Regulator of cyclin A1
The genetic data indicate that Rca1 inhibits APC-Cdh1Fzr function. To test whether Rca1 is in a physical complex with Cdh1Fzr, coimmunoprecipitation experiments were performed. Extracts from 6-8 hr old embryos were used, in which HA-Rca1 was overexpressed using the arm-Gal4 driver line. A single band, corresponding to the HA-Rca1 protein, can be detected on Western blots of this extract using HA antibodies (Grosskortenhaus, 2002).
Coprecipitation of CycA, Cdc20Fzy, Cdh1Fzr, and Cdc27, a subunit of the APC, was examined. CycA coprecipitating with HA-Rca1 could not be detected, indicating that they are not elements of a stable complex under these experimental conditions. No interaction with Cdc20Fzy was seen. In contrast, coprecipitations were seen with HA-Rca1 and Cdh1Fzr and Cdc27. Both proteins are present only in the HA precipitations using extracts from HA-Rca1-expressing embryos but not from wt embryos, although Cdh1Fzr and Cdc27 are present in this wt extract (Grosskortenhaus, 2002).
In vitro-translated Cdh1Fzr and Rca1 also interact. This indicates that no other Drosophila protein is required for the complex formation between Rca1 and Cdh1Fzr. However, other proteins present in the reticulocyte lysate may be required for this interaction. In summary, these data demonstrate that Rca1 is in a physical complex with Cdh1Fzr and an APC component, Cdc27 (Grosskortenhaus, 2002).
In the developing eye of Drosophila, cells become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex (rux) mutants, cells enter S phase precociously because of ectopic activation of a Cyclin A/Cdk complex in early G1. This leads to defects in cell fate and pattern formation, and results in abnormalities in the morphology of the adult eye. A screen for dominant suppressors of the rux eye phenotype led to the identification of mutations in cyclin A, string (cdc25), and new cell cycle genes. One of these genes, Regulator of cyclin A (Rca1), encodes a novel protein required for both mitotic and meiotic cell cycle progression. Rca1 mutants arrest in G2 of embryonic cell cycle 16 with a phenotype very similar to cyclin A loss of function mutants. Expression of Rca1 transgenes in G1 or in postmitotic neurons promotes Cyclin A protein accumulation and drives cells into S phase in a Cyclin A-dependent fashion (Dong, 1997).
Embryos mutant for Rca1 undergo normal embryonic development and cell proliferation until the extended germband stage (Dong, 1997). At this time, epidermal cells in wild-type (wt) embryos undergo the 16th mitotic cycle and then enter a G1 phase for the first time of embryogenesis. Rca1 mutants fail to perform this division, resulting in embryos with reduced cell number compared with those of wt (Dong, 1997 and Grosskortenhaus, 2002). No mitotic spindles are seen at the time when cells would normally undergo mitosis 16 (Dong, 1997) and the DNA is decondensed. Thus, Rca1 mutants arrest during interphase of cycle 16. These features are identical to those of CycA mutants, but it was reported that no differences in CycA levels were detectable in Rca1 mutant embryos (Dong, 1997). In contrast, it has been found that CycA is markedly reduced shortly before embryos would normally enter mitosis 16 (Grosskortenhaus, 2002). In wt, this mitosis occurs in a stereotypic way that is reflected in the degradation pattern of CycA. This pattern is segmental, and cells expressing high levels of CycA (G2 cells of cycle 16) can be distinguished from G1 cells with low levels of CycA. In Rca1 embryos, only uniformly low levels of CycA can be detected in the epidermis at this stage, and mitotic figures are seen only occasionally. Most of the cells show a size typical for cell cycle 16 with the DNA in a decondensed state (Grosskortenhaus, 2002).
To determine when Rca1 mutant cells deviate from the wt situation, embryos were examined that lack functional Rca1 in every other segment. A haemagglutinin (HA)-tagged Rca1 cDNA under the control of the UAS promoter was constructed. HA-Rca1 was expressed in every second segment in the Rca1 mutant background by a paired-Gal4 (prd-Gal4) driver line. HA-Rca1 was detected using HA antibodies. High magnifications show that Rca1 is nuclear, confirming a nuclear localization sequence between amino acids 115 and 133. HA-Rca1-expressing segments undergo mitosis 16, visualized by staining for phosphorylated histone 3 (PH3). This demonstrates that HA-Rca1 is able to rescue the mitotic failure in Rca1 mutants. Indeed, HA-Rca1-expressing segments have almost twice as many cells as the mutant segments, when analyzed in older embryos (Grosskortenhaus, 2002).
The degradation pattern of two mitotic cyclins, CycA and CycB, was examined. In HA-Rca1-expressing segments, CycA and CycB levels are high compared with Rca1 mutant segments. No differences were detected in slightly younger embryos between adjacent segments, and both cyclins accumulate normally at the beginning of interphase 16. It is therefore concluded that the disappearance of mitotic cyclins in Rca1 mutant cells is caused by their premature destruction in late interphase of cell cycle 16. The remaining cyclin levels are apparently not sufficient to drive cells into mitosis (Grosskortenhaus, 2002).
CycA is essential for mitosis, but Rca1 mutants might also lack other components required for mitotic induction. To address this question, additional CycA was expressed in Rca1 mutants using an UAS-HA-CycA construct and the prd-Gal4 driver line. This construct does not disturb mitotic progression in a wt background and is able to rescue the CycA mutant phenotype. In the Rca1 mutant background, HA-CycA-expressing segments have higher cell numbers. Thus, overexpression of CycA can overcome the mitotic block of Rca1 mutant embryos. This shows that CycA is the only essential mitotic function missing in the Rca1 mutants (Grosskortenhaus, 2002).
The defect in Rca1 mutants can be observed in cell cycle 16 at a time shortly before the first G1 phase in Drosophila embryogenesis. To test if Rca1 function is restricted to embryogenesis, Rca1 mutant cells were generated in imaginal disc epithelia using the FLP/FRT system. Cells homozygous for Rca1 were identified by the absence of a nuclear GFP that was expressed from the sister chromosome (Grosskortenhaus, 2002).
In these imaginal discs, only small Rca1 mutant clones could be seen, while their twin clones were significantly larger. Thus, Rca1 mutant clones have a growth disadvantage. Presumably, mutant cells have a limited proliferation potential and stop dividing after a few cell divisions. The clone size of mutant cells is also affected by cell elimination, since clones that contain two copies of the GFP gene were found without the corresponding Rca1 mutant twin clone. In all imaginal discs analyzed, clones were found throughout the disc and were not restricted to a particular region. Thus, Rca1 function is generally required for maintaining mitotic cyclin levels in proliferating cells. Interestingly, nuclei of the mutant cells were enlarged and appear to contain higher DNA levels. However, quantification on single cells would be required to determine the exact DNA content of these cells. In conclusion, clonal analysis shows that Rca1 function is not only needed during embryogenesis, but that it is a general factor required for proliferation (Grosskortenhaus, 2002).
The degradation of CycA in Rca1 mutants apparently occurs in late interphase before cells would enter mitosis. Normally, CycA is stable in cellularized embryos during interphase. CycA degradation starts in metaphase and persists through the reminder of mitosis and G1. The activities that cause CycA degradation are still poorly understood. Genetic data indicate that Cdc20Fzy and Cdh1Fzr are required for the degradation of CycA in mitosis and G1, respectively. While Cdc20Fzy is expressed throughout embryogenesis, Cdh1Fzr transcript is upregulated during stage 11, consistent with its function during G1. In fzy mutants, ectodermal cells arrest with high CycA levels in metaphase of cycle 16. fzr mutants are unable to keep mitotic cyclin levels low during G1, resulting in an additional S phase and mitosis (Sigrist, 1997). Overexpression of Cdc20Fzy does not perturb cell cycle progression (Sigrist, 1997), while overexpression of Cdh1Fzr causes premature cyclin degradation (Sigrist, 1997). The overexpression of Cdh1Fzr by the prd-Gal4 driver line results in premature CycB and CycA degradation (Sigrist, 1997). Cells fail to execute mitosis 16, and, consequently, fewer cells are visible in those segments. This phenotype is strikingly similar to the Rca1 phenotype. Thus, a possible explanation of the Rca1 mutant phenotype would be an abnormal activity of Cdh1Fzr in interphase of cell cycle 16 (Grosskortenhaus, 2002).
Whether Rca1 suppresses the effect of ectopic Cdh1Fzr was tested. HA-Rca1 and fzr transgenes were expressed using the prd-Gal4 driver line. In these embryos, CycA is no longer degraded prematurely in the fzr-expressing segments, and the overall pattern of CycA degradation looks identical to that of wt embryos. In older embryos, similar cell numbers were found in all segments, indicating that all cells were able to undergo a normal proliferation program. Thus, Rca1 overexpression counteracts the effect of Cdh1Fzr overexpression (Grosskortenhaus, 2002).
Cdh1Fzr is a regulatory subunit of the APC. The inhibition of ectopic Cdh1Fzr activity by Rca1 overexpression could be explained if Rca1 inhibits either Cdh1Fzr function or the activity of the core APC. Accordingly, in the absence of Rca1 function, either Cdh1Fzr-dependent APC activity or APC activity regulated by other means would cause the premature cyclin degradation (Grosskortenhaus, 2002).
To distinguish between these possibilities, fzr;Rca1 double mutants were analyzed. If Rca1 affects APC activity independently of Cdh1Fzr, it would be expected that the double mutants continue to degrade cyclins prematurely. In the case that Rca1 is specifically preventing Cdh1Fzr-associated activity during interphase 16, the double mutant should enter mitosis 16, since APC-Cdh1Fzr activity is absent (Grosskortenhaus, 2002).
The only available fzr mutation was used, a small deficiency [Df(1)bi-D3] removing fzr and also the gene hindsight (hnt), that is required for the retraction of the germband but has no influence on cell cycle progression (Sigrist, 1997). The cell density of wt, Rca1, and fzr mutants was compared with those of fzr;Rca1 double-mutant embryos at the beginning of germband retraction, when all epidermal cells in wt embryos have completed mitosis 16. At this stage, fzr mutants also had completed mitosis 16, and the cell density was comparable to that of wt. In contrast, Rca1 mutant embryos displayed reduced cell numbers, since they failed to enter mitosis 16. Finally, in fzr;Rca1 double-mutant embryos, cell density was again similar to wt embryos, indicating that these embryos had gone through mitosis 16 (Grosskortenhaus, 2002).
Thus, in fzr;Rca1 double mutants, the Rca1 phenotype is suppressed, indicating that the premature degradation observed in Rca1 mutants is mediated by Cdh1Fzr. This suggests that Rca1 functions by inhibiting APC-Cdh1Fzr activity during interphase. In Rca1 mutants, this complex would be active prior to mitosis and cause premature mitotic cyclin degradation, resulting in a failure to enter mitosis. In the double mutant, APC-Cdh1Fzr activity is not present, and Rca1 is not required to prevent premature cyclin destruction (Grosskortenhaus, 2002).
The activity of Cdh1Fzr is negatively controlled by Cdk-mediated phosphorylation. Both Cdk1 and Cdk2 kinase activities have been implicated in Cdh1 phosphorylation. During the first 15 divisions, CycE/Cdk2 kinase activity is present throughout the cell cycle. It declines during cell cycle 16, caused by the downregulation of CycE transcription and the upregulation of the Cdk2-specific inhibitor dacapo (dap). Thus, Cdh1Fzr is not inhibited by CycE/Cdk2 activity during later stages of the 16th cell cycle. However, overexpressed CycE is able to suppress the effects of ectopic Cdh1Fzr during cell cycle 16 (Sigrist, 1997). To analyze whether CycE is also able to compensate for the lack of Rca1 function, CycE was overexpressed in Rca1 mutant embryos using the prd-Gal drive line. In segments overexpressing CycE, higher cell densities are observed. This demonstrates that CycE is able to suppress the Rca1 mutant phenotype, presumably by its negative influence on Cdh1Fzr activity (Grosskortenhaus, 2002).
These data would suggest that CycE and Rca1 have overlapping functions in Cdh1Fzr inhibition. Thus, the requirement for Rca1 only becomes visible when CycE levels decline during the 16th cell cycle. However, CycE and Rca1 are only partially redundant. This can be concluded from Rca1;CycA double-mutant embryos. In CycA mutants, very low levels of CycA are already present during the 15th cell cycle but these levels are still sufficient to execute mitosis 15, and cells arrest before mitosis 16. Thus, cell numbers are similar to Rca1 mutant embryos and reduced compared with those of wt. Interestingly, Rca1;CycA double mutants have even fewer cells. Apparently, these double-mutant embryos fail to execute mitosis 15, likely caused by a further reduction in CycA levels due to the absence of Rca1. This shows that Rca1 is active at earlier cell cycles and becomes essential when CycA levels are reduced. Under these circumstances, CycE that is present during cycle 15 is apparently not sufficient to prevent excessive CycA degradation (Grosskortenhaus, 2002).
To see whether Rca1 overexpression influences normal cell cycle progression, the prd-Gal4 driver line was used to express HA-Rca1 in alternating segments. An Rca1 antibody was used to detect the expressed transgene. The antibody recognizes Rca1 in Western blots from embryonic extracts and overexpressed HA-Rca1 in embryos, but it fails to detect the endogenous protein, probably due to relatively low expression levels (Grosskortenhaus, 2002).
CycA levels and cell cycle progression in segments that overexpress HA-Rca1 were compared with wt segments. There was no deviation in CycA protein levels or cell cycle progression between neighboring segments. Thus, Rca1 does not promote CycA stability when overexpressed. In addition, the pattern of CycA degradation during mitosis is not changed by elevated Rca1 levels. CycA degradation starts in metaphase and persists through anaphase and telophase. Metaphase cells with CycA as well as those in which CycA is already degraded are visible in the HA-Rca1-overexpressing segment. In telophase cells, CycA is completely degraded (Grosskortenhaus, 2002).
The arm-Gal4 driver line that forces ubiquitous expression in all embryonic and imaginal tissues was used. High levels of HA-Rca1 were present in embryos undergoing the 15th cell cycle. However, no change in normal mitotic progression was observed, and the mitotic degradation pattern of CycA was normal. HA-Rca1 was present throughout the cell cycle without signs of degradation. However, during the 16th cell cycle, HA-Rca1 degradation started in a pattern that was similar but not identical to that of CycA. HA-Rca1 and CycA were absent in most G1 cells of the epidermal cell layer at later stages. Degradation of Rca1 and CycA were compared in a region of the embryo that undergoes mitosis 16. HA-Rca1 was present throughout the right part of this magnified region, where cells in different stages of mitosis were visible. Degradation of CycA was seen in a group of cells that had completed mitosis 16 and were in G1 of cycle 17, but only a fraction had degraded HA-Rca1. It is concluded that Rca1 is not degraded during mitosis but disappears during G1 (Grosskortenhaus, 2002).
The embryos in which HA-Rca1 was expressed throughout development using the arm-Gal4 driver line hatch, develop larvae to the pupal stage, but flies fail to eclose, typical for defects during imaginal disc development. Thus, while Rca1 overexpression does not influence cell cycle progression during embryogenesis, it does perturb development at later stages (Grosskortenhaus, 2002).
Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the Drosophila embryo. In one of the well-studied neuronal lineages in the ventral nerve cord (the NB4-2->GMC-1->RP2/sib lineage), Notch (N) signaling interacts with asymmetrically localized Numb (Nb) to specify sibling neuronal fates to daughter cells of GMC-1. The NB4-2 is delaminated in the second wave of NB delamination during mid stage 9 (~4.5 hours) of embryogenesis and is located in the 4th row along the anteroposterior axis and 2nd column along the mediolateral axis within a hemisegment. The NB4-2 generates its first GMC (GMC-1, also known as GMC4-2a) ~1.5 hours after formation. The GMC-1 divides ~1.5 hours later to generate two cells, the RP2 and the sib. The RP2 cell eventually occupies its position in the anterior commissure along with the other RP neurons (RP1, RP2, RP3 and RP4) and projects its anteroipsilateral axon to the intersegmental nerve bundle (ISN) and innervates muscle #2 on the dorsal musculature. The sib cell migrates to a position posterior and more dorsal to RP2. The DiI tracing of the NB4-2 lineage indicates that the sib has no axonal projection at mid stage 17 of embryogenesis; thus, its ultimate fate has not been determined. In this study, loss-of-function mutations in N and nb, cell division mutants cyclinA (cycA), Regulator of cyclin A1 (Rca1) and string/cdc25 phosphatase (stg/cdc25 phosphatase), and the microtubule destabilizing agent, nocodazole, were all used to investigate asymmetric cell fate specifications by N and Nb in the context of cell cycle. Mutation in Rca1 gene was initially identified as a dominant suppressor of roughex (rux) eye phenotype. In rux, the cells enter S-phase precociously due to ectopic activation of a CycA/Cdk complex in early G1 (Dong, 1997). In embryos lacking the Rca1 activity, the cells appear to arrest in G2 of the cell cycle (at stages 15-16) similar to cycA mutants (Wai, 1999 and references).
The loss of cycA, Rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1 exclusively adopts an RP2 identity. The requirement of cycA or Rca1 for cell division in the CNS is lineage specific. Anti-Eve staining of cycA or Rca1 mutant embryos indicates that loss of these gene products does not affect all the Eve-positive lineages in the ventral nerve cord. Eve is expressed in other neuronal lineages such as the CQs, the Us and the ELs. The CQs are formed from NB7-1, an S1 neuroblast. The GMC for these neurons are formed at the same time as the GMC for the aCC/pCC neurons (generated from another S1 neuroblast, NB1-1) and divide at the same time as GMC for the aCC/pCC lineage. The NB7-1 in cycA or Rca1 mutants does not divide to generate an Eve-positive GMC for the CQs. However, the effect on CQs is partially penetrant in both the mutants. Thus, ~75% of the hemisegments had missing CQs in cycA mutants; in Rca1 mutants, this figure is ~50%. The effect on the generation of U neurons is as follows: in cycA mutants, the effect is fully penetrant; whereas, in Rca1 mutants, 65% of the hemisegments are missing the Us. It must be pointed out that in those hemisegments where these neurons (Us and CQs) are formed, the number of these neurons is fewer than normal. Finally, the effect of the loss of cycA or Rca1 on another Eve-positive lineage, the EL neurons, is minimal. The EL neurons are formed from NB3-3, an S4 neuroblast (the formation of this neuroblast extends between S3-S5). None of the hemisegments have missing EL neurons, in either the cycA mutants or the Rca1 mutants. The above result indicates that the loss of Rca1 or cycA does not affect the division of all neuroblasts. One possibility for this result is that the maternal deposition of these gene products is masking the zygotic loss of these gene products in these lineages. However, this seems unlikely since the GMCs for the aCC/pCC or the RP2/sib lineages are generated earlier than the GMCs for the EL neurons. Moreover, the maternal deposition of CycA, for example, is completely exhausted before stage 7 and none of the neuroblasts have delaminated from the neuroectoderm at this stage of development. Thus, these results indicate that the effect of loss of cycA or Rca1 is lineage specific and every neuronal lineage is not sensitive to the loss of these cell division genes. It is most likely that some other cyclins (i.e., Cyclin B) complement the loss of CycA in these lineages (Wai, 1999).
While the loss of N leads to the specification of RP2 fates to both progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1 in the double mutant between nb and cycA assumes a sib fate. While the GMC-1 fails to divide to generate two cells in these double mutants, the GMC-1 assumed a sib fate. About ~35% of the hemisegments show this phenotype. This penetrance of the phenotype is slightly higher than the phenotype observed in nb single mutants alone. This suggests that cycA mutation has an enhancing effect on the nb phenotype. This would argue that normally a small amount of the Nb protein segregates into a sib cell and that, in the absence of cell division, all of Nb is accumulated in one cell, and therefore, is much more effective in blocking the N signaling. Moreover, since the nb phenotype is epistatic to the cell division mutant phenotype, Nb must be acting downstream of these genes. This result is consistent with the finding that Nb becomes localized during metaphase and is not localized in stg mutants. Thus, in Rca1 or cycA mutants, the absence of a localized Nb prevents the N signaling from specifying sib fate and, as a result, the GMC-1 assumes an RP2 fate. These epistasis results indicate that both N and nb function downstream of cell division genes and that progression through cell cycle is required for the asymmetric localization of Nb. In the absence of entry into metaphase, the Nb protein prevents the N signaling from specifying sib fate to the RP2/sib precursor. These results are also consistent with the finding that the sib cell is specified as RP2 in N;nb double mutants. Finally, these results show that nocodazole-arrested GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib fate specification (Wai, 1999).
In the central nervous system (CNS) of Drosophila embryos lacking either cyclin A or Regulator of cyclin A (Rca1) several ganglion mother cells (GMCs) fail to divide. Rca1 is novel 412 amino acid protein required for both mitotic and meiotic cell cycle progression, Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). The Rca1 mutation was initially identified and characterized from a screen for aberrant expression patterns of Even- skipped (Eve) protein in the embryonic CNS (I. Orlov, R. Saint, N. Patel, unpublished results cited by Lear, 1999). Eve is normally expressed in the nuclei of several cells in the CNS; these include GMC 1-1a and its progeny, aCC and pCC; GMC 4-2a and one of its progeny, RP2, as well as the EL, U, and CQ neurons. In cycA and Rca1 mutants, Eve is expressed in fewer cells per hemisegment than wild-type. In the position where the siblings aCC and pCC normally sit, a single Eve-positive nucleus that is larger than the wild-type aCC or pCC is observed. In the position of RP2, there is still one Eve-positive nucleus, but again it often appears larger than normal. A loss of Eve expression is also observed where the U and CQ neurons normally sit, as well as a decrease in the number of Eve-positive EL neurons (Lear, 1999).
The GMC 4-2a and GMC 1-1a lineages received the closest focus because of their well-characterized development and because various molecular markers exist that label these GMCs and their progeny. In wild-type embryos, GMC 4-2a divides early in stage 11, and two Eve-expressing nuclei are initially observed upon this division. Eve expression is quickly shut off in the smaller RP2 sibling nucleus but remains on in RP2. In cycA or Rca1 mutants, Eve expression turns on normally in GMC 4-2a; however, two nuclei are rarely observed during stage 12, and the single Eve-expressing nucleus remains large. Likewise, GMC 1-1a normally divides during stage 10 in wild-type embryos to generate the Eve-positive neurons aCC and pCC. In cycA or Rca1 mutants, GMC 1-1a expresses Eve as in wild-type but rarely divides. Instead, this GMC comes to reside in the same dorsal plane and posterior position where aCC and pCC sit in wild-type embryos. Other Eve-expressing lineages, including the U/CQ neurons and the EL neurons, appear to be affected as well in cycA and Rca1 mutants. Notably, even the most severe alleles of cycA and Rca1 examined do not show complete expressivity of CNS phenotypes in all lineages (Lear, 1999).
Having observed that GMCs acquire the fate of the 'B' sibling neuron in cycA or Rca1 mutants, it was next determined whether GMCs could acquire the 'A' fate through activation of the Notch pathway. If Delta signal must be provided from a sibling neuron, then GMCs, which lack a true 'sibling', may not have the potential to acquire the 'A' fate through extracellular signaling. The Rca1 mutation was combined with either a zygotic numb mutation or an activated form of Notch in order to examine this question. In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In Rca1;numb double mutant embryos, binary cell fate changes ('B' to 'A') in several GMCs as well. GMC 4-2a frequently adopts the 'A' fate of the RP2 sibling in Rca1;numb or hs-N intra;Rca1 embryos. In contrast, GMC 4-2a always acquires the 'B' fate of RP2 in Rca1 mutants alone. Notably, it was observed that the 'B' to 'A' fate change (RP2 to RP2 sibling) occurs with greater frequency in Rca1;numb double mutants than the RP2/sib ('A/B') to sib/sib ('A/A') fate change that occurs in numb mutants alone (Lear, 1999).
Thus GMCs in cycA and Rca1 mutants differentiate as neurons: they assume the 'B' fate normally taken by one of their sibling progeny. These GMC fate decisions correspond to Notch pathway mutants ('B/B'), and they oppose the fate changes observed in embryos lacking numb ('A/A'). The loss of zygotic numb or constitutive activation of Notch in a Rca1 background allows for a binary fate switch in GMCs: GMCs often differentiate as the 'A' sibling in the context of these mutations. These results indicate that activation of the Notch pathway causes GMCs to adopt the 'A' neuronal fate. Thus, fate choice in non-dividing GMCs appears to occur in much the same way that binary fate decisions occur in sibling neurons. In some models of asymmetric division, a specific factor required to attain one of the sibling fates is produced only upon progression of the cell cycle. The observation that GMCs can attain the fate of either sibling neuron indicates that gene products dependent upon GMC division are not required in this fate decision (Lear, 1999).
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date revised: 12 February 2002
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