Expression first appears at stage 6 in the procephalic neuroectoderm. Immediately thereafter, transcripts are detected in the central nervous system neuroblasts of the first segregation wave, but only after they have begun to delaminate. One striking feature of the INSC protein is that the protein is polarly localized to the membrane of at least two cell types. An apical localization of the protein (refering to the apex of cells located on the "outside" of the embryo in contrast to basal location) is obvious in the neuroblasts, on the side of the cell that retains contact with the neuroectoderm epithelium, and in cells of the posterior midgut primordium. Thus INSC is found in 'necks' of delaminating neuroblasts remaining behind in the neuroectoderm before the delaminatinon is fully completed (Kraut, 1996a).

At stage 10, INSC is detected in two cells per segment in the lateral epidermis, although not always synchronously. Presumably, these are the first two cells that give rise to developing lateral peripheral sense organs. Expression is lost from the developing sensory organs by the time of dorsal closure and remains in the primordium of the CNS until after the CNS has condensed. During stages 10-13, expression is seen in several scattered mesodermal cells in each segment. These cells might correspond to muscle precursors. Expression is present in some ganglion mother cells. Expression in anterior and posterior midgut primordia, in cells corresponding to adult midgut precursors, continues until fusion of both primordia. At stage 15 expression is evident in pharynx, gastric caecae, hindgut tracheal branches and Malpighian tubules (Kraut, 1996a).

Embryonic: Inscuteable localization and junctional integrity

The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).

Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001 and references therein).

The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?

To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).

Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).

After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).

Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).

In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).

Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).

To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homolog of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore, the function of E-APC in epithelial cell division was tested (Lu, 2001).

In about 60% of E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells. Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).

To test whether E-APC functions with EB1 to orient the mitotic spindle, RNAi was performed on a closely related fly homolog of EB1 (dEB1). In dEB1(RNAi) embryos, the epithelial divisions are also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. The penetrance of dEB1(RNAi) phenotype (20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologs in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain, which is required for interaction with EB1, and no direct interaction between E-APC and EB1 could be detected in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orient spindles (Lu, 2001).

These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orient their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in an insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behavior of the adherens junction (Lu, 2001).

When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orienting axis division may have its precedent in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlight the importance of tumor suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).

Par-6 is apically localized in asymmetrically dividing neuroblasts. To test whether the protein is required for asymmetric cell division, the distribution of Bazooka and Inscuteable were analyzed in neuroblasts of Par-6GLC embryos (embryos deficient for both maternal and zygotic par-6). Seventy-three per cent of the Par-6GLC mutant neuroblasts revealed homogeneous cytoplasmic distribution of Bazooka. In 27% of the mutant neuroblasts, Bazooka still shows some weak apical localization, but the strong apical crescents that are observed in 97% of the control neuroblasts were never seen. Whereas Inscuteable localizes asymmetrically at the apical cortex in 94% of the control neuroblasts, only 23% of the Par-6GLC mutant neuroblasts show clear Inscuteable crescents. In 44% of the mutant neuroblasts, the protein is partially delocalized, and in 32% Inscuteable is cytoplasmic. Thus, Par-6 is required for correct localization of both Inscuteable and Bazooka, even though the effect on Bazooka localization is stronger. Both Bazooka and Inscuteable are required for spindle orientation and asymmetric localization of Numb and Miranda (Petronczki, 2001).

Whether Par-6 is required in these processes was examined by staining Par-6GLC embryos for DNA and Miranda or Numb. Metaphase plates are frequently misoriented indicating a defect in spindle orientation. Statistical analysis showed that 25% of the neuroblast metaphase plates were misoriented by more than 60° relative to the horizontal plane, and 37% of the metaphase plates were misorientated between 30° and 60°. Although in control embryos Miranda localizes into a basal cortical crescent in 100% of all metaphase neuroblasts, no signs of asymmetric localization were detected in 80% of metaphase neuroblasts from Par-6GLC embryos. In 20% of Par-6 mutant metaphase neuroblasts, Miranda was excluded from the apical-most quarter of the neuroblast cortex, but a basal cortical crescent was never detected in these mutants. During anaphase and telophase, Miranda maintained its basal localization and segregated into the basal daughter cell in 100% of the control neuroblasts. In Par-6 mutant anaphase neuroblasts, Miranda concentrated at the cleavage furrow (77% or was actually indistinguishable from wild type (23%), indicating that there is a second, Par-6-independent mechanism involved in Miranda localization during late mitosis. Similar observations were made for Numb. Thus, Par-6 is required in neuroblasts for spindle orientation, for apical localization of Bazooka and Inscuteable, and for basal localization of Numb and Miranda during mitosis (Petronczki, 2001).

The bipotential ganglion mother cells, or GMCs, in the Drosophila CNS asymmetrically divide to generate two distinct post-mitotic neurons. The midline repellent Slit (Sli), via its receptor Roundabout (Robo), promotes the terminal asymmetric division of GMCs. In GMC-1 of the RP2/sib lineage, Slit promotes asymmetric division by down regulating two POU proteins, Nubbin and Mitimere. The down regulation of these proteins allows the asymmetric localization of Inscuteable, leading to the asymmetric division of GMC-1. Consistent with this, over-expression of these POU genes in a late GMC-1 causes mis-localization of Insc and symmetric division of GMC-1 to generate two RP2s. Similarly, increasing the dosage of the two POU genes in sli mutant background enhances the penetrance of the RP2 lineage defects whereas reducing the dosage of the two genes reduces the penetrance of the phenotype. These results tie a cell-non-autonomous signaling pathway to the asymmetric division of precursor cells during neurogenesis (Mehta, 2001).

The symmetric division of GMCs in sli mutants is similar to that observed in insc, Notch or rapsynoid (raps; also known as pins) mutants and opposite that of nb. Previous results show that the cytoplasmic adaptor protein Insc is required for the asymmetric division of GMC-1 into RP2 and sib. During GMC-1 division, Insc protein localizes to the apical side and Nb to the basal side. The Nb-negative daughter cell becomes specified as sib by Notch signaling whereas the cell that inherits Nb becomes an RP2 owing to the blocking of Notch signaling by Nb. Thus, in insc mutants, both cells inherit Nb and are specified as RP2 while in nb mutants both progeny becomes sib. Given the similarity of sli, sim and insc mutant phenotypes, the relationship between Sli and Insc was examined. First, in sli mutants the localization of Insc in GMC-1, when examined, is not asymmetric. About 7% of the hemisegments show this phenotype. A similar non-localization of Insc was also observed in GMC1-1a of the aCC/pCC lineage. In raps mutant embryos, Insc is also not localized and as in sli the GMC-1 divides symmetrically to generate two RP2s. Thus, failure to localize Insc in these GMCs in sli mutants is responsible for their symmetric mitosis. In insc;nb double mutants both the daughters of GMC-1 are specified as sib by Notch signaling. In sli;nb (or sim;nb) double mutant embryos also, both the progeny of GMC-1 adopt a sib fate. Thus, Sli is required upstream of Nb during the asymmetric division of GMC-1. Since the GMC-1 symmetrically divides to yield two RP2s in Notch;nb double mutants, and two sibs in sli;nb double mutants, Sli is also upstream of Notch signaling during the asymmetric division of GMC-1. These results also indicate that when the GMC-1 in sli mutants symmetrically divides, both daughters inherit Nb (Mehta, 2001).

The loss-of-function effects of sli on the distribution of Insc in GMC-1 (and thus the symmetrical division of GMC-1) could be due to this lack of down regulation of Miti and Nub in GMC-1. To test this possibility, the miti transgene was ectopically expressed from the hsp70 promoter. A 25-minute induction of miti was sufficient to alter the localization of Insc and the distribution of Insc in these embryos resembled the distribution of Insc in sli embryos (Mehta, 2001).

The above results indicate that the symmetrical division of GMC-1 in sli mutants is due to the up regulation of the two POU genes and that these two POU genes are the targets of Sli signaling in GMC-1; however, the partial penetrance of these phenotypes in sli mutants indicate that additional pathways also mediate this very same process and regulate the levels of the two POU proteins in GMC-1. Since the penetrance in insc mutants is also partial, additional pathways must exist to mediate the asymmetric division of GMC-1 to partially complement the loss of the Insc/Sli pathway (Mehta, 2001).

The following picture emerges from this study. The Sli-Robo signaling down regulates the levels of Nub and Miti in late GMC-1, allowing the asymmetric localization of Insc and the asymmetric division of GMC-1. The possibility is entertained that loss of sibling cells in sli mutants would mean that some projections will be duplicated, while others are eliminated. Depending upon the extent, this might have an overall bearing on the pathfinding defects in sli mutants. Since Sli signaling is conserved in vertebrates, it is possible that this signaling may regulate generation of asymmetry during vertebrate neurogenesis as well (Mehta, 2001).

Embryonic: Inscuteable localization and cell cycle regulation

Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. These findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence (Tio, 2001).

The embryonic central nervous system (CNS) of Drosophila is derived from progenitors called neuroblasts (NBs). NBs undergo repeated asymmetric divisions, budding off a series of ganglion mother cells (GMCs) from their basal/lateral surfaces; GMCs can divide asymmetrically to produce progeny with distinct neuronal fates. Both the NB and GMC asymmetric divisions are mediated, in part, by a protein localization machinery that directs the preferential segregation of Prospero (Pros) or Numb to the more basally located daughter. Mitosis is driven by activation of the Cdc2 protein kinase, which, during the first 13 embryonic divisions, depends on dephosphorylation by the product of maternal string (cdc25). NB divisions occur after depletion of maternal string and depend on zygotic string. However, NBs, although arrested at G2 of cycle 14, do form in embryos lacking zygotic string. In contrast, loss of zygotic cdc2 does not substantially affect embryonic development, and lethality occurs during postembryonic development (Tio, 2001).

From a mutant screen two lines were identified that exhibit defective localization of Pros and Inscuteable (Insc) in NBs. Genetic mapping, complementation and DNA sequencing reveal that the phenotypes associated with both mutants were caused by the same mutation in cdc2, resulting in a glutamic acid to glutamine change at amino-acid 51. Embryos homozygous for cdc2E51Q show late embryonic lethality and exhibit various abnormalities that are also seen in insc and partner of inscuteable (pins) mutants, which can be explained by defects in asymmetric divisions (Tio, 2001).

To illustrate these defects, a focus was placed on the first GMC produced from NB4-2, GMC4-2a, which divides to generate two daughter neurons, RP2 and its sibling RP2sib. The Even-skipped (Eve) protein is expressed in the GMC4-2a sublineages. Eve is initially expressed in both RP2 and RP2sib; however, its expression is extinguished in RP2sib, such that late in embryonic development only one Eve+ neuron can be seen at the RP2 position in each wild-type hemineuromere. Two types of defects are seen in cdc2E51Q homozygotes. Fourteen per cent of the mutant hemisegments exhibit near the RP2 position a single Eve+ cell that has all of the characteristics of the RP2 neuron but is larger than the wild-type RP2 neuron. In cell-division mutants that prevent GMC4-2a division, GMC4-2a differentiates into an RP2-like cell that is larger than normal. More notably, 33% of the mutant hemisegments possess two cells near the RP2 position, both of which show characteristics of the RP2 neuron as judged by marker expression (Tio, 2001).

Duplication of the RP2 neuron is caused by an RP2sib -> RP2 transformation, indicating that the normally asymmetric division of GMC4-2a (GMC4-2a -> RP2 + RP2sib), has been converted to a symmetric division (GMC4-2a -> RP2 + RP2) in the mutant. This defect in asymmetric division is not restricted to the CNS; the normally asymmetric division of muscle progenitor, P15, which in wild type produces a single Eve-expressing muscle DA1 and a second daughter of unknown fate, can also become symmetric in mutant embryos, leading to the duplication of muscle DA1 (Tio, 2001).

The localization of Insc, Partner of Numb (Pon, which colocalizes with Numb) and Miranda (which colocalizes with Pros) was assessed in mutant neural progenitors which are clearly undergoing mitosis. Dividing wild-type GMC4-2a always localizes Insc as an apical crescent and Pon as a basal crescent. In dividing cdc2E51Q GMC4-2a, defective localization is observed of Insc (39%) and Pon (37), in the form of cortical distribution or misplaced crescents, and misorientation of the mitotic spindle (Tio, 2001).

These defects are not restricted to GMCs. Mislocalization of Insc (25%), Bazooka (38%), Pon and Miranda, and defective spindle orientation are also seen in mutant mitotic NBs. These data suggest that the underlying cause of the abnormal progenitor divisions may be the failure to localize the apical components during mitosis, and consequently localization of the basal determinants is also defective. Consistent with this notion, the duplicated RP2 neurons show identical nuclear size -- a phenotype characteristic of insc and pins mutants (Tio, 2001).

Embryonic lethality and defects in asymmetric division are seen in cdc2E51Q embryos but embryos totally lacking zygotic cdc2 function develop essentially normally, owing to the maternal contribution of cdc2. Several observations indicate that cdc2E51Q acts as a maternal effect dominant-negative allele that can antagonize the maternally inherited wild-type cdc2. Strong mutant phenotypes are seen in genotypically hemizygous (cdc2E51Q/deficiency) embryos only if the cdc2E51Q allele is inherited from the (cdc2E51Q/CyO) mother, and not if it comes from the father. Moreover an earlier arrest of cell divisions, resulting in a marked increase in the frequency of undivided GMC4-2a cells, is seen by overexpressing cdc2E51Q (from a uas-cdc2E51Q transgene) (Tio, 2001).

To show that defects in asymmetric cell divisions and protein localization are not peculiar to cdc2E51Q, a stock was used that is homozygous for an amorphic allele, cdc2B47, and in addition contains four copies of a transgene carrying a temperature-sensitive allele, cdc2A171T, of cdc2 (referred to as cdc2ts4X). Immunoprecipitates from cdc2ts4X embryonic extracts exhibit kinase activity that is highly temperature sensitive. Under appropriate temperature-shift conditions, these embryos can exhibit all of the phenotypes seen in cdc2E51Q including RP2 and muscle DA1 duplications, and defective protein localization in dividing progenitors (Tio, 2001).

These results suggest that the levels of Cdc2 activity determine whether and how a progenitor divides. When the level of Cdc2 is low, neural progenitors fail to divide; at intermediate levels they can divide, but mitotic NBs often fail to correctly localize asymmetric components, and GMC divisions can become symmetric with respect to segregation of cell-fate determinants and the size and fate of their daughters; only at higher levels of cdc2 do normal asymmetric divisions take place (Tio, 2001).

A binary expression system was used to express wild-type and mutated forms of cdc2 in the neural progenitors of cdc2E51Q embryos. Expression of a wild-type cdc2 transgene rescues the cdc2E51Q phenotypes, whereas the expression of enzymatically dead versions of cdc2, cdcT161A and cdc2K33R/T161A, which do not exhibit dominant-negative properties, did not rescue those phenotypes, suggesting that kinase activity is required for asymmetric divisions. Since Cdc2 kinase activity appears to be required to maintain apical Insc localization during mitosis, might it also be required for the apical localization of Insc during interphase? In embryos lacking zygotic string, NBs form but arrest at G2 during interphase of cell-cycle stage 14 and fail to enter mitosis because mitotic kinase activation does not occur. Insc, Pins and Bazooka form normal apical crescents, indicating that the initial apical localization of the apical components during interphase does not require mitotic kinase activation. Moreover, temperature upshifts can induce similar string-like phenotypes in cdc2ts4X embryos, and in these embryos the NBs arrested at G2 show apical localization of the apical components. These data suggest that Cdc2 kinase activity is required to maintain the apical complex proteins during mitosis but not for their establishment during interphase (Tio, 2001).

If Cdc2 activity is responsible for maintaining the apical components, premature inactivation/reduction of its activity -- at a point in mitosis when its activity is normally high -- should lead to premature delocalization of apical components like Insc. cdc2ts4X and wild-type control embryos were arrested at metaphase using colcemid treatment at 21°C for 30 min; half of the embryos were shifted to 31°C for 45 min, while the other half were kept at 21°C for 45 min, then both groups were fixed and stained for Insc. cdc2ts4X NBs arrested at metaphase and maintained at 21°C showed normal apical crescents of Insc (98%); similarly, colcemid-treated wild-type controls that were shifted to 31°C show 100% apical localization. cdc2ts4X NBs arrested at metaphase and upshifted to 31°C showed defective localization of Insc (only 7% have normal apical crescents) and Miranda. These results show that downregulating Cdc2 activity in NBs arrested at metaphase can cause delocalization of the apical (and basal) component proteins and provide direct evidence that elevated Cdc2 activity is required to maintain the localization of apical components during mitosis (Tio, 2001).

There are three known mitotic cyclins in Drosophila -- cyclin A, B and B3; these need to be destroyed for mitotic exit to occur. Is a subset of these cyclins preferentially required for maintaining the apical components? The temporal profile of Insc localization was followed with respect to the time of cyclin A (metaphase), cyclin B (early anaphase) and cyclin B3 (late anaphase) destruction. The results from anti-Insc/anti-cyclin-A/DNA-stain triple-labelling experiments show that Insc remains apically localized at metaphase/anaphase after destruction of cyclin A. Supporting the idea that cyclin A is dispensable, no evidence of defective Insc localization was detected in cyclin A single mutants and cyclin A;cyclin B3 or cyclin A;cyclin B double mutants. In wild-type NBs, Insc delocalization occurs after chromosome separation, coinciding with the time when cyclin B3 becomes undetectable. Single mutants in cyclin B and cyclin B3 do not show defects in Insc localization; however, in cyclin B;cyclin B3 double mutants, mislocalization of Insc can be seen in most mitotic NBs (72% for prophase; 71% for metaphase). These results indicate that whereas cyclin A appears dispensable, the B-type cyclins are required to maintain asymmetric localization of Insc during mitosis (Tio, 2001).

These data show that a key cell-cycle regulator is involved in mediating asymmetric cell divisions. Phosphorylation mediated by Cdc2 is likely to be important in maintaining the correct localization of the apical complex of asymmetry proteins during mitosis; however, Cdc2 probably does not act directly on Insc as the putative Cdc2 phosphorylation sites of Insc can be removed without affecting its function in overexpression paradigms. These observations provide an explanation for the normal temporal profile of the localization of molecules like Insc. Since long as there is Cdc2 kinase activity during mitosis, apical/cortical localization of molecules such as Insc is maintained; however, when kinase (and B-type cyclins) is destroyed towards the end of mitosis, the apical components become delocalized. These findings indicate that the tight temporal correlation of asymmetrically localized components important for mediating asymmetric divisions to the cell cycle may be because the two processes share key regulator(s) like cdc2 (Tio, 2001).


The division of postembryonic neuroblasts (Nbs) has been studied in the outer proliferation center (OPC) and central brain anlagen of Drosophila. Attention has been focussed on three aspects of these processes: the pattern of cellular division; the topological orientation of these divisions, and the expression of asymmetric cell fate determinants. Although larval Nbs are of embryonic origin, the results indicate that their properties appear to be modified during development. Several conclusions are summarized: (1) in early larvae, Nbs divide symmetrically to give rise to two Nbs while in the late larval brain most Nbs divide asymmetrically to bud off an intermediate ganglion mother cell (GMC) that very rapidly divides into two ganglion cells (GC); (2) symmetric and asymmetric divisions of OPC Nbs show tangential and radial orientations, respectively; (3) this change in the pattern of division correlates with the expression of Inscuteable, which is apically localized only in asymmetric divisions; (4) the spindle of an asymmetrically dividing Nb is always oriented on an apical-basal axis; (5) Prospero does not colocalize with Miranda in the cortical crescent of mitotic Nbs; (6) Prospero is transiently expressed in one of the two sibling GCs generated by the division of GMCs (Ceron, 2001).

In simple geometric terms, one may describe the OPC as a germ neuroepithelium forming a ring-like structure that covers the most lateral side of the lobe. Nbs occupy the external layer, close to the outside surface, and their progeny ganglion cells lay inside it, forming a thicker layer. This layered structure, which can be observed in frontal sections of optic lobes, allows an easy identification of the different cell types. If sectioning is similarly applied to BrdU-labeled optic lobes, one may observe that different time pulses give rise to different patterns of cell labeling in the OPC. Thus, short pulses result in preferential labeling of medium-size nuclei located just below Nbs that in turn are very often unlabeled. In contrast, longer pulses yield extensive labeling of large Nb nuclei and abundant small GC. Different pulse periods do not result in differential labeling of central brain (CB) Nbs and their progeny. Thus, short pulses yield pairs of labeled cells that consist of one Nb and a single daughter cell, while longer pulses produce labeling of one Nb together with a couple of daughter cells (Ceron, 2001).

The incorporation of BrdU in the progeny of Nbs during short pulses and the frequent observation of two labeled nuclei apparently undergoing cytokinesis very close to a Nb suggest the existence of GMCs that have a cell cycle shorter than their parent Nbs. Direct evidence for the existence of mitosis in those daughter cells was obtained by applying several immunochemical tools. Medium-size mitotic cells are detected just below the layer of OPC Nbs. Also, in the CB, where individual Nbs and their progeny can be observed, medium-size mitotic cells are detected immediately close to each Nb. In this case, all daughter cells are located at the same side of the Nb but no more than one is in mitosis. Interestingly, even in interphase Nbs, the centrosome is always located at the pole opposite that of the budding cells and the mitotic spindle of daughter cells is most often oriented at an oblique angle relative to that of the parent Nb. Altogether, labeling experiments with BrdU and mitotic markers demonstrate the presence of GMC-like cells in postembryonic proliferative anlagen (Ceron, 2001).

OPC Nbs stop producing more Nbs and begin to generate the final neuronal progeny around the third-instar larval period. This change in proliferative behavior could be explained by a change from an initial symmetric pattern of division to a late asymmetric one. Since asymmetric divisions of embryonic Nbs follow an apical/basal orientation, it would be also interesting to find out whether symmetric and asymmetric divisions of postembryonic Nbs have different orientations. This is indeed the case. The divisions of mitotic Nbs in the OPC of early third-instar larvae are preferentially oriented on an axis tangential to the surface, whereas those observed in late third-instar larvae show almost exclusively a radial orientation. Larval ventral ganglion Nbs, which divide asymmetrically, contain unequal centrosomes during mitosis. The larger centrosome is segregated into the resulting Nb and the smaller is inherited by the GMC. Radially oriented divisions of OPC Nbs have asymmetric centrosomes with the larger one close to the optic lobe surface, whereas tangentially oriented divisions have symmetric centrosomes. The metaphase plate of asymmetrically dividing Nbs is located close to the smaller (basal) centrosome. In contrast to the epithelial sheet-like organization of the OPC anlagen, CB Nbs are distributed in the most medial part of the optic lobe and each one shows a different direction of asymmetric division. Nevertheless, all the progeny of each Nb appear to be released by the same side and the interphase centrosome is maintained at the opposite side of the progeny (Ceron. 2001).

To determine whether the regulation of asymmetric divisions and the segregation of cell fate determinants of postembryonic Nbs follow a pattern similar to that described for embryos, the expression and localization of Insc, Mir, Numb, and Pros were examined in whole mounts of third-instar larval brain. Immunostaining of Insc is not detected in Nbs at early larval stages, when most divisions are symmetric and oriented tangentially to the surface of the optic lobe. At late stages of larval development, Insc is detected in asymmetrically dividing Nbs. Its pattern of localization changes during cell division. Thus Insc shows a low expression in interphase Nbs, but is apically localized, both in the OPC and in the CB at early mitotic phases. After metaphase, Insc labeling becomes undetectable. No Insc protein is detected in GMC of the CB but polarized Insc is consistently observed in medium-size cells (i.e., smaller than Nb but larger than GCs) that are undergoing mitosis inside the OPC (Ceron, 2001).

A study of APC1 and APC2 examines asymmetric protein localization in larval neuroblasts

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. The functions of the Drosophila APCs is further explored using the larval brain as a model. Both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. These experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins (Akong, 2002).

One striking feature of the asymmetric localization of APC2 is that it is present throughout the cell cycle and is particularly strong during interphase. During embryonic neuroblast divisions, most asymmetric markers are localized only during mitosis. However, less is known about their localization in larval neuroblasts. Several asymmetric markers in larval neuroblasts were examined, and their localization was compared with that of APC2. In embryonic neuroblasts, the transcription factor Prospero (Pros) and its mRNA are GMC determinants that are asymmetrically localized to the GMC daughter. Pros protein then becomes nuclear and helps direct cell fate. In larval neuroblasts, a similar localization is observed. Pros is not detectable in interphase neuroblasts, when the cortical APC2 crescent is strongest. A small amount of Pros transiently localizes to an asymmetric crescent during mitosis. Pros is present at low levels in GMC nuclei and at higher levels in the nuclei of ganglion cells (Akong, 2002).

Mira is basally localized in embryonic neuroblasts, and required there for localization of Pros protein and mRNA. In central brain neuroblasts, Mira is diffusely cytoplasmic during interphase, when the APC2 crescent is the strongest. As cells enter mitosis, Mira first becomes cortical and then begins to accumulate asymmetrically on the side of the neuroblast where the daughter will be born. By metaphase, Mira asymmetry is very pronounced. The center of the Mira crescent is always precisely aligned with one spindle pole. As a result, in cells with the spindle pointing toward the center of the APC2 crescent, the Mira and APC2 crescents substantially overlap, while in cells in which the spindle points to the edge of the APC2 crescent, the two crescents are offset. Mira is partitioned into the GMC during anaphase, while APC2 relocalizes to the cleavage furrow. Mira could still be detected in some GMCs, which are thought to be those that were recently born (Akong, 2002).

In contrast to Mira and Pros, Inscuteable (Insc) and Bazooka (Baz) localize to the apical sides of embryonic neuroblasts, where they play essential roles in asymmetric divisions. Insc is asymmetrically localized in larval neuroblasts. Insc localizes to the side of the neuroblast opposite that of APC2 through much, if not all, of the cell cycle. Interestingly, there is a weak Insc crescent during interphase, that becomes stronger through prophase and metaphase. During anaphase, Insc localizes to the neuroblast cortex but not the GMC daughter. Baz localization was similar to that of Insc, though no cortical localization during interphase was detected. During prophase and metaphase, Baz localizes to a crescent opposite APC2, and as the chromosomes begin to separate, Baz localizes to a tight cap opposite the future GMC. Together, these data confirm that larval and embryonic neuroblasts asymmetrically localize many of the same proteins, and that APC2 localizes on the GMC side (basal) of the neuroblast, overlapping Mira and opposite Baz and Insc, which localize apically (Akong, 2002).

Arm also localizes asymmetrically in neuroblasts. Extending this, an examination was made of the localization of Arm's adherens junction partners DE-cadherin and ß-catenin. When central brain neuroblasts undergo a sequential series of asymmetric divisions, the GMCs remain associated with their neuroblast mother, resulting in a cap of GMCs in association with each neuroblast. APC2 localizes strongly to the boundary between the neuroblast and each GMC, and more weakly to the borders between the GMCs. APC2 is present at lower levels in ganglion cells and differentiating neurons (Akong, 2002).

The adherens junction proteins DE-cadherin, Arm, and ß-catenin all show a striking and asymmetric localization pattern in central brain neuroblasts. All precisely colocalize both at the boundary between neuroblasts and GMCs and at the boundaries between GMCs. DE-cadherin, Arm, and ß-catenin are also all expressed in epithelial cells of the outer proliferation center. The localization of DE-cadherin and the catenins is consistent with the idea that cadherin-catenin-based adhesion could help ensure that GMCs remain associated with each other, via association with their neuroblast mother (Akong, 2002).

To further explore this, how successive GMCs are positioned relative to their older GMC sisters was examined using two different approaches. First Mira was used to mark the newborn GMCs and DE-cadherin was used to mark the neuroblast and all of her GMC daughters. Mira localizes to a crescent on the side of the neuroblast where the daughter will be born (basal side), and then is segregated into the daughter. Mira persists for some time in newborn GMCs, and it remains detectable in the other GMCs as well, thus allowing the position of newborn GMCs to be examined relative to their older sisters. In many cases, new GMCs are clearly born at the edge of the cluster of older GMCs. This is particularly striking in neuroblasts with many progeny. It is worth noting that the cluster of daughters is three-dimensional, comprising a 'cap' of daughters in three dimensions rather than a two-dimensional line of daughters. It is thus suspected that new daughters are born near the edge of this cap (Akong, 2002).

These data suggest that neuroblasts and their GMC progeny remain closely associated. The GMCs then divide to form ganglion cells and ultimately neurons. The data further suggest that these latter cells may also remain associated and send their axons together toward targets in the central brain. When sections were made more deeply into the brain, below each cluster of neuroblasts and GMCs, structures that appear to be axons were detected projecting from these groups of cells. These axons label with Arm, DE-cadherin, and APC1. Arm also localizes to the axons of the neuropil, while DE-cadherin and APC2 are present at low levels or are absent from this structure (Akong, 2002).


In the wing disc, INSC protein is detected in pupal wings older than roughly 20 hours after puparium formation. INSC appears in the anlage of the intervein space but is excluded from the veins themselves (Kraut, 1996a).


INSC is seen in nurse cell-associated follicle cells and in those cells covering the anterior part of the oocyte. At oogenesis stage 9 expression is evident in follicle cells migrating over the anterior end of the oocte chamber, and fades posteriorly. This gradient retracts, and by late stage 10 covers only the centripetal cells at the border between the nurse cells and oocyte (Kraut, 1996a).

Effects of Mutation or Deletion

In a search for mutations affecting embryonic muscle development in Drosophila a mutation caused by the insertion of a P-element was identified and termed called not enough muscles (nem) (currently termed inscuteable). The phenotype of the P-element mutation of the nem gene, suggests that it may be required for the development of the somatic musculature and the chordotonal organs of the PNS, while it is not involved in the development of the visceral mesoderm and the dorsal vessel. Mutant embryos are characterized by partial absence of muscles, monitored by immunostainings with mesoderm-specific anti-beta 3 tubulin and anti-myosin heavy chain antibodies. In addition to these muscle distortions, defects in the peripheral nervous system are found, indicating a dual function for the nem gene product. Ethyl methane sulfonate-induced alleles for the P-element mutation were created for a detailed analysis. One of these alleles is characterized by unfused myoblasts that express beta 3 tubulin and myosin heavy chain, indicating the state of cell differentiation (Burchard, 1995).

Each larval hemisegment comprises ~30 uniquely specified somatic muscles. These derive from muscle founders that arise as distinct sibling pairs from the division of muscle progenitor cells. The progenitor cell divisions of three mesodermal lineages (P2, P15, and P17) that generate muscle (and pericardial cell) founders have been analyzed. Each of these progenitors divides once at a specific stage in development to give rise to the founders for the Eve+ pericardial cells (EPCs), the Eve+ Kr+ dorsal acute 1 (DA1 = m1) muscle, and the Kr+ dorsal oblique 1 (DO1 = m9) muscle, respectively, and a sibling cell of unknown fate. These progenitors were examined because mutations in insc and nb have strong effects on the development of the EPC as well as DA1 and DO1, and suitable markers are available for the analyses of these cells and the division of their progenitors (Carmena, 1998).

Inscuteable and Numb proteins are localized as cortical crescents on opposite sides of dividing progenitor cells. Asymmetric segregation of Numb into one of the sibling myoblasts depends on inscuteable and is essential for the specification of distinct sibling cell fates. In contrast to the nervous system where Insc protein crescents are localized to the apical cortex of neuroblasts, there does not appear to be a fixed orientation for the Insc crescent in the different progenitors of the mesoderm. This appears to be due to the fact that unlike neuroblasts that align their mitotic spindles along the apical/basal axis, these progenitors do not divide with a fixed orientation. However, for any given type of progenitor, the Insc crescent accumulates at a similar position relative to the anterior/posterior or dorsal/ventral axis. Despite these differences in the orientation of the Insc crescent, there appears to be a tight correlation between the position of the Insc protein crescent and the orientation of the progenitor cell division as deduced from the staining of DNA; the location of the Insc crescent appears to center on one of the mitotic spindle poles (Carmena, 1998).

Loss of numb results in opposite cell fate transformations from loss of inscuteable - loss of either prevents sibling myoblasts from adopting distinct identities, resulting in duplicated or deleted mesodermal structures. Embryos homozygous for nb3, a putative amorphic allele of numb, were stained with anti-myosin heavy chain (MHC), which labels all somatic muscles, and anti-Eve, which stains DA1 and EPC. The loss of nb has a general effect causing many of the dorsal and ventral somatic muscles to be lost. However, the expressivity of the phenotype varies for each muscle, and not all somatic muscles are affected. Nevertheless, in nb3 homozygotes, DA1 is almost always absent, whereas DO1 is lost from >50% of the mutant hemisegments. In contrast, the number of EPC is increased. The overexpression of Nb can lead to the duplication of DA1 as well as the loss of EPC, effects that are opposite those caused by the loss of nb. However, because of the multiplicity of extra dorsal muscles associated with this overexpression paradigm, DO1 could not be scored. These observations are consistent with the notion that nb can act in a necessary and sufficient manner to specify mesodermal cell fate (Carmena, 1998).

A model is presented and tested for insc and nb loss- (and gain-) of-function phenotypes. In wild-type embryos, P15 (and P17) divide such that one progeny becomes the founder for DA1 (and DO1), henceforth referred to as FDA1 (and FDO1), as a consequence of inheriting all of the asymmetrically localized Nb protein. Its sibling cell does not inherit Nb and adopts an alternative (unknown) fate. In the absence of insc (or when nb is overexpressed), Nb is no longer asymmetrically distributed so both daughter cells derived from the P15 (and P17) division inherit Nb and both adopt an FDA1 (and FDO1) identity at the expense of its sibling. This leads to the duplication of DA1 (and DO1), which in fact is observed in insc mutants. In the case of EPC, for the wild-type P2 cell division, it is the progeny that fails to inherit Nb that becomes the FEPC, whereas its sibling (FEPCsib), which inherits Nb, adopts an alternative but unknown fate. Hence, in insc mutants (or when nb is overexpressed) both of the P2 progeny are Nb+ and adopt the fate of FEPCsib at the expense of the FEPC, leading to the loss of EPC. Conversely, in the absence of nb, both siblings derived from the progenitor cell division adopt the identity of the sibling which would normally not inherit Nb. As a result, the opposite cell fate transformations occur, leading ultimately to the loss of DA1 and DO1 and the gain of EPC (Carmena, 1998).

Because insc and nb mutants show opposite mesodermal phenotypes, a double mutant was made and its phenotype examined to ascertain the hierarchical relationship between insc and nb. The insc, nb double homozygous embryos show loss of DA1 and DO1, as well as gain of EPC. Although qualitatively similar to those shown by nb mutant embryos, the double mutant embryos exhibit these phenotypes with higher levels of expression. These results suggest that nb acts downstream of insc, consistent with data showing that insc is required for wild-type Nb localization (Carmena, 1998).

Roughly 5% of all mutants eclose from the pupal cases. About half of these adult escapers have blistered wings. The cuticle of mutant embryos exhibit a relatively mild pattern of defects in the ventral denticles (that are occasionally missing or disordered) and in the head skeleton. Obvious early defects are seen in the neuroblast pattern. The neuroblasts in insc mutants are arranged in a disorderly manner, are slightly smaller than in wild type, and have an abnormal shape. Some of them do not segregate completely out of the neuroectoderm. There are mild defects in the CNS, particularly in the pattern of commissures and connectives. The organization and number of sense organs is variably faulty. Chordotonal organs are either absent or disorganized. In over half of the hemisegments in insc mutants, the RP2 neural precursors are either duplicated or absent. The number of neurons in the aCC/pCC/CQ and EL clusters also appears to be altered (Kraut, 1996a)

Miranda protein can interact with both Prospero and Numb. The regions of Miranda that interact with these two proteins are distinct from each other and are mapped to the central and C-terminal portions of the Miranda protein, respectively (Shen, 1997).

In embryos deficient for miranda, Prospero is not associated with the membrane, but stays in the cytoplasm in prophase. Prospero remains in the cytoplasm in metaphase and anaphase and then is segregated into both daughter cells. Shortly after cell division, Prospero is translocated into the nuclei of both daughter cells. The orientation of the mitotic spindle in neuroblasts and cells of the procephalic neurogenic region is normal in miranda deficient embryos. A transduced miranda can rescue the Prospero localization defects in neuroblasts of miranda indicating that miranda is required for the correct positioning of Prospero in neuroblasts during mitosis (Shen, 1997).

The Numb protein is asymmetrically localized to the basal cell membrane in neuroblasts and cells of the procephalic neurogenic region during mitosis, in contrast to the cytoplasmic distributions of Prospero. After cell division, Numb is segregated into only the basal daughter cell, whereas Prospero is translocated into the nuclei of both cells (Shen, 1997).

The asymmetric localization of Miranda in neuroblasts of prospero and numb mutants is indistinguishable from that of wild-type embryos. Therefore, the asymmetric localization of Miranda does not require prospero or numb. In embryos homozygous for a null allele of inscuteable, both Miranda and Prospero are unable to form crescents or they form crescents that are randomly localized along the cell membrane. Therefore Miranda crescent formation and localization requires inscuteable (Shen, 1997).

not enough muscles (nem) (currently termed inscuteable) mutants of Drosophila reveal defects in the development of embryonic muscles, a subset of pericardial cells, the CNS and derivatives of the PNS. The nem mutation reveals reduction of the Drosophila embryonic muscle pattern. The molecular analysis of the nem locus shows a complex genomic structure. One transcription unit is identified as inscuteable (insc). Within the first intron of insc is found another independent gene, skittles (sktl), which is not affected in nem mutants. insc transcripts are localized apically in neuroblasts and may prefigure the localization of the protein. The skittles mRNA is ubiquitously distributed during early embryogenesis due to maternal contribution. Later, some enrichment of sktl is observed in the nervous system and the mesoderm. The muscle phenotype shows deletions as well as duplication of specific muscles, all reflected in a reduction in the number of even-skipped (eve) expressing paracardial cells. insc mutants have a variable phenotype in Kruppel expressing muscle 1 and muscle 9 cells. These data suggest a role for insc in the specification process of a subset of muscle progenitors/founders. In insc mutants the eve expressing pericardial cells of the developing heart are significantly reduced in numbers. It is thought that Insc works as a cytoskeletal adaptor protein in muscle progenitors similar to its functions in the PNS and CNS (Knirr, 1997).

Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells

Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of asymmetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).

The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).

Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).

Staining of wild-type embryos with anti-Insc antibody reveals that Insc is expressed in many, and possibly all, GMCs. During interphase, Insc protein is found cortically. In dividing GMCs, anti-Insc immunoreactivity is always seen as an apical/near apical crescent with respect to the surface of the embryo, that is, Insc is localized in the part of the cell located toward the ventral (outside) portion of the embryo. These observations suggest that GMCs possess apical-basal polarity and that their division may be asymmetric in an insc-dependent manner. If the asymmetry of GMC division leads to the generation of pairs of sibling cells that are nonequivalent, then which are the cell fate determinants that are segregated differentially? One such candidate is Numb: it is widely expressed in the developing CNS and has been shown to act as an intrinsic fate determinant in the SOP and MP2 lineages. Moreover, asymmetric Numb localization has been shown to be Insc-dependent in NBs (Buescher, 1998).

To see if Numb is asymmetrically localized in GMC1-1a and GMC4-2a in an Insc-dependent manner, wild-type and insc mutant embryos were stained with anti-Numb, anti-Eve, and DNA stain. In wild-type embryos, Numb always forms a crescent at or near the basal cortex of dividing GMCs, whereas in insc mutant embryos, crescents are either not formed or appear in basal and occasionally in lateral or apical positions. Although it is difficult to directly visualize the orientation of the mitotic spindle in GMCs, the positioning of the metaphase plate in wild-type strongly suggests a perpendicular orientation with respect to the apical surface of the embryo. Consistent with a horizontally placed cleavage plane, the newborn RP2/RP2sib cells are always oriented perpendicular or nearly perpendicular to the apical surface, with the larger cell located in the more dorsal (basal) position. It is concluded that during GMC4-2a division, Numb will be segregated predominantly or exclusively to the future RP2 neuron. In addition, the orientation of metaphase plate in dividing GMCs other than GMC4-2a is also horizontal with respect to the apical surface, indicating that the division of many, possibly all, GMCs are stereotyped in the same manner as NB divisions. Consistent with its function in NBs, loss of insc alters the orientation of GMC division: In 74% of the samples the metaphase plate is oriented perpendicular or close to perpendicular to the apical surface. Accordingly, the newborn RP2/RP2sib are frequently oriented horizontally with respect to the apical surface. Taken together, these results indicate that the apical-basal polarity, which is found in NBs is maintained in their daughter cells, the GMCs, and that Numb localization as well as spindle orientation are coordinated to ensure asymmetric segregation of Numb into one sibling only. So far, insc appears to be the only gene that is required for the apical-basal polarity of GMCs: the analysis of mam, spdo, and N mutant embryos reveals that none of these mutations alter the plane of GMC division. This observation is in agreement with the notion that these genes act later than insc in sibling neuron fate determination (Buescher, 1998).

It has been shown that the the correct positioning of Numb requires Inscuteable; Numb acts downstream of Insc. Taken together, the data show that asymmetric fate determination in the GMC4-2a lineage involves the same components as fate specification in the MP2 and SOP lineages and may be accomplished through productive (effective) N signaling in one sibling (RP2sib) and inhibited (ineffective N signaling) in the Numb-containing sibling (RP2). However, in the case of asymmetric fate determination in GMCs, Notch function is determined through an intracellular, Numb based mechanism, and not through a lateral inhibition mechanism. Consistent with this notion, the ectopic expression of N-intra (a constitutively active form of N) causes the same RP2 to RP2sib fate transformation as numb loss of function, implying that N-intra ectopic expression can override the effects of Numb (Buescher, 1998).

The loss of size asymmetry in insc mutant embryos is not restricted to the RP2/RP2sib lineage but is also observed in muscle progenitor cell divisions. For example, when the muscle progenitor P15 divides, the daughter cells are not equal in size; it is the larger of the two daughter cells that preferentially inherits the Numb that is asymmetrically localized in the dividing muscle progenitor. Similar to the RP2/RP2sib situation, removing insc function appears to equalize the size of the daughter cells derived from the P15 cell division. In contrast, this equalizing effect does not occur in NBs. NB division is highly asymmetric: each division generates a new NB and a GMC that is several times smaller than the NB. In insc mutants, NBs often bud off GMCs in lateral (rather than basal) positions, but the size asymmetry is retained. At present, it is not understood how size asymmetry is generated during progenitor cell division and if, and how, it might be linked to spindle orientation (Buescher, 1998).

Modes of protein movement that lead to the asymmetric localization of partner of Numb during Drosophila neuroblast division

The proper localization of Numb depends on its interaction with the adapter protein Partner of numb (Pon). In pon mutant embryos, the formation of Numb crescent is delayed in neuroblasts and is disrupted in muscle progenitor cells (Lu, 1998). Pon was isolated on the basis of its physical interaction with Numb. Pon is asymmetrically localized during mitosis and colocalizes with Numb. Ectopically expressed Pon responds to the apical-basal polarity of epithelial cells and is sufficient to localize Numb basally. It is proposed that PON is one component of a multimolecular machinery that localizes Numb by responding to polarity cues conserved in neural precursors and epithelial cells (Lu, 1998 and Lu, 1999).

In principle, the asymmetric localization of Numb/Pon can be accomplished by one or a combination of the following mechanisms: localization and local translation of their mRNAs; active transport of the proteins by motor molecules along the cytoplasmic or cortical cytoskeleton; passive diffusion (3D in the cytosol or 2D along the cortex) and trapping of the proteins by basally localized anchor molecules, or protein targeting to the membrane followed by selective degradation at one side of the cortex. The available method to detect protein localization by immunostaining of fixed embryos only provides static images of the proteins in different cells and is inadequate to distinguish among the above possibilities (Lu, 1999).

The mechanism of asymmetric Pon localization has been shown to operate at the protein level. The asymmetric localization domain of Pon has been mapped to its C-terminal region. Using a fusion between this localization domain and GFP, the entire process of Pon localization was monitored in neuroblasts of living embryos. This in vivo analysis reveals that the asymmetric localization of Pon is a dynamic, multistep process. The protein is first recruited from the cytosol to the cell cortex, a step that requires cell cycle progression into mitosis. Cortically recruited Pon then moves on the cortex and is later restricted to the basal side to form a crescent. The crescent disintegrates upon exit from mitosis. Photobleaching experiments reveal both apical and basal movements of Pon on the cortex. These movements can still occur when myosin motor activity is inhibited by drug treatment. Genetic and pharmacological analyses further reveal that the formation and anchoring of the Pon crescent at the basal cortex require actomyosin and Inscuteable (Lu, 1999).

Inscuteable coordinates mitotic spindle orientation and asymmetric protein localization in neuroblasts. To gain more insight into the mechanism by which Inscuteable directs the asymmetric localization of Pon, Pon-GFP was introduced into an insc mutant background. In insc mutant neuroblasts, cortical recruitment of Pon-GFP is normal, but the Pon-GFP crescent fails to form in 70% of the cases, and, in the remaining 30% of the neuroblasts, the crescent forms but it is randomly positioned. These observations are consistent with previous findings about protein localization in insc mutants. However, the ability to continuously monitor Pon-GFP in living embryos revealed a previously unrecognized function of Inscuteable. In wild-type embryos, the Pon-GFP crescent is stably anchored to the basal cortex once the crescent is formed. In contrast, in insc mutant embryos, the crescent is not stably anchored on the cortex, and it shifts position gradually. Depending on the orientation of the mitotic spindle and the position of the crescent at the time of cytokinesis, the protein can be segregated equally or unequally between the two daughters. Therefore, Inscuteable is required to provide positional cues that direct the formation and the anchoring and maintenance of Pon-GFP crescent at the basal cortex (Lu, 1999).

Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. By labelling mitotic spindles in living Drosophila embryos, it has been shown that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. Daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes (Kaltschmidt, 2000).

Inscuteable, a novel protein encoding a putative SH3 (Src homology 3) target site and a PDZ-binding domain, is both necessary and sufficient to direct apical-basal cell division in neuroblasts. Inscuteable also directs the axis of cell division in epithelial cells of the procephalic neurogenic region (PNR). In inscuteable mutant embryos, the orientation of the neuroblast spindles becomes random, as is the site of formation of a crescent of Prospero; in contrast, spindle reorientation simply does not occur in epithelial cells of the PNR (Kaltschmidt, 2000).

To investigate how the loss of Inscuteable interferes with normal apical-basal cell division, neuroblast spindle rotation was followed in living inscuteableP72 embryos. In these embryos, 20% of neuroblasts have spindles that fail to rotate. This percentage is in agreement with the number of misorientated neuroblast cell divisions that were counted in fixed inscuteableP72 embryos stained for DNA and Neurotactin, which labels the cell membrane. Because Inscuteable is expressed in ovaries, Inscuteable activity is also inhibited by RNA interference (RNAi). In principle, RNAi should block both maternal and zygotic messenger RNA and would be expected to produce a more severe phenotype than does the loss of zygotic Inscuteable alone. In embryos injected with inscuteable double-stranded RNA, the mitotic spindle initially forms normally, perpendicular to the apical-basal axis. In 10% of neuroblasts, the spindles seesaw but fail to rotate, a phenotype also seen in inscuteableP72 embryos. Seesawing is characteristic of epidermoblasts before cell division (Kaltschmidt, 2000).

It is concluded that the asymmetry of neuroblast cell division is dictated by the spindle itself becoming asymmetric at anaphase. Microtubules on the apical side of the cell elongate, while those on the basal side become shorter. As the astral microtubules become longer, and seemingly more abundant, the apical aster enlarges. The basal aster is concomitantly reduced in size. This process is independent of Inscuteable, since the spindle remains asymmetric even when it fails to rotate in an inscuteable mutant. Earlier reports have highlighted the existence of asymmetric spindle poles/centrosomes in yeast and C. elegans. However, in none of these cases were asymmetric midzone microtubules observed (Kaltschmidt, 2000).

Astral microtubules have been proposed to be involved in specifying the site of the cleavage furrow at cytokinesis. The results presented here are consistent with this model. It is found that, during neuroblast cell division, the apical astral microtubules elongate dramatically and grow toward the emerging GMC before the cell membrane invaginates. The overlapping apical and basal astral microtubules, which are distinctly different in length, may specify the asymmetric site of the cleavage furrow. It has been suggested that astral microtubules are not required for cytokinesis. Spermatocytes from an asterless mutant are still able to undergo cytokinesis: therefore, it has been suggested instead that the midbody specifies the site of the cleavage furrow. The results presented here do not distinguish between these two hypotheses; however, it is interesting that in neuroblasts the midbody moves basally towards the cleavage site only after the cell membrane has started to invaginate. It will be interesting to discover what, in addition to Inscuteable, regulates the rotation of the mitotic spindle. In C. elegans, rotation of the nucleus-centrosome complex depends upon dynein and dynactin. Dynactin is proposed to tether the dynein motor to the cortex of the cell, where it reels in one centrosome by movement along the astral microtubules. The speed of spindle rotation in Drosophila neuroblasts (less than 60 s) indicates that this process could also be mediated by dynein (Kaltschmidt, 2000).

There is no precedent for a cleavage furrow being placed asymmetrically between the spindle poles. The asymmetric position of the midbody may be characteristic of cells in the nervous system, or may in the future be detected in other cell types as imaging of live cells becomes more prevalent. Spindle asymmetry is cell-cycle regulated, but does not depend on previous spindle rotation. It will be interesting to identify the factors that induce spindle asymmetry in neuroblasts, and to discover how they are regulated during the cell cycle (Kaltschmidt, 2000).

Mechanism of glia-neuron cell-fate switch in the Drosophila thoracic neuroblast 6-4 lineage

During development of the Drosophila central nervous system, neuroblast 6-4 in the thoracic segment (NB6-4T) divides asymmetrically into a medially located glial precursor cell and a laterally located neuronal precursor cell. To understand the molecular basis for this glia-neuron cell-fate decision, the effects of some known mutations on the NB6-4T lineage were examined. prospero mutations lead to a loss of expression of Glial cells missing; this is essential to trigger glial differentiation in the NB6-4T lineage. In wild-type embryos, Pros protein is localized at the medial cell cortex of dividing NB6-4T and segregates to the nucleus of the glial precursor cell. miranda and inscuteable mutations alter the behavior of Pros, resulting in failure to correctly switch the glial and neuronal fates. These results suggest that NB6-4T uses the same molecular machinery in the asymmetric cell division as other neuroblasts in cell divisions producing ganglion mother cells. Furthermore, outside the NB6-4T lineage most glial cells appear independently of Pros (Akiyama-Oda, 2000).

In a null allele of pros no cells express Gcm or Repo in the NB6-4T lineage. In contrast, in a null allele of miranda all cells of the lineage express the glial proteins. The double mutant pros;mira produces no glial cells in the NB6-4T lineage, the same result obtained with the pros mutation. These results indicated that both pros and mira are involved in a pathway leading to the glia-neuron cell-fate switch, and that pros is epistatic to mira in this pathway. The effects of the insc mutation on the glia-neuron cell-fate switch in the NB6-4T lineage are slightly different from those of the pros or mira mutations. In insc mutants, both glial and non-glial cells are generated from NB6-4T in many of the hemisegments examined, but glial fate arises randomly from either of the daughter cells. These involvements of pros, mira and insc suggest an analogy between the first cell division of NB6-4T and NB cell divisions that produce GMCs and no glia. Pros, Mira and Insc proteins behave similarly during the first division of NB6-4T to the usual NB divisions producing GMCs. In the analyses of wild-type and mutant embryos, the high levels of expression of the earliest glial protein Gcm, and of the later glial protein Repo, are correlated with the nuclear localization of Pros in NB6-4T daughter cells. Consistent with this, in a pros mutant in which the mutant Pros protein does not enter the nucleus even after cell division, no glial cells are observed in the NB6-4T lineage. These observations suggest an important role for Pros in the onset of glial differentiation in the NB6-4T lineage (Akiyama-Oda, 2000).

Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. The asymmetric divisions that give rise to these cells have been visualized in developing pupae using green fluorescent protein fusion proteins. Spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorients to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, it is proposed that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila (Roegiers, 2001).

Both Miranda and Inscuteable (Insc) are expressed in the pIIb cell and Insc is required for proper apical-basal orientation of the mitotic spindle. The planar polarity gene fz is required for proper positioning of the Pon crescent at the anterior cortex of the pI cell during mitosis. Loss of Fz function has no effect on the proper apical-basal orientation of the pIIb division. This observation, along with the spindle asymmetry observed at anaphase in the pIIb division led to an exploration of the possibility that the pIIb cell division is a neuroblast-type cell division. Neuroblasts divide in the apical-basal orientation and require Insc to reorient the spindle along the apical basal axis. In addition, neuroblasts express Miranda, a linker protein that is asymmetrically localized to the basal cortex during mitosis. pIIb cells express Miranda and Inscuteable during mitosis, and Miranda forms a basal crescent. Insc is localized to the apical cortex of the pIIb cell (Roegiers, 2001).

Since Insc has a central role in orienting the spindle in the neuroblast division, it was of interest to examine whether Insc is also involved in spindle orientation in the pIIb cell. Previous studies did not detect a role for Insc in the SOP lineage, but these studies did not focus on the pIIb division. Mitotic clones of the inscp72 mutant, which has been shown to disrupt asymmetric divisions in the embryonic neuroblast lineage, were generated within the SOP lineage to determine whether insc is required for proper apical-basal spindle orientation in the pIIb cell. The widely used p72 allele of insc is a small deletion, which removes the inscuteable gene product as well as removing skittles, a kinase involved in the phosphoinositol cycle. The skittles mutant has no effect on asymmetric divisions in the embryonic neuroblast lineage, thus the phenotypes observed are most probably caused by the loss of insc. In most inscp72 mutant clones in pIIb cells (5/6 cases), the spindle orients within the plane of the epithelium. In the remaining case, the spindle orients roughly along apical-basal axis. Interestingly, in the five cases mentioned above the spindle showed a clear bias along the A-P axis, and in each of these divisions the spindle was observed to reorient with respect to the initial orientation of the duplicated centrosomes. Loss of Insc function had no effect on spindle positioning in the pI cell division. Together, these results indicate that the pIIb cell division strongly resembles that of an embryonic neuroblast, but there appears to be residual cue in the absence of insc that orients the spindle along the A-P axis (Roegiers, 2001).

These results suggest that there are two fundamental types of asymmetric divisions in the developing Drosophila nervous system. During Drosophila development these two types of divisions are reiterated in different tissues at different times to generate cell-fate diversity. The divisions of the sensory organ precursor cell provide a unique system for studying different types of asymmetric cell divisions within the same lineage and how they might be coordinated. The orientations of the divisions are tightly regulated: two divisions occur along the A-P axis, and two divisions occur in the apical-basal orientation. In the pI division, which occurs along the A-P axis, the spindle is symmetric and reorients to align perpendicular to the crescent of Pon-GFP, and fz is important for the proper orientation of the crescent and appears to contribute to the coordination of spindle orientation and crescent positioning. In contrast, the spindle in the pIIb cell orients along the apical-basal axis and exhibits a strong size asymmetry. insc, a gene of central importance in coordinating spindle orientation and crescent formation in embryonic neuroblast divisions, also has an important role in orienting the mitotic spindle in the pIIb cell. These findings provide strong evidence that the pIIb division is a neuroblast-like division. It will be interesting to know whether other genes known to be involved in controlling the asymmetric divisions of neuroblasts, such as bazooka or partner of inscuteable, are also required for the pIIb division. The results may reveal general mechanisms for generating cell-fate diversity in Drosophila as well as in other species (Roegiers, 2001).

ineage, cell polarity and inscuteable function in the peripheral nervous system of the Drosophila embryo

The stereotyped pattern of the Drosophila embryonic peripheral nervous system (PNS) makes it an ideal system to use to identify mutations affecting cell polarity during asymmetric cell division. However, the characterization of such mutations requires a detailed description of the polarity of the asymmetric divisions in the sensory organ lineages. The pattern of cell divisions generating the vp1-vp4a mono-innervated external sense (es) organs is described. Each sensory organ precursor (SOP) cell follows a series of four asymmetric cell divisions that generate the four es organ cells (the socket, shaft, sheath cells and the es neuron) together with one multidendritic (md) neuron. This lineage is distinct from any of the previously proposed es lineages. Strikingly, the stereotyped pattern of cell divisions in this lineage is identical to that described for the embryonic chordotonal organ lineage and for the adult thoracic bristle lineage. This analysis reveals that the vp2-vp4a SOP cells divide with a planar polarity to generate a dorsal pIIa cell and a ventral pIIb cell. The pIIb cell next divides with an apical-basal polarity to generate a basal daughter cell that differentiates as an md neuron. Inscuteable specifically accumulates at the apical pole of the dividing pIIb cell and regulates the polarity of the pIIb division. This study establishes for the first time the function of Inscuteable in the PNS, and provides the basis for studying the mechanisms controlling planar and apical-basal cell polarities in the embryonic sensory organ lineages (Orgogozo, 2001).

The external sensory organ cells are arranged in a segmental, highly stereotyped fashion, and each es organ cell can be reliably identified using anti-Cut antibodies in stage 16 embryos. In order to describe the pattern of cell divisions in the es organ lineage, the divisions of the Cut-positive es precursor cells were followed between stages 11 and 16. Analysis focused on the five mono-innervated es organs located in the ventral region, vp1-vp4a, because this region is particularly outstretched following germ-band elongation, thus facilitating the identification of each es organ cell. In stage 16 embryos, the vp1, vp2, vp3, vp4 and vp4a (vp1-vp4a) organs are arranged in a circular arc. Each organ is composed of four Cut-positive cells. The socket and shaft cells, which lie within the epithelium, are strongly labelled by anti-Cut antibodies, whereas the neuron and the sheath cell, which are subepidermal, are more weakly labeled. Elav and Pros proteins accumulate specifically in the neuron and in the shaft cell, respectively. The vp4a organ is found relatively close to the weakly Cut-positive anterior ventral md neuron called vdaa. The vdaa and vp4a cells are born from the same md-es lineage. In the center of the ventral region, the four vdaA-D and the ventral bipolar (vbp) md neurons, which are clustered together, are also weakly labeled by anti-Cut antibodies (Orgogozo, 2001).

At stage 11, five isolated cells that accumulate Cut appear at stereotyped positions in the ventral region. Based on their position, these cells correspond to the pI cells of the five vp1-vp4a organs.The analysis of the positions of the two pI daughter nuclei at telophase indicates that pI divides within the plane of the epithelium. Numb localizes asymmetrically in pI at metaphase and is inherited by one of the two daughter cells at telophase. The pI daughter cell inheriting Numb is the pIIb cell and its sister is pIIa. In the case of the vp2-vp4a organs, the two daughter nuclei are positioned along the dorsal-ventral (d-v) axis, and Numb forms a ventral crescent at metaphase and segregates to the ventral daughter cell at telophase. It is concluded that, at the vp2-vp4a position, pI divides with a stereotyped d-v planar polarity. In contrast, the division of the vp1 pI cell is randomly oriented within the plane of the epithelium with Numb segregating in only one daughter cell (Orgogozo, 2001).

pIIb divides asymmetrically with an apical-basal polarity. At stage 12, the anterior-ventral cell of each pIIa-pIIb cell pair seen at the vp2-vp4a position enters mitosis. The position of the daughter nuclei relative to the surface of the embryo at telophase indicates that pIIb divides roughly perpendicular to the plane of the epithelium. Numb, Pon, Miranda and Pros, which are first detected in the dividing pIIb cell, localize to the basal pole of the pIIb cell at metaphase and segregate to the basal daughter cell. Noticeably, at telophase, the basal daughter cell appears to be significantly smaller than its apical sister. This indicates that pIIb generates two cells of different size. However, following pIIb division, no difference in nuclear size is detected using the Pros and Cut markers. It is concluded that, at the vp2-vp4a position, the pIIb division is polarized along the apical-basal axis of the epithelium (Orgogozo, 2001).

At the vp1 position, one of the two pI daughter cells expresses Pros and divides with an apical-basal polarity to generate a basal cell that inherits both Numb and Pros. Based on these observations, it is concluded that the second cell division observed at the vp1 position is the pIIb division, as shown for the vp2-vp4a positions. The small basal pIIb daughter cell that has specifically inherited Pros has been termed X, and pIIIb is its apical sister. Soon after the pIIb division, the only es cell to accumulate Pros is the X cell. In early stage 13 embryos, in which all pIIb cells have divided, two Pros-positive cells are observed: the basal highly Pros-positive X cell and the apical weakly Pros-positive pIIIb cell (Orgogozo, 2001).

pIIa divides next to generate the socket and shaft cells. At early stage 13, a Pros-negative pIIa cell entering mitosis can be observed, while clusters of four cells are seen at the corresponding position in adjacent hemisegments. These clusters contain the highly Pros-positive X cell, the weakly Pros-positive pIIIb cell and two Pros-negative cells. It is concluded that the two Pros-negative cells are the pIIa daughter cells. These cells are localized in the superficial epidermal layer and are strongly Cut positive. These two strongly Cut-positive cells are observed in the epidermis at the vp1-vp4a positions from stage 13 onward. At stage 16, these two cells express A1-2-29, a socket and shaft cell marker. These observations indicate that the division of pIIa generates the socket and shaft cells. At late stage 13, the weakly Pros-positive pIIIb cell enters mitosis. Pros is asymmetrically localized in dividing pIIIb and is inherited by only one daughter cell at telophase. The X and pIIIb cells both accumulate Elav, a neuronal marker. The X cell can be easily identified as it accumulates a higher level of Elav. In contrast to Pros, Elav segregates equally into the two pIIIb daughter cells at telophase. Following the pIIIb division, the vp1-vp4a clusters are composed of five cells: the socket and shaft cells, the two pIIIb daughter cells and the X cell (Orgogozo, 2001).

At stage 13, each X cell occupies a stereotyped position. The vp4a X cell is located dorsally between the vp4a and vp4 clusters, and each of the vp1-vp4 X cells is found nearest the center of the circular arc formed by the vp1-vp4a cells. The accumulation of Elav in the X cell indicates that X may become a neuron. To determine the fate of the X cell, the positions of the Pros- and Elav-positive X cells were compared in adjacent hemisegments of late stage 13 embryos. This analysis suggests that the vp1-vp4 X cells migrate towards the center of the vp1-vp4a circular arc, while the vp4a X cell migrates dorsally. Consistent with a migratory behaviour, the X cells display long cytoplasmic extensions at this stage. The level of Pros accumulation in the migrating Cut- and Elav-positive X cells appears to decrease over time, and becomes undetectable when these cells cluster in the center of the circular arc at stage 14. At this stage, these Cut-positive X cells can still be identified on the basis of their stereotyped position and of their high level of Elav accumulation. These cells occupy the positions of the vdaA-D/vbp cluster and of the vdaa neuron and, from stage 14 onwards, express the E7-2-36 md marker. These data indicate that the vp4a X cell migrates dorsally and becomes the vdaa md neuron, whereas the four vp1-vp4 X cells migrate towards the center of the circular arc to form four of the five vdaA-D/vbp neurons. The fifth vdaA-D/vbp neuron corresponds to the additional Cut-, Pros-and Elav-positive cell that migrates (together with the vp1 md neuron) toward the center of the circular arc (Orgogozo, 2001).

This fifth md neuron probably originates from a Cut-positive precursor cell detected anterior to vp1. This precursor cell divides asymmetrically at late stage 12 to generate a Pros- and Elav-positive cell that migrates dorsally (Orgogozo, 2001).

The es neuron and sheath cell are born from the pIIIb cell. From stage 14 onwards, one of the two pIIIb daughter cells accumulates a higher level of Elav, and is therefore identified as the es neuron. Its sister cell accumulates a high level of Pros and is thus identified as the sheath cell. No additional division is observed in the vp1-vp4a lineages after the pIIIb division (Orgogozo, 2001).

In summary, this analysis shows that the vp1-vp4 es SOPs produce four md neurons that most likely correspond to the four vdaA-D organs. The vp4a SOP follows an identical lineage and generates the vdaa md neuron. In this novel md-es lineage, the md neuron is generated by the division of the pIIb cell. This study of the vp1-vp4a lineages rules out all three previously proposed models for the md-es lineage. Also, the pattern of cell divisions is identical in the vp1- vp4a, chordotonal and adult bristle lineages. It is therefore proposed that the lineage described here for the vp1-vp4a lineages applies to all mono-innervated es organs in the embryo (Orgogozo, 2001).

This detailed analysis of the vp1-vp4a lineages allowed for an investigation of the mechanisms regulating cell polarity in these lineages. Previous studies have indicated that insc is expressed in pI, suggesting a role for insc in regulating cell polarity in these lineages. The expression pattern of insc was examined in the vp1-vp4a lineages. Insc protein is not detectable in dividing pI, pIIa and pIIIb cells, but specifically accumulates in an apical crescent in dividing pIIb cells. The lack of insc expression in pI is further confirmed by the analysis of an insc-lacZ enhancer-trap marker. The expression of insc-lacZ is not detectable in pI and pIIa. However, it is first detected in the pIIb cell as it divides and specifically accumulates in both pIIb daughter cells. insc regulates the apical-basal polarity of the pIIb division The role of insc in regulating cell polarity was examined in the vp1-vp4a lineages. In insc mutant embryos, the vp1-vp4a pI divisions occur within the plane of the epithelium. The vp2, vp4 and vp4a pI cells divide with a d-v orientation with Numb localiz ing asymmetrically to the ventral pole of pI. Furthermore, the cell that divides next is always found at an antero-ventral position in both wild-type and insc mutant embryos, suggesting that the pIIa and pIIb cells are correctly specified. It is concluded that the loss of insc activity does not affect the polarity of the pI division. This is entirely consistent with the observation that the Insc protein is not present in the pI cell (Orgogozo, 2001).

To analyse the role of insc in the dividing pIIb cell, the asymmetric distribution of Miranda, an adaptor protein for Pros, was examined. In wild-type embryos, Miranda accumulates to the basal pole of pIIb at metaphase. In contrast, Miranda localizes asymmetrically to the basal pole in only 32% of insc mutant pIIb cells at metaphase. In the other pIIb cells, Miranda is either partly (52%) or largely (16%) delocalized around the cell cortex. This shows that insc is required for the basal localization of Miranda (Orgogozo, 2001).

The distribution of Pros, which is the earliest marker for the fate of the md and pIIIb cells in the vp1- vp4a lineages, was examined. An equal level of Pros accumulation was found in 27% of the pIIb daughter cells in insc mutant embryos. This indicates that insc is required to regulate the unequal segregation of Pros during the pIIb division and/or to establish a fate difference between the two pIIb daughter cells (Orgogozo, 2001).

The role of insc in regulating cell fate decisions in the vp1-vp4a lineages was examined. Attention was focused on the vp4a organ because this lineage generates one md neuron that migrates very little, which greatly facilitates the identification of all the cells produced by the vp4a lineage. Cut, Elav and E7-2-36 were used as cell fate markers to identify by stage 16 the vp4a socket, shaft and sheath cells, and the es neuron and the vdaa md neuron. At all vp4a positions in insc mutant embryos, the socket and shaft cells are always present. In some segments, however, the vdaa md neuron is duplicated and the vp4a es neuron and sheath cell are missing. This suggests that the pIIIb cell has been transformed into a second md neuron. In some other segments, a single Elav-positive, E7-2-36-negative cell is seen at the position of the vp4a es neuron and sheath cell. This suggests that the pIIIb cell has failed to divide. In yet other segments, the two cells at the position of the es neuron and sheath cell express variable levels of Elav, indicating that the two pIIIb daughter cells are not correctly specified. It is concluded that insc regulates the fate of the pIIb daughter cells (Orgogozo, 2001).

This analysis was extended to the vp1-vp4 lineages. Socket and shaft cells are always detected, while the neuron and sheath cells form properly in only 66% (n=124) of the vp1-vp4 organs. In 22% of the cases, the two cells at the position of the sheath cell and es neuron express a similar level of either Elav, or Pros, or both Pros and Elav. In 6% of the es organs, only one Elav-expressing cell is detected. Finally, in the remaining 6%, the es neuron and sheath cell are both missing. This defect is always associated with the presence of an additional md neuron at the vdaA-D/vbp position (7 cases out of 7. This indicates that the pIIIb cell has been transformed into an md neuron (Orgogozo, 2001).

In conclusion, the data show that the loss of insc activity results in cell polarity defects in the pIIb cell, as revealed by the mislocalization of Miranda at metaphase. This phenotype correlates with the abnormal accumulation of Pros into the apical pIIb daughter cell and with the mis-specification of the pIIIb cell (Orgogozo, 2001).

This study provides the first detailed description of each asymmetric cell division in an md-es lineage. The division of the vp2-vp4a pI cell is planar and takes place with a d-v polarity, revealing for the first time the existence of a planar polarity orienting asymmetric cell divisions in the embryo. Similarly, in the pupa, the pI cell divides in the plane of the epithelium and along the a-p axis. The polarity of this division is controlled by the Fz signaling pathway. In both pupae and embryos, the pIIb cell divides with an apical-basal polarity, with Numb, Pros and Miranda segregating to the basal cell. Moreover, Insc forms an apical crescent in the pIIb cell in the pupal lineage. This suggests that Insc regulates also the apical-basal polarity of the pIIb cell in the adult bristle lineage. It is clear, however, that a detailed analysis of the function of insc in regulating cell polarity in the adult PNS would have been much more difficult and time-consuming because insc mutations are embryonic lethal. In conclusion, this study clearly illustrates that the regulation of both planar and apical-basal polarities can now be studied in the embryonic PNS. This detailed analysis therefore provides the basis for future studies addressing the function of various candidate genes known to affect the development of the embryonic PNS (Orgogozo, 2001).

Drosophila atypical protein kinase c associates with Bazooka and controls polarity of epithelia and neuroblasts

Atypical protein kinase C (aPKC) from Drosophila shows very high sequence similarity to PKClambda and PKCzeta from vertebrates and PKC-3 from C. elegans. Drosophila aPKC and Baz coimmunoprecipitate and directly bind to each other in a yeast two-hybrid assay. In embryos, both proteins colocalize in the apical cortex of almost all epithelial tissues and in neuroblasts. Apical localization of DaPKC in epithelia and neuroblasts is abolished in baz mutants, and vice versa: Baz is delocalized in DaPKC mutants. The phenotype of aPKC loss-of-function mutants resembles that of baz mutants, consistent with a close functional interdependence of both proteins. Together, these data provide in vivo evidence for an essential role of an atypical protein kinase C isoform in establishment and maintenance of epithelial and neuronal polarity (Wodarz, 2000).

It has been shown before that Baz is required for apical localization of Insc in neuroblasts and that Insc is required for stabilization of Baz in neuroblasts after delamination. A test was performed to see whether Baz and Insc are also required for localization of aPKC in neuroblasts. aPKC localization is indistinguishable from wild type in neuroblasts of inscP49/CyO heterozygous embryos, but is neither cortical nor apical in neuroblasts of inscP49 homozygous mutant embryos. In embryos lacking maternal Baz but carrying a paternal wild-type allele of baz (partial paternal rescue), asymmetric cortical localization of aPKC is detected in most neuroblasts at metaphase. However, aPKC crescents and metaphase plates are often misoriented with respect to the surface of the embryo, a phenotype that has also been observed at low penetrance in embryos lacking only zygotic expression of Baz. In embryos lacking both maternal and zygotic expression of Baz (baz null), aPKC is completely delocalized in neuroblasts and epithelial tissues. These results indicate that Baz is absolutely required for apical localization of aPKC in neuroblasts and epithelial tissues, while Insc is required for localization of aPKC only in neuroblasts. Baz levels are strongly reduced in neuroblasts of insc mutant embryos, most likely because Insc is required for stabilization of Baz. Thus, the effect of Insc on DaPKC localization is probably indirect and can be explained by the loss of Baz in insc mutant neuroblasts (Wodarz, 2000).

The function of Bazooka in neuroblasts, at least in part, is to localize Inscuteable to the apical cortex. Bazooka is strictly required for Inscuteable localization, but Inscuteable is dispensable for Bazooka localization even though Bazooka crescents become weaker in Inscuteable mutants. Therefore Par-6 localization was analyzed in inscuteable mutants. Whereas 88% of metaphase control neuroblasts showed a strong apical crescent, normal localization of Par-6 was only observed in 14% (n = 42) of inscuteableP72 mutant neuroblasts. In 52% of these neuroblasts, Par-6 was localized into an apical crescent that was weaker and extended further to the lateral cortex than in control embryos and in 33% of the metaphase neuroblasts, Par-6 was not asymmetrically localized. Thus, although Bazooka is strictly required for Par-6 localization, absence of Inscuteable only causes a partially penetrant defect in Par-6 localization (Petronczki, 2001).

Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions

Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled has remained unclear. Mitotic spindle geometry and unequal daughter cell size were shown to be controlled by two parallel pathways (Bazooka/DaPKC and Pins/Gαi) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequally sized neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/Galphai localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle (Cai, 2003).

Thus members of the NB apical protein complex control the generation of daughter cells of unequal size. There are two redundant pathways: (1) Baz/DaPKC/ (and presumably DmPar6) as well as (2) Pins/Gαi, either of which, when asymmetrically localized to the NB cortex, can lead to the formation of an asymmetric mitotic spindle through the preferential elongation of the proximal spindle arm and the displacement of the spindle toward the distal cell cortex, resulting in the production of unequal-sized daughter cells. In addition, in NBs, Insc is required for the function of the Baz/DaPKC/(DmPar6) pathway. When both pathways are inactivated/attenuated, spindle asymmetry and displacement fail to occur and equal-sized daughter cells are produced at high frequency. In the PNS progenitor, pI, where Baz/DaPKC are localized to the posterior cortex and Pins/Gαi are localized to the anterior cortex, the mitotic spindle is symmetric. Consistent with this hypothesis that both pathways can act to cause the preferential elongation of the proximal spindle arm relative to the distal spindle arm, removing posterior baz function without abolishing the localization and function of the anterior components results in the production of an asymmetric spindle with an anterior bias; removing anterior pins function without affecting the function of the posterior components results in a posteriorly biased asymmetric spindle; if components of both pathways are localized to the anterior cortex through the ectopic expression of Insc, an anteriorly biased asymmetric spindle results. These findings suggest that DaPKC and hetrotrimeric G protein signaling work in conjunction in the NB to produce an asymmetric spindle and in opposition in pI to produce a symmetric spindle (Cai, 2003).

Several lines of evidence suggest that localized signaling is essential to generate an asymmetric spindle and daughter cells of unequal size. (1) When both signaling pathways are abolished/attenuated (e.g., in insc/pins double mutant) or when signaling is uniform, which is assumed to be the case when Baz/DaPKC/Pins/Gαi are all uniformly localized throughout the cell cortex (e.g., in the case of Gαi overexpression in wt NBs), equal-sized daughters are generated. (2) When pins function is removed and DaPKC/Baz is asymmetrically localized (e.g., in pins mutant NB) or when Pins/Gαi are uniformly cortical but DaPKC/Baz are asymmetrically localized (e.g., in 69% [n = 51] of wt NBs overexpressing C-Pins), the site of the DaPKC/Baz localization coincides with the position where the larger daughter forms. (3) When Pins/Gαi is asymmetrically localized but baz/DaPKC function has been compromised (e.g., in insc mutant) or when Pins/Gαi is asymmetrically localized but Baz/DaPKC is uniformly cortical (in the case of NBs with basal Pins-C-Pon crescents), the site of localization coincides with the larger daughter and the extended spindle arm. These observations indicate that just one localized signal source, mediated presumably by either heterotrimeric G protein or DaPKC, is sufficient to cause proximal spindle arm elongation and the generation of unequal-sized daughters (Cai, 2003).

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).

In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).

Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).

The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).

It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).

In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).

Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).

Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).

Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).

In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).

The PDZ protein Canoe regulates the asymmetric division of Drosophila neuroblasts and muscle progenitors; Cno functions downstream of Insc and Pins-Gαi and upstream of Mud during asymmetric NB division

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).

Neuralized mediates asymmetric division of neural precursors by two distinct and sequential events: promoting asymmetric localization of Numb and enhancing activation of Notch-signaling

In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1 --> RP2/sib lineage, it was found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. It was further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. These results tie Numb and Notch-signaling through a single player, Neur, thus giving a more complete picture of the events surrounding asymmetric division of precursor cells. It was also shown that Neur and Numb are interdependent for their asymmetric-localizations (Bhat, 2011).

The results in this paper tie the localization of Numb and the signaling-processing of Notch through a single upstream player, Neur. This gives a more complete picture of the events that surround asymmetric division of neural precursor cells. The E3 Ubiquitin ligase protein Neur regulates asymmetric division of Numb and Notch-sensitive neural precursor cells in the CNS via two distinct, sequential mechanisms: first, by promoting the asymmetric localization of Insc and Numb in GMCs and second, via non-cell autonomously potentiating or enhancing the activation of Notch signaling in the Numb-negative daughter cell. While Neur is known to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch, an earlier role for it in asymmetric division via Insc and Numb localization has not been discovered. In fact, these results show that this is the primary role for Neur in generating asymmetry in the CNS. That Neur plays a secondary role or a role which is not absolute in the potentiation or enhancement of Notch signaling is indicated by the finding that in neur; numb double mutants, both sibling cells often but not always adopt a mixed fate as opposed to an RP2 fate seen in Notch; numb double mutants. If the role of Neur in Notch potentiation in this lineage is an absolute one, the same result would have been seen in neur; numb as N; numb mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch, however, the penetrance of this effect is weak (Bhat, 2011).

Previous studies had shown that the RP2-sib binary fate decision is regulated by unequal segregation of the Notch regulator Numb. The simplest interpretation of the current results would suggest that Neur is required for sib fate specification via Notch. However, the results indicate that the requirement of Neur for sib-specification to a daughter cell of a GMC-1 via regulating Notch is preceded by its requirement in GMC-1 for Numb localization, where Neur itself is expressed and becomes asymmetrically localized to the basal Numb-domain. Thus, the loss of sib identity in neur mutants appears to be mainly due to the non-asymmetric localization of Insc and Numb in GMC-1. Moreover, the levels of Pdm1 are responsive to both loss of function neur (Pdm1 level is up-regulated) and gain of function neur (the Pdm1 level is down-regulated), which are more likely a consequence of Neur function within GMC-1. This regulation of Insc and Numb localization appears to be via regulation of Pdm1 levels inside GMC-1, whereas regulating Notch processing is later and the source of Neur is from outside. By regulating asymmetric localization of Numb, Neur ensures that one of the two daughters is free of Numb, thus, later on the activation of Notch-signaling in that cell can occur. The source of Neur for this Notch processing appears to be from outside of the lineage since a division-arrested GMC-1 in numb; cyc A double mutant can still adopt a sib fate. Thus, the two roles of Neur in this lineage are distinct and separable. But then is it possible Notch has a role in the asymmetric localization of Numb and this activity of Notch is regulated by Neur? It certainly is possible but then one would have to disregard the presence of asymmetrically localized Neur in a GMC-1 as anything but of no consequence to the asymmetric division of GMC-1. It should also be pointed out that the identity of GMC-1 per se in neur is not altered, if it did, two neurons of some other identities would have been seen, not RP2s (or sibs) (Bhat, 2011).

A previous study in the sensory system of the PNS indicated that Neur protein localizes asymmetrically in the pI cell of SOP. It then segregates to pIIb, where it is thought to enhance the endocytosis of Dl to promote N activation in the pIIa cell. This represents a trans-differentiation mechanism to specify different cell fates. The results confirm the findings in SOP lineage but at the same time extends the data on SOP lineage in that this trans-determination process is a potentiation step to mediate a more efficient Notch-signaling-processing, but it is not necessarily a deterministic one. What is new and different from the SOP lineage is that Neur controls not only the asymmetric localization of Numb during mitosis, but also controls the localization of Insc, an apical cue that controls spindle orientation and participates in Numb basal localization. In neur mutant cells, Insc is no longer asymmetric indicating that Neur is somehow needed to localize Insc. The fact that Neur is somehow needed for Insc localization is also consistent with the finding that genetically insc is epistatic to neur, therefore that it is downstream of neur (Bhat, 2011).

Finally, while insc is epistatic to neur in the RP2 lineage defect in insc; neur double mutants, as for the neurogenic phenotype, neur is epistatic. This is not surprising since epistasis relationships are lineage/cell-type/tissue specific, depending upon whether or not the two genes in question are expressed in the same lineage and if the two single mutants give the same (or opposing) phenotype. Insc has no role during the neural versus ectodermal fate decisions and loss of function for insc does not cause a neurogenic phenotype, hence, the neurogenic phenotype of neur mutants is not expected to be present (epistatic) in the double mutant (Bhat, 2011).

It is clear from the results that Neur regulates asymmetric division of GMCs in the CNS. This was examined in at least two different GMCs, the GMC of the RP2/sib lineage (GMC-1 or GMC4-2a of NB4-2) and the GMC of the aCC/pCC lineage (GMC-1 or GMC1-1a of NB1-1). In neur, these GMCs symmetrically divide to generate two of the same cells, RP2 neurons in the case of GMC-1 and aCC neurons in the case of GMC1-1a. It is thought that many more GMC lineages are affected by loss of function for neur. Being a neurogenic protein, Neur is also involved in selecting neural versus ectodermal fates for the neuroectodermal cells. Due to its neurogenic property, the mutant will generate extra copies of many of the NBs in the nerve cord, which in turn, will generate more of the GMCs and neurons. Several lines of evidence indicate that symmetric division of a GMC indeed occurs at a high frequency in the CNS in neur mutants. For example, GMC-1 normally generates an RP2 and a sib, RP2 is larger than the sib and the two have distinct gene expression profiles and patterns. This is also the case for aCC/pCC pairs—they also have distinct gene expression profiles. These specific criteria were used to separate the ones that are generated by the symmetric division from those generated due to a neurogenic effect of neur mutation (Bhat, 2011).

Several additional pieces of evidence indicate a role for Neur in generating asymmetry. These include the asymmetric localization of Neur in GMCs, non-asymmetric localization of Numb in GMC-1 in neur mutants, non-asymmetric localization of Neur in numb mutants, genetic interaction results and effect on downstream players such as Pdm and Numb. All these results point to a specific role for Neur in regulating asymmetric mitosis of precursor cells (Bhat, 2011).

The results show that Neur itself is asymmetrically localized in GMC-1 to the Numb-domain and opposite to that of the Insc-domain (Neur is also localized to the basal end of several NBs, the significance of which is not known). In neur mutants, both Insc and Numb are not localized but found uniformly distributed along the cell cortex. This suggests that Neur is upstream of Insc and Numb localization but not their expression per se. The levels of Numb or Insc are also not affected in neur mutants indicating that Neur does not participate in Numb degradation (via ubiquitination, or otherwise). There is no evidence that Neur has a direct role in the localization of Numb. Do these results therefore mean Neur basically regulates the identity or the fate (i.e. gene expression program) of the GMC-1 prior to its division and therefore that Neur has only one function, which is potentiating Notch signaling? The GMC-1 was examined in neur mutants with several different GMC-1 markers (Eve, Pdm1, Zfh-1, Spectrin, etc.) and with the exception of a higher than normal Pdm1 in a late GMC-1, none of these markers were affected. A higher than normal levels of Pdm1 does not change the identity of a GMC-1. Indeed, several studies have shown that high levels of Pdm1 or its sister protein Pdm2 will induce a GMC-1 to undergo symmetric division to produce two GMC-1s and then two RP2s and two sibs. In order for a GMC-1 to change its identity, many of its genes should be turned off and a new set of genes has to be initiated. Such a drastic change does not occur in GMC-1 of neur mutants. Similarly, an identity change should result in this GMC-1 in neur mutants to produce different sets of neurons, which it does not. Instead, it produces two RP2s. Given these results and that Neur is necessary for the normal localization of Numb, whether this is directly mediated or indirectly mediated, the conclusion that Neur regulates asymmetric division at two different levels during the lineage development is based on firm grounds (Bhat, 2011).

The main question is how might Neur regulate Insc and Numb localization. A clue to this question comes from previous studies. It was shown that over-expression of Pdm POU transcription factors (Pdm1 or Pdm2) in GMC-1 causes non-localization of Insc and Numb and their segregation to both daughter cells of GMC-1; these cells then adopt an RP2 fate, with Numb blocking the N-signaling from specifying a sib fate. Pdm1 was up-regulated in GMC-1 in neur mutants and down-regulated with over-expression of Neur. This shows that the localization of Insc and Numb is altered in neur mutants indirectly via the up-regulation of Pdm protein. At the moment, it is not clear how an up-regulation of Pdm alters Insc or Numb localization. A most likely possibility is that Pdm proteins, being transcription factors, their over-expression may cause changes in the expression of genes that are needed for the proper localization of Insc and Numb but without altering the cell-identity itself (since this cell still produces RP2 neurons and not some other neurons). These conclusions are all consistent with the overall expression pattern and mutant effects of pdm genes: Pdm proteins are down-regulated in GMC-1 prior to its division, loss of function for Pdm causes loss of GMC-1 identity (Bhat, 2011).

The gain of function for these pdm genes indicates that the GMC-1 division is quite sensitive to varying levels and timings of expression of these POU proteins. For example, a high level of pdm gene expression in a GMC-1 from pdm transgenes causes a symmetric division of GMC-1 into two GMC-1s and then each of these GMC-1s generates an RP2 and a sib. In contrast, a symmetric division of GMC-1 into two RP2s can also be observed in these embryos. In this case, the cells from the GMC-1 express Zfh1; a GMC-1 does not continually express (a late GMC-1 about to divide does express Zfh1 at a very low level), a sib transiently expresses Zhf-1, and an RP2 stably expresses Zfh-1. Moreover, both these cells inherit Insc and Numb. No more cells are produced from these two cells, and each of these cells generates a projection as that of an RP2. When these genes are over-expressed for a prolonged period of time, a GMC-1 divides multiple times producing a GMC-1 and a differentiated progeny: First two unequal sized cells are observed. Only one of the two (the smaller cell) expresses markers such as Zfh1. Later on, three cells, and then five cells, etc., are sequentially seen, all in a tight cluster; from these clusters, as many as 5 RP2s are formed. Indeed, with this prolonged over-expression of pdm genes for 90 min from a heat shock promoter causes hemisegments with all the above types of divisions depending upon the time of over-expression. In contrast, it is not clear what the sensitivity range of GMC-1 is to varying concentrations in terms of the kind of division pattern generated. One clue to this comes from an earlier study, that GMC-1 in embryos carrying a duplication chromosome for the chromosomal region containing the two POU genes undergo a single self-renewing asymmetric division of GMC-1. This suggests that when the copy numbers for these genes are doubled, this presumably results in producing twice the amount of these proteins (from their own promoters), and causes a single self-renewing division. Having said that, it was also found that in neur mutants a GMC-1 rarely divides symmetrically into two GMC-1s and then each produces a sib and an RP2, or a GMC-1 dividing more than once with self-renewing asymmetric division as in pdm-GOF situations (Bhat, 2011).

Based on these results with gain of function for pdm genes, a loss of function for pdm genes should suppress the neur defects. However, this experiment is not possible to do since loss of function for the pdm genes causes loss of GMC-1 identity (GMC-1 becomes some other GMC) and therefore GMC-1 is undetectable with GMC-1 markers (Bhat, 2011).

While the exact mechanism as to how the level of Pdm1 is up-regulated in GMC-1 of neur mutants or down-regulated when Neur is over-expressed in GMC-1, is not known, one possibility is that Neur is involved in the degradation of Pdm1 in GMC-1. This scenario is most likely since Neur has the RING domain, one of the signature domains for E3 Ubiquitin-ligase proteins involved in protein degradation. Neur has also been shown to ubiquitinate proteins in vitro. One indication that Neur might be involved in the degradation of Pdm1 is the result that while ectopic or over-expression of full length neur from a transgene down-regulated Pdm1 and resulted in the same phenotype as loss of function for pdm genes, a similar ectopic or over-expression of a neur transgene missing the RING domain (Hs-neurΔRF) did not result in a down-regulation of Pdm1 or resulted in any phenotypes. Pdm1 appears to be specifically affected in GMC-1 of the RP2/sib lineage and not in other cells where Pdm proteins are present. Even if the up-regulation of Pdm proteins in neur mutants is via an indirect mechanism, say via factor X or Y, the results define a major role for Neur in regulating asymmetric division prior to the Notch-potentiation role of Neur: regulating Numb localization via down-regulating (directly or indirectly) Pdm proteins (Bhat, 2011).

Results from the analysis of neur, neur; numb double mutant embryos and neur gain-of-function embryos show that Neur functions to increase the efficiency of Notch-signaling but not essential for it. None of the previous studies have made this important distinction. Previous results have indicated that Neur activates Notch-signaling via endocytosis of Delta and the Delta-bound extracellular domain of Notch. However, in neur null mutants (embryos homozygous for a deficiency that removes neur completely), sib specification still occurs in ~ 10% of the hemisegments. While this may arguably be due to a partial redundancy for neur, there is another line of evidence that suggests a role for Neur in enhancing the efficiency of Notch signaling. That is, while in Notch; numb double mutants both daughter cells of a GMC-1 adopt an RP2 fate (note that for the specification of an RP2 fate Numb is needed only when there is an intact Notch-signaling), in neur; numb double mutants the daughters often adopt a mixed identity. This result indicates that Notch is still able to specify some features of a sib identity (i.e., reduced levels of Eve expression) in the absence of Neur activity. If Neur is absolutely needed for Notch signaling, the double mutant results would have been exactly the same as Notch; numb double mutants where both daughters adopt an unambiguous RP2 fate (Bhat, 2011).

In contrast, the results from Neur over-expression experiments indicate that when present at high levels Neur is able to overcome the Numb block and induce both the progeny of GMC-1 to adopt a sib fate. This phenotype is strikingly similar to the phenotype observed with the over-expression of the intracellular domain of Notch or the phenotype in numb mutants. These results suggest that over-expression of Neur leads to processing of Notch in the cell that has Numb. It is also pointed out that the source of Neur for the trans-effect on Notch-signaling need not be only from the “RP2” cell, but may also be from the neighboring cells. This is indicated by the previous result that while the GMC-1 in embryos mutant for cyclin A adopts an RP2 fate, the same GMC-1 in cyclin A; numb double mutants adopts a sib fate (Bhat, 2011).

These results show that the asymmetric basal localization of Numb in neur mutants and Neur in numb mutants is affected. This shows the interdependence of localization of these two proteins. Whether there is any initial localization of Numb or Neur in the two mutants was examined to determine if the localization of the one protein falls apart in the absence of localization of the other. However, no such initial localization was observed for either of the two proteins. It is possible that both Neur and Numb control the same pathway(s) that directly or indirectly mediates localization of the other. Perhaps Neur and Numb interact physically with each other in the cytoplasm prior to any localization and it is this Neur-Numb complex that gets localized to the basal pole; in the absence of either of the two proteins, no such complex is formed, and no localization occurs. This model has not been tested due to lack of appropriate reagents. In contrast, loss of Numb-localization in neur could be due to loss of Insc localization; loss of Neur localization in numb mutants could be more direct where Neur is downstream of Numb and Numb mediates directly or indirectly the localization of Neur. The function of Neur in GMC-1, however, appears to be required for the down-regulation of Pdm and allow localization of such proteins as Insc. Thus, Neur is both upstream and downstream of Numb in GMC-1. Another important distinction between Neur and Numb is that while non-asymmetric localization of Numb in GMC-1 will lead to both daughters of GMC-1 inheriting Numb and adopting RP2 fates, a non-asymmetric localization of Neur and inheritance of Neur by both daughters will not make them adopt an RP2 fate, but a sib fate (Bhat, 2011).

In numb mutants, the localization of Neur is affected in such a way that both daughters inherit Neur. Does this have a consequence? The results argue that unlike Numb there is no consequence to the non-asymmetric localization and segregation of Neur to both daughters. For instance, in wild type the sib cell does not inherit Neur, thus, the potentiation of Notch in this cell by Neur occurs in a cell non-autonomous mechanism (removing the extracellular domain of Notch bound by Delta) and there is no role for Neur in the sib itself. Thus, in numb mutants although both daughters inherit Neur, they still adopt a sib fate (Bhat, 2011).

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. This assumption was tested in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. These findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions (Bergstralh, 2015).

Previous work demonstrated that metaphase spindles in the cuboidal follicle epithelium are oriented between 0° and 35° relative to the plane of the layer, roughly perpendicular to the apical-basal axis of the cell. Metaphase spindle orientation in this tissue relies on the canonical factors Mud and Pins, and mutants in either gene randomize spindle orientation. Unexpectedly, this study found that the organization of the epithelium is maintained in mud and pins mutants. This is not due to post-metaphase correction of division angles, as vertically oriented spindles persist into telophase in mud mutants (Bergstralh, 2015).

To disrupt spindle orientation more severely, Inscuteable was ectopically expressed in follicle cells. In neuroblasts, this protein recruits Pins and Mud to the apical cortex of neuroblasts so that mitotic spindles are oriented along the apical-basal axis. It has a similar effect on spindle orientation when ectopically expressed in follicle cells. Rather than randomizing spindle orientation as in pins and mud mutants, Inscuteable orients almost all spindles perpendicular to the epithelial plane. Divisions are thus horizontal and produce an apical and a basal daughter. Like spindle randomization, this has no effect on tissue organization. In the neuroblast, spindle orientation controls cell fate by ensuring the asymmetric segregation of fate determinants to one daughter cell. Inscuteable expression in the follicle epithelium does not confer neural cell fate, because it does not cause expression of the transcription factor Deadpan. It was also observed that female flies expressing UAS-Inscuteable under the control of the strong follicle cell driver Traffic Jam-Gal4 are fertile, indicating that reorienting most divisions in the follicular epithelium does not disrupt egg chamber development (Bergstralh, 2015).

In the imaginal wing disc, misoriented cell division is associated with basal cell extrusion and apoptosis. The possibility was therefore considered that the apically misplaced cells produced by horizontal divisions in the follicle cell layer are also eliminated by programmed cell death. However, misplaced cells show neither cleaved caspase-3 immunoreactivity nor pyknosis. Furthermore, expression of the apoptotic inhibitor p35 has no effect on follicular epithelia expressing Inscuteable or containing pinsp62 mutant clones. Live imaging reveals that rather than dying, misplaced daughter cells simply reintegrate back into the epithelial monolayer (Bergstralh, 2015).

The findings prompted a closer examination of mitosis in wild-type follicle cells. These cells divide only during the early stages of egg chamber maturation, switching from mitosis to endocycling at stage 6. Live imaging reveals that the monolayer has an uneven, 'bubbly' appearance in early stages. This is because mitotic cells round up, exhibiting a concomitant increase in cortical phospho-myosin, and often move apically, pulling away from the basement membrane. Daughter cells are frequently born detached from the basement membrane. These cells then reinsert into the monolayer. These results are consistent with the earlier observation that metaphase spindle angles, which determine the angle of division, are not strictly parallel to the plane of the tissue. They also show that in the follicle epithelium reintegration is not only a backup mechanism, but occurs as a normal feature of division. It is speculated that apical movement and angled cell divisions may help to relieve local tension caused by cell expansion and division, which crowds the tightly packed neighbouring cells (Bergstralh, 2015).

Reintegration of newly born epithelial cells has previously been observed in two specific developmental contexts. In mammalian ureteric buds, cells move apically into the lumen to divide and one daughter cell then re-inserts into the epithelium at a distant site. This may contribute to branching. Second, neuroepithelial cells of the zebrafish neural keel normally orient their spindles vertically, and the apical daughter then intercalates into the opposite side of the neural tube in a process that depends on planar cell polarity signalling. In both of these cases, reintegration occurs at a distant site. In contrast, reintegration in the follicle epithelium is always local, and therefore acts to maintain, rather than to alter, epithelial architecture (Bergstralh, 2015).

As local reintegration can be detected only by live imaging, it is possible that it is a general feature of epithelial tissues that has been largely overlooked. To test this possibility, two other types of Drosophila epithelium were examined: the columnar epithelium of the early embryonic ectoderm and the neuroepithelium of the developing optic lobe. It has previously been shown that ectopic expression of Inscuteable reorients spindles in these tissues without affecting tissue integrity. The neuroepithelium is pseudostratified and undergoes interkinetic nuclear migration before division. Expression of Inscuteable in this tissue efficiently reorients divisions, producing one daughter cell that protrudes apically from the layer, as in the follicular epithelium. Live imaging reveals that these apical cells then reintegrate into the epithelium over the next 30min. Inscuteable expression also causes misoriented divisions in the columnar cells of the early embryonic ectoderm, resulting in misplaced daughter cells that lie below, rather than above, the monolayer. Three-dimensional tracking over time shows that these basally misplaced daughter cells can move apically to reintegrate (Bergstralh, 2015).

Reintegration seems to be an active process, because cells undergo a series of shape changes as they reinsert into the monolayer. One possibility is that this is a cell migration process driven by actomyosin constriction at the rear (the apical surface), which squeezes the basal side of the cell back into the epithelium. However, no obvious enrichment of the Myosin Regulatory Light Chain (Spaghetti Squash) or Heavy Chain (Zipper) was observed at the apical surface of reintegrating cells. Myosin is most obviously enriched at the adherens junctions. This correlates with a planar constriction of the reintegrating cell at this level, which would be predicted to hinder rather than help reintegration. Furthermore, reintegrating cells often show a large, transient expansion of their apical free surface, which suggests that the apical membrane is pushed out to accommodate the compression of the basal side of the cell as it squeezes between its neighbours. This behaviour is incompatible with a reintegration mechanism initiated by a contractile force at the rear of the cell, although myosin may play a role in retracting the apical projection during the final stages of reintegration (Bergstralh, 2015).

These observations raise the question of how cells born above or below the monolayer are induced to move in the correct direction to reintegrate. The apical polarity factors aPKC, Bazooka and Crumbs have been observed to disappear from the apical cortex of the follicle cells during mitosis, so it is unlikely that they act as polarity cues for reintegration. Similarly, misplaced cells have no obvious attachment to the basement membrane, and there is no evidence that they form basal stalks, which in any case would be inherited only by the basal daughter of a horizontal division. In Drosophila, cadherin-based adherens junctions localize to the apical side of the lateral membrane, in contrast to mammals where they lie more basally. Cells born apical to the epithelium remain attached to the monolayer by these apical adherens junctions, as revealed by Armadillo (Drosophila β-catenin) staining. In wild-type tissues, both daughter cells inherit part of the apical belt of adherens junctions from the mother cell, whereas the more apical daughter inherits all of the adherens junctions following a horizontal division. Live imaging reveals that the basal cell generates a new junction with its sister and a transient junction that extends along its lateral cortex. Thus, adherens junctions link both apical and basal daughters to cells within the epithelium (Bergstralh, 2015).

To test for a role for adherens junctions in reintegration, the strong hypomorphic allele armadillo3 (previously called armXP33) was used, that encodes a truncated protein and causes intermittent gaps in the epithelium. No misplaced cells or multilayering was observed in armadillo3 clones expressing Inscuteable and no cell death was observed. Reintegration of an armadillo3 mutant cell expressing Inscuteable was also observed directly. These results argue against a major role for adherens junctions in this process (Bergstralh, 2015).

In addition to their apicolateral adherens junctions, follicle cells adhere laterally through functionally redundant homophilic adhesion molecules, such as the IgCAM Neuroglian167 (Nrg167) and the N-Cam-like protein Fasciclin II (Fas2). Both Nrg167 and Fas2 are highly expressed along the length of follicle cell lateral membranes during the first half of oogenesis, when follicle cells are dividing, but their expression is downregulated in post-mitotic stages. This pattern of expression suggests that these proteins are important during division. They are also expressed along lateral membranes in the embryonic epithelium and neuroepithelium. Furthermore, Nrg is localized along the cortex throughout the course of reintegration. In agreement with earlier work, short hairpin RNA (shRNA)-mediated depletion of Nrg167 causes the appearance of occasional follicle cells lying apical to the epithelial monolayer, which is otherwise unperturbed. Apical cells are also observed in mutant clones of Fas2G0336, a P-element allele that behaves as a protein null. Similar phenotypes have been previously attributed to the loss of apical-basal polarity, but the Nrg shRNA and Fas2 mutant cells within the monolayer seem to have normal polarity, as shown by the wild-type distributions of aPKC, Par-6, Bazooka, DE-cadherin, Arm and Dlg. It was therefore reasoned that the apically extruded cells represent failed reintegrations. To test this possibility, the number of cells born above the layer was increased by overexpressing Inscuteable in Nrg knockdown or Fas2 mutant cells. Inscuteable expression increased the mean number of apically positioned cells more than twofold when combined with Nrg shRNA and more than tenfold in Fas2G0336 mutant egg chambers. Live imaging confirmed that cells born apically remain above the epithelium and never reintegrate. Cumulatively, these results show that normal levels of lateral adhesion are required for reintegration (Bergstralh, 2015).

On the basis of these results, it is proposed that tissue surface tension drives reintegration by acting to maximize cell-cell adhesion. As this process is driven by lateral adhesion, it should be able to pull cells back into the monolayer from either side of the epithelium, and this may explain how misplaced cells in the embryonic ectoderm reintegrate from the basal side, whereas follicle and optic lobe cells reintegrate from the apical side. Although these three epithelia reintegrate misplaced cells, this does not seem to be the case in the wing disc epithelium. This difference may arise because lateral adhesion molecules such as Neuroglian are concentrated in apical septate junctions in the wing disc, rather than along the entire lateral membrane as seen in most other mitotic epithelia. These lateral adhesion proteins will therefore segregate into only the apical daughter of a horizontal division in the wing disc, thereby preventing the basal daughter from integrating by maximizing lateral adhesion (Bergstralh, 2015).

Contrary to expectation, spindle misorientation does not disrupt the organization of typical cuboidal, columnar or pseudostratified epithelia in Drosophila. Instead, misplaced cells reintegrate, providing a robust mechanism to protect epithelial monolayers from the consequences of misoriented divisions. Indeed, this mechanism may act more generally to safeguard epithelia against any processes that might disrupt their organization. It will therefore be interesting to investigate whether reintegration also occurs in vertebrate epithelia, where the main lateral adhesion molecule is E-cadherin, and whether a role in reintegration contributes to E-cadherin’s function as a tumour suppressor (Bergstralh, 2015).

inscuteable: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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