the apical cortex with Pins: it either forms cortical crescents over both spindle poles or is uniformly cortical. In mud null mutants, larval neuroblasts have virtually no detectable Mud protein, confirming the antibody specificity. It is concluded that Mud and Pins form apical cortical crescents during late interphase and prophase (this is the time when spindle orientation is established in larval neuroblasts), and that Mud is also detected at the basal cortex later in mitosis, as well as on spindle poles and microtubules. The minor differences in Mud localization between embryonic and larval neuroblasts may be due to differences in fixation/visualization or in cell types. Thus, Drosophila Mud, C. elegans Lin-5 and mammalian NuMA all share a common localization profile of cell cortex, spindle poles and spindle microtubules (Siller, 2006).
Whether Mud and Pins rely on each other for apical cortical localization was tested. In mud mutant larval neuroblasts, normal apical-basal localization of Pins-Galphai and all other tested cortical polarity proteins was found. By contrast, pins or Galphai maternal-zygotic null-mutant neuroblasts always lacked apical enrichment of Mud: it was either cytoplasmic or cytoplasmic with residual uniform cortical localization, although centrosome/spindle-pole localization was unaffected. In addition, the C-terminal truncated Mud protein that is encoded by the mud allele fails to localize to the cortex or spindle poles in larval neuroblasts. It is concluded that Pins recruits Mud to the neuroblast apical cortex, probably via interaction with the Mud C-terminal domain (Siller, 2006).
The function of Mud in spindle orientation was investigated. Because Mud is maternally provided and required during meiosis, spindle orientation was analyzed in larval neuroblasts. Wild-type larval neuroblasts invariably aligned their metaphase spindle within 15° of the centre of the Pins apical crescent or the Mira basal crescent. By contrast, mud mutant neuroblasts showed significant defects in metaphase spindle alignment with the apical Pins crescent. Also, formation of bent spindles were observed in 29%-40% of all mud mutant neuroblasts, but these are not correlated with spindle-orientation defects and arise after spindle orientation is fixed. It is concluded that Mud is required for metaphase spindle orientation. Despite severe defects in metaphase spindle orientation, it was found that the mitotic spindle and cortical polarity markers were nearly always re-aligned by telophase in mudmutant neuroblasts. In the rare neuroblasts in which 'telophase rescue' of spindle-cortex alignment failed to occur, and the spindle axis remained nearly perpendicular to the cell polarity axis, it was found that the neuroblast division was invariably symmetric with regards to cortical polarity and sibling cell size. Thus, Mud specifically regulates spindle orientation, but spindle orientation defects can affect the asymmetry of cell division. It is concluded that: (1) Mud is required to align the mitotic spindle with Pins cortical polarity at metaphase; (2) a Mud-independent mechanism can rescue spindle-cortex alignment at telophase, and (3) proper spindle-cortex alignment is necessary to promote asymmetric cell division of larval neuroblasts (Siller, 2006).
Time-lapse imaging of larval neuroblasts was used to address two important questions: when do the spindle orientation defects arise in mud mutants, and how are the spindle orientation defects 'rescued' at telophase? Mitotic larval neuroblasts were imaged in whole brain explants expressing a spindle marker labelled with green fluorescent protein (transgenic line G147) and/or an enhanced yellow fluorescent protein (EYFP)-Baz apical cortical marker. In wild-type neuroblasts, it was found that the two fully separated centrosomes were always aligned along the future apical-basal axis by the end of prophase. Thus, in contrast to embryonic neuroblasts in which spindle rotation is reported to occur at metaphase, larval neuroblasts fix spindle-pole/centrosome alignment at prophase and maintain spindle orientation up to the end of telophase. Analysis of the EYFP-Baz apical cortical marker revealed that cortical polarity was always established prior to fixation of centrosome position and accurately predicted the final axis of spindle orientation. This is consistent with the tight alignment of centrosomes and cortical polarity axes that were observed from the end of prophase to telophase in fixed preparations. It is concluded that wild-type neuroblasts establish cortical polarity by prophase, establish centrosome position by the end of prophase and maintain tight spindle-cortex alignment during telophase (Siller, 2006).
In mud mutant neuroblasts, it was found that spindle orientation was also established at prophase, with little or no spindle movement through telophase. However, significant defects were found in the alignment of the mitotic spindle with the EYFP-Baz cortical crescent, including neuroblasts in which the spindle and cortical polarity axes were nearly perpendicular. When examined mud mutant neuroblasts were examined during anaphase/telophase, movement of the mitotic spindle to bring it into alignment with the EYFP-Baz cortical polarity axis was never observed, despite data from fixed preparations showing that the majority of metaphase spindle orientation defects are corrected by telophase. Finally, it was observed that mud mutants could divide asymmetrically or symmetrically. Symmetric divisions occurred only when the spindle was nearly orthogonal with the cortical polarity axis and it was inferred that these neuroblasts correspond to the equally dividing neuroblasts that were seen in fixed preparations. Three conclusions were drawn from the live imaging experiments. First, the mud spindle-orientation defects are due to a failure in centrosome/spindle-pole positioning at prophase, prior to the formation of the metaphase spindle. This further supports the conclusion that metaphase spindle morphology defects are not the source of the spindle-orientation defects. Second, mud mutants do not rotate their spindle towards the cortical polarity axis at anaphase-telophase, indicating that the observed 'telophase rescue' of spindle-cortical polarity occurs by modification of cortical protein distribution to match the spindle axis. Third, Mud does not directly promote asymmetric cell division, but it does regulate spindle orientation relative to cortical polarity, and only when the spindle is orthogonal to the cortical polarity axis does the cell division become symmetric. Thus, spindle orientation dictates whether the cell division is symmetric or asymmetric (Siller, 2006).
This study has shown that Mud has the properties of a functional orthologue of the vertebrate NuMA and C. elegans Lin-5 proteins. All three proteins contain coiled-coil regions and an adjacent NLM domain (found only in NuMA-related proteins), and all three proteins directly interact with similar Galpha-binding proteins (Pins, LGN, GPR1/2). In addition, all three proteins are localized to the cell cortex, spindle poles and spindle microtubules, and at least Mud and Lin-5 have some role in spindle orientation and generating unequal daughter cell size. However, there are differences. NuMA and Lin-5 cortical association depends on LGN and GPR1/2, respectively, whereas Mud can localize to the cortex (albeit uniformly) in the absence of cortical Galphai and Pins. Pins-independent Mud cortical localization may be mediated by the Mud C-terminal putative transmembrane domains, which are absent in NuMA and Lin-5 proteins. Conversely, NuMA and Lin-5 facilitate cortical localization of LGN and GPR1/2, respectively, whereas Mud is not required for Pins localization. Finally, it is unknown how Mud interacts with the mitotic spindle. NuMA directly binds tubulin through a domain containing the NLM motif, raising the possibility that the Mud NLM domain mediates microtubule association. Alternatively, Mud may associate with the spindle via dynein/dynactin, as has been shown for NuMA (Siller, 2006).
Pins and Galphai regulate cortical polarity, spindle orientation, spindle asymmetry and the establishment of sibling cell size differences. Previously, all Drosophila mutants in cortical polarity proteins either severely disrupted cortical polarity, thereby precluding analysis of cortical-spindle alignment, or had no effect on spindle orientation. Reduction in Mud or Khc-73 levels affects spindle orientation without altering cortical polarity; each has a partially penetrant phenotype, so they may function redundantly. mud mutants affect only spindle orientation without directly regulating any other known Pins-Galphai-dependent functions, such as regulation of cortical polarity or sibling cell size. Only when the spindle is aligned orthogonally to the Pins-Galphai crescent are there defects in sibling cell size, presumably due to the equalized activity of Pins-Galphai in both siblings. Whether each of the many essential Pins-Galphai functions has a unique effector protein, similar to the role of Mud in regulation of intrinsic spindle orientation, will be an interesting question for the future (Siller, 2006).
Asymmetric cell division is a conserved mechanism to generate cellular diversity during animal development and a key process in cancer and stem cell biology. Despite the increasing number of proteins characterized, the complex network of proteins interactions established during asymmetric cell division is still poorly understood. This suggests that additional components must be contributing to orchestrate all the events underlying this tightly modulated process. The PDZ protein Canoe (Cno) and its mammalian counterparts AF-6 and Afadin are critical to regulate intracellular signaling and to organize cell junctions throughout development. Cno functions as a new effector of the apical proteins Inscuteable (Insc)-Partner of Inscuteable (Pins)-Gαi during the asymmetric division of Drosophila neuroblasts (NBs). Cno localizes apically in metaphase NBs and coimmnunoprecipitates with Pins in vivo. Furthermore, Cno functionally interacts with the apical proteins Insc, Gαi, and Mushroom body defect (Mud) to generate correct neuronal lineages. Failures in muscle and heart lineages are also detected in cno mutant embryos. These results strongly support a new function for Cno regulating key processes during asymmetric NB division: the localization of cell-fate determinants, the orientation of the mitotic spindle, and the generation of unequal-sized daughter cells (Speicher, 2008).
NBs delaminate from the neuroectoderm inheriting the apicobasal polarity of the neuroectodermal cells, in which the PDZ proteins Bazooka (Baz)/Par-3 and DmPar-6 and the kinase DaPKC localize apicolaterally. After delamination, NBs maintain the apical localization of Baz/DmPar-6/DaPKC. The cytoplasmic PDZ protein Cno localizes at the adherens junctions of some epithelial cells, and it was asked whether Cno was also present in the neuroectoderm and in the delaminated NBs. Double immunofluorescences with antibodies against Cno and Baz showed that these proteins colocalize both apicolaterally at the adherens junctions of neuroepithelial cells and apically in the delaminated metaphase NBs (mNBs). At later phases of the NB division, Cno was no longer detected (Speicher, 2008).
Apical proteins, such as Baz/Par-3, are critically involved in regulating cell-fate determinants localization and spindle orientation at metaphase. Given that Cno was detected in an apical crescent in mNBs, it was asked whether Cno was also required for modulating those events. In control embryos, the cell-fate determinant Numb was basally located in 95.4% of mNBs. In cno2 zygotic mutants, Numb was uniform or undetectable or was present in nonbasal crescents in 47.9% of the mNBs analyzed. cno2 has been defined as the strongest allele of cno, although the particular lesion associated is unknown. However, cno2 is probably a null allele because cno2 over the Df(3R)6-7 (covering the cno gene) showed a similar percent of Numb localization failures. Additionally, cno3, another strong allele of cno considered as a null displayed defects in Numb localization in comparable cases. The basal distribution of the scaffolding protein Miranda (Mira) was also altered in 16.9% of mNBs of cno2 mutants. Indeed, the localization of two Mira cargo proteins, the cell-fate determinants Prospero (Pros) and Brain Tumor (Brat), was affected in mNBs. The variable penetrance of the cno2 mutant phenotype observed for the different proteins analyzed may reflect, at least in part, the different sensitivity of the antibodies used (Speicher, 2008).
Intriguingly, the orientation of the mitotic spindle in mNBs of cno2 mutants was randomized in 18.3% of the cases. In control embryos, the spindle is tightly aligned with the center of Numb crescents in mNBs. In cno2 mutants, the spindle was uncoupled with the Numb crescent in 7.7% of the mNBs that showed these crescents (either basal or at other incorrect localizations). The maternal contribution of cno might reduce the penetrance of these phenotypes (Speicher, 2008).
The overexpression of Cno also caused Numb localization failures (45.8%) and aberrant spindle orientations (39%) in mNBs. Hence, the results showed that Cno regulates essential processes during asymmetric NB division: the basal localization of cell-fate determinants and the proper orientation of the mitotic spindle (Speicher, 2008).
Another characteristic feature of asymmetric NB division is the different cell size of the progeny. Hence, whether Cno was also regulating this process was analyzed. Control telophase NBs (tNBs) showed unequal-sized daughter cells in 100% of the cases analyzed. In cno2 mutants, equal-size divisions were observed in 21.3% of tNBs. Two redundant pathways, Baz/DaPKC/Insc and Pins-Gαi, regulate cell size and mitotic-spindle asymmetry at the NB apical pole . Only when both pathways are compromised is the different size of the daughter cells affected. The data suggested that Cno functions downstream of Gαi. Thus, Cno might belong to the Pins-Gαi pathway. Indeed, when both insc and cno were eliminated, 85.2% of tNBs showed equal-sized daughter cells, a much more penetrant phenotype than those displayed by each single mutant. Moreover, ΔGαi, cno2 double mutants showed a much lower percentage of equal-sized divisions (30.4%) than the inscP49; cno2 double mutants. Hence, these results strongly suggest that Cno participates within the Pins-Gαi pathway to regulate NB progeny size (Speicher, 2008).
Given the defects observed in cno2 mutant embryos during NBs division, it was asked whether neuronal lineages were altered in cno2 mutants. The lineage of the ganglion mother cell (GMC) 4-2a has been extensively studied. This GMC expresses the transcription factor Even-Skipped (Eve) and divides asymmetrically to give rise to two different neurons called RP2 and RP2 sibling. Both maintain the expression of Eve initially; however, at later stages of embryogenesis, only the RP2 neuron keeps expressing Eve . In control embryos, 0.9% of the segments analyzed showed defects in the number of RP2s. In cno2 mutants, two or no RP2s were detected per hemisegment in 5.7% of the segments analyzed. Such a result suggested failures in the GMC 4-2a asymmetric division. This phenotype was also observed in cnomis1 hypomorph mutants (4.6%) as well as in mutants for genes that are critical during asymmetric cell division. For example, homozygotes for DaPKCk06403, inscP49, ΔGαi, and mud4 (zygotic null mutant embryos) showed defects in the GMC 4-2a lineage in 6.4%, 13.8%, 2.5%, and 8.3% of the segments analyzed, respectively. Hence, it was next investigated whether Cno was interacting with these proteins to properly generate the GMC 4-2a neuronal lineage. Double heterozygotes DaPKCk06403/+; cno2/+ showed defective RP2 number in 0.8% of segments. This result is consistent with a lack of functional interactions between DaPKC and Cno. However, double heterozygotes inscP49/+; cno2/+ and ΔGαi, +/+, cno2 showed an altered RP2 lineage in 14.4% and 7.6% of the segments analyzed. In addition to the analysis of double heterozygotes, it was found that the cnomis1 phenotype was significantly enhanced in a mud4 zygotic null mutant background. Altogether, these results indicated that Cno functionally interacts with the apical proteins Insc, Gαi, and Mud during the asymmetric cell divisions that generate specific neuronal lineages in the CNS (Speicher, 2008).
Since Cno functionally interacts with Insc, Gαi, and Mud, the epistatic relationships between them were analyzed. To investigate whether Cno was acting upstream of the apical proteins, the localization of Baz, Insc, and Gαi was examined in cno2 mutant embryos. The distribution of all these proteins was normal in cno2 mutants. This result suggested that Cno acts either downstream or in parallel to Baz, Insc, and Gαi. To clarify this point, the distribution of Cno was analyzed in loss- and gain-of-function (lof and gof) mutants for several apical proteins. In inscP49 lof mutants, Cno was untraceable or showed a wrong orientation in 78.8% of the mNBs analyzed. Insc overexpression also caused failures in Cno localization (76%); Cno was either undetectable (13/21) or present in not-apical crescents (3/21). Likewise, in Gβ13F maternal and zygotic null mutant embryos, in which Gαi is lost, Cno was mislocalized or undetectable in 94% of the mNBs. Moreover, the overexpression of Gαi caused a striking mislocalization of Cno in 100% of the mNBs analyzed. The NuMA-related protein Mud binds the apical protein Pins and functions downstream of Pins-Gαi to regulate spindle orientation. In mud mutant NBs, the spindle fails to tightly align with the basal crescent, and this failure is also shown by cno2 mutant NBs. Additionally, Cno and Mud interacted genetically. Hence, it was asked whether Cno functions along with Mud to regulate spindle orientation. In control embryos, Mud localized at the apical cortex of mNBs (97%) and at the two centrosomal regions (100%). In cno2 lof mutants, Mud failed to accumulate apically in 49% of mNBs, and 15% of NBs showed Mud localization in one or none of the two centrosomes. cno gof also caused failures in Mud localization (38%). Altogether, these results strongly support a function of Cno downstream of Insc and Pins-Gαi and upstream of Mud during asymmetric NB division (Speicher, 2008).
Given the functional relationships found between Cno and apical proteins during asymmetric NB division, it was asked whether Cno was physically interacting with some of these proteins. Coimmunoprecipitation experiments from Drosophila embryo extracts showed that Cno is forming a complex with Pins. Cno did not physically interact with DmPar6, Baz, DaPKC, or other apical proteins tested such as Insc, Gαi, and Mud (Speicher, 2008).
Pins also forms a complex in the delaminated metaphase NBs with the tumor-suppressor protein Discs Large (Dlg) and the kinesin Khc-73, an astral microtubule-binding protein. First, at prophase, the DmPar6/Insc pathway is required to polarize Pins/Gαi at the apical pole of the NB. Then, at metaphase, the Pins/Gαi/Dlg/Khc-73 complex forms, and it is key for tightly coupling cortical polarity with spindle orientation. Hence, it was asked whether Cno was also forming part of this complex. Experiments showed that neither Dlg nor Khc-73 coimmunoprecipitate Cno in embryo extracts. This result indicated that Cno is not forming part of the Dlg/Khc-73 complex (Speicher, 2008).
Altogether, a working network of protein interactions is proposed. Analysis of epistatic relationships between apical proteins and Cno showed that Cno is acting downstream of Insc-Pins-Gαi and upstream of Mud. Indeed, genetic analysis suggests that, at least for the control of daughter cells size asymmetry, Cno functions within the Pins-Gαi pathway, in parallel to the DaPKC-Baz-Insc pathway. Accordingly, Cno was found to form a complex with Pins in vivo. Cno did not coimmunoprecipitate with Gαi, though. One possibility is that Cno and Gαi are mutually exclusive in the complex that each of them forms with Pins. Additionally, transient or labile interactions between Cno and Gαi may occur that were not possible to detect. Another Pins interacting partner, the microtubule-binding protein Mud contributes to coordinate spindle orientation with cortical polarity. Given the functional relationships that were found between Cno and Mud, Cno could act in a complex with Pins to modulate Mud localization and, consequently, spindle orientation (Speicher, 2008).
Finally, it was asked whether the function of Cno during asymmetric cell division was conserved in different tissues. Since the NBs of the CNS, the Drosophila somatic muscle and heart progenitors divide asymmetrically to give rise to two different founder cells. Cno is present in the somatic mesoderm and is required for muscle and heart progenitor specification. Hence, it was aked whether Cno was also functioning during the asymmetric division of muscle and heart progenitors. For this analysis, focus was placed on two dorsal progenitors called P2 and P15 that express the transcription factor Eve and whose lineages have been characterized in detail. In this study, it was found that the transcription factor Seven-up (Svp), a characteristic marker of a subset of cardial cells, was expressed in a dorsal founder cell of unknown identity until now, which is here named founder of Svp cardial cells (FSvpCs). With all these markers, specific for individual derivatives, whether dorsal muscle and cardial lineages were altered in cno2 mutants was analyzed. It was found that at late stages (stage 14), 3.1% of hemisegments (n = 96) showed simultaneously either loss of EPCs and gain of DO2 muscle or gain of EPCs and loss of the DO2 muscle (P2 lineage). In control embryos, this phenotype was not observed in any of the hemisegments analyzed. Indeed, Numb localization, which was basal in 100% of the metaphase P2s analyzed in control embryos, was altered in 93% of metaphase P2s in cno2 mutants. Hemisegments showing duplication of DA1 muscle and loss of SvpCs or DA1 muscle loss and gain of SvpCs (P15 lineage) were also detected in cno2 mutants. Hence, Cno was required for the asymmetric division of progenitor cells both in the CNS and in the mesoderm (Speicher, 2008).
In conclusion, the discovery of new modulators of asymmetric cell division, as described in this study, for the PDZ protein Cno, is key to complete understanding of this intricate process. Especially challenging in the future will be unraveling the complete network of connections between all the players required for an accurate asymmetric cell division (Speicher, 2008).
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).
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date revised: 1 November 2010
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