Effects of Mutation or Deletion (part 3/3)

Numb and the Central Nervous System

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 were 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).

Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system

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 and a decrease in the number of Eve-positive EL neurons (Lear, 1999).

The GMC 4-2a and GMC 1-1a lineages recieved 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 undergoes a change ('B' to 'A') in several GMCs as well. GMC 4-2a frequently adopts the 'A' fate of 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).

Finally, expression of Delta in the mesoderm is sufficient to attain both sibling fates. In Dl mutant embryos, misspecification of neuroectodermal cells results in an excess of neuroblasts and their resulting neuronal progeny. Additionally, binary cell fate alterations are observed at the sibling neuron level. In wild-type embryos, Vnd protein is expressed in pCC but not aCC; in Dl mutants, the numerous GMC 1-1a progeny all lack Vnd expression, indicating that all of these neurons acquire the aCC fate. The twist-GAL4 line was used to drive Dl expression in the embryonic mesoderm. In a wild-type background, expression of ectopic Dl using the twist- GAL4 line appears to have little effect on the embryo; significantly, no effect in CNS cell fate specification is observed. When Dl is expressed in the embryonic mesoderm of a Dl homozygous mutant using twist-GAL4, many sibling neuron pairs attain differential fates ('A/B'). Specifically, at least one aCC and one Vnd-expressing pCC is observed in each thoracic/abdominal hemisegment of these embryos. Thus, cell fate specification of the aCC/pCC sibling pair is rescued by expression of Dl in the mesoderm. Thus, these results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, these results indicate that Delta signal can be received from outside the sibling pair (Lear, 1999).

A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions

In the Drosophila CNS glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. The genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. SPGs share direct sibling relationships with neurons and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, Notch signaling positively regulates glial cells missing expression in the context of SPG development (Udolph, 2001).

In order to better understand how a complete lineage of a specific NGB with all its progeny, including its glial cells, might be created, NB1-1 was chosen for a detailed analysis. NB1- 1 has been extensively used for cell fate specification studies and a sound basis of information about this NB lineage is available. NB1-1 is a NB that develops differential lineages in the thoracic versus the abdominal segments. Focus was placed on the abdominal NB1-1A because only these abdominal NB1-1 lineages contain glia. In addition to the aCC/pCC sibling neurons, which are the progeny of the first GMC produced from this lineage, NB1-1A generates 2 to 3 glial cells and 4 to 5 clustered interneurons (cN), yielding a total of 9 to 10 cells. The three glial cells belong to the group of subperineurial glia (SPG) that lie at the periphery of the nerve cord and enwrap the entire ventral nervous system. Two of the glia, the A- and B-SPGs, can be found in dorsal positions, with a third glia, the LV-SPG, located at ventral positions of the nerve cord. All SPGs, including the A- and B-SPG and LV-SPG of NB1-1A, are specifically labelled by two enhancer trap lines, M84 and P101 (Udolph, 2001 and references therein).

As a first step toward elucidating the origin of the glial cells of the NB1-1A lineage, the effects of loss of function mutants in several genes, Notch, mastermind (mam) and numb, which are known to affect the resolution of distinct sibling cell fates, were tested for their effect on the development of A-, B- and LV-SPGs. Embryos hemizygous/homozygous for a conditional Notch allele, Nts1, and also carrying one copy each of M84 and P101 (Nts1/M84/P101) were subjected to the non-permissive temperature of 29°>C after 6 hours of development. This regime allows Notch to function during the singling out of NBs and removes Notch during the crucial period when it is required for sibling cell fate resolution. Double staining with anti-Eve and anti-ß-gal was performed. As expected, in most hemisegments, Nts1/M84/P101 embryos duplicate the RP2 neuron at the expense of its sibling cell. Moreover, in 96% of the hemisegments, M84/P101+ cells could not be found in typical dorsal or ventral positions. It is concluded that Notch function is required for the specification of the M84/P101 positive A-, B- and LV-SPGs. In wild-type embryos, M84/P101 is expressed in about eight SPGs per hemisegment, including the A- and B-SPGs and the LV-SPG (Udolph, 2001).

mastermind, which has been linked to the Notch signaling pathway by its genetic interactions with Notch and its strikingly similar phenotype in early and late neurogenesis, was tested. mam acts downstream of Notch during sibling cell fate specification in the embryonic nervous system. The hypomorphic mam345 allele used in this study shows only a mild hypertrophy of the nervous system but clearly has an effect on sibling cell fate specification. A severe reduction (94%) of P101+ cells was observed in mam345;P101 embryos similar to that seen with Nts1/M84/P101 embryos. These data suggest that both genes are strictly required for the specification of SPGs, most likely in a linear pathway. However, it is unclear how Notch acts in the specification of the SPGs. The possibility is considered that SPG glial cells could arise from a series of asymmetric cell divisions, with Notch being required to specify the glial daughters of these divisions (Udolph, 2001).

Based on its function as a negative regulator of Notch signaling, the expected numb phenotype is opposite that of Notch in terms of sibling cell fate transformation. The P101 expression pattern was tested in the background of a strong numb mutation. In contrast to Notch and mam, additional P101+ cells were found in the vicinity of the aCC/pCC position. In most of the examined hemi-neuromeres, up to four ß-gal-positive cells were detected in dorsal positions close to aCC/pCC. This is indicative of a duplication of the A- and B-SPGs. Additional P101+ cells with glial morphology were found in lateral and ventral positions of the nerve cord, presumably duplications of other SPGs. These findings are consistent with an asymmetric cell division model for the genesis of the SPGs (Udolph, 2001).

Slit signaling promotes the terminal asymmetric division of neural precursor cells in the Drosophila CNS

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).

Role of cortical tumor-suppressor proteins in asymmetric division of Drosophila neuroblast

In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumor-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).

In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch signaling, associate with their respective adapter proteins, Miranda and Partner of numb (Pon), and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz) and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of the basal determinants. However, the mechanisms that underlie the asymmetric protein sorting in neuroblasts are not known. To address this issue, chromosomal deficiencies have been sought that affect the subcellular distribution of Miranda. Such screening identified the lgl tumor-suppressor gene that encodes a protein containing WD40 repeats. In wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the basal cortex upon mitosis after a transient spread into the cytoplasm. In germline clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda does not localize asymmetrically in mitotic neuroblasts, but rather is distributed uniformly throughout the cortex as well as in the cytoplasm, where it is concentrated along microtubule structures. Consequently, Miranda segregates into both the daughter neuroblast and the ganglion mother cell (GMC). Numb and Pon are also distributed uniformly at the cortex and in the cytoplasm (Ohshiro, 2000).

It would be expected that the abnormal distribution of Numb and Miranda in lgl mutant neuroblasts results in incorrect determination of neural cell fate. Given the difficulty of monitoring neural cell fate in severely distorted lglGLC embryos, this prediction was tested by analyzing the lineage of the external sensory organ in the notum, in which all cell divisions are asymmetric and sibling cells adopt distinct fates as a result of the asymmetric inheritance of Numb. Sensory organ precursor cells in this lineage segregate Numb into a daughter cell pIIb, which subsequently generates three inner cells (a glial cell, a neuron and a sheath cell). The sibling pIIa cell divides into two outer cells constituting the external sensory structure, a hair and a socket. Exposure of lglts3 mutant larvae to 29°C during external sensory organ development mislocalizes Numb in mitotic precursor cells, as observed in neuroblasts, and often transforms inner cells into outer cells resulting in duplicated external sensory structures, a phenotype expected from loss of numb function. Indeed, this notum phenotype is enhanced by reducing the numb gene dosage by half. Equal partition of Numb between sibling cells would result in numb gain of function phenotypes because the half dose of numb is enough for correct cell-fate decisions. The observed numb loss-of-function phenotype therefore suggests that a reduction in lgl activity does not only equalize Numb distribution between sibling cells but also attenuates numb function, consistent with the observation of cytoplasmic Numb in the lgl mutants. Conversely, the presence of an extra numb gene induces opposite phenotypes under the lgl mutant condition. The outer cells are frequently transformed into the inner cells, resulting in the loss of the external sensory structure. This appearance of the numb gain of function phenotypes is simply explained by the fact that the partition of additional Numb from the transgene into both sibling cells raises numb activities over the threshold necessary to suppress Notch function in both cells. These data thus indicate that Lgl is essential in neural fate decisions through cortical targeting of cell-fate determinants (Ohshiro, 2000).

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 us 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).

Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

Notch signaling mediates multiple developmental decisions in Drosophila. This study examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, these results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells (Wang, 2011).

This study find that Notch signaling plays an essential role in the maintenance and expansion of neuroepithelial cells in the optic lobe; it also inhibits medulla neuroblast formation. Clonal analyses of several pathway components indicate that this dual function bifurcates downstream of Su(H) with E(spl)-C only partly involved in the inhibition of medulla neuroblast formation but not the maintenance and expansion of neuroepithelial stem cells (Wang, 2011).

In the optic lobe, Notch signaling plays a role analogous to lateral inhibition during embryonic CNS development. However, the selection of neuroblasts in the OPC neuroepithelium is an all-or-none process rather than selecting individual neuroblasts from the neuroepithelium. Medulla neuroblasts are generated in a wave progressing in a medial to lateral direction in the OPC neuroepithelium with all cells at a particular position along the medial-lateral axis differentiating into neuroblasts. Interestingly, this wave of medulla neuroblast formation coincides with the down-regulation of both Delta and Notch expression in the medial cells in the OPC, which might reduce Notch signaling activity, thereby allowing medulla neuroblasts to form. What factors drive the recession of both Delta and Notch expression in the OPC neuroepithelium along the medial-lateral axis is not known. When Notch signaling is inactivated, neuroepithelial cells in the OPC change cell morphology and differentiate into medulla neuroblasts prematurely. The results indicate that Notch signaling actively controls neuroepithelial integrity, possibly by regulating the adherens junction (AJ), since in Notch pathway mutant mosaic clones in the OPC, the apical determinants PatJ, Crumbs and aPKC are cell autonomously reduced or lost and the mutant cells change to rounded or irregular morphology. Further experiments will be needed to determine how Notch signaling activity affects the maintenance of neuroepithelial integrity, particularly the stability of the adherens junction (Wang, 2011).

Is neuroblast formation also actively inhibited by Notch signaling or simply a default state of neurogenic epithelial cells? In the latter model, Notch signaling may only maintain neuroepithelial integrity and promote their expansion while medulla neuroblasts form when the neuroepithelial integrity is disrupted. The argument against this model is that changes in neuroepithelial integrity are not always accompanied with cell fate changes. In N, Dl or Su(H) mosaic clones located in the OPC neuroepithelium, it was found that in about 25% of the clones, the mutant cells changed morphology or lost apical marker expression but did not become neuroblasts (Dpn-negative), whereas in E(spl)-C mosaic clones, Dpn+ cells were prematurely induced, which indicate that the cells begin to differentiate into neuroblasts, but these cells still retained columnar epithelial cell morphology and apical marker expression. This suggests that the suppression of neuroblast formation by Notch signaling activity is separable from the maintenance of neuroepithelial integrity and that medulla neuroblast formation is actively suppressed by Notch signaling. A possible scenario is that activation of the Notch pathway turns on the E(spl)-C genes, which in turn suppress proneural gene expression in the optic lobe neuroepithelia. Indeed, at least one member in the E(spl)-C genes, E(spl)m8, appears to be activated in the neuroepithelial cells by the Notch pathway, as the E(spl)m8-lacZ reporter is expressed in a pattern similar to Delta and Notch expression in the OPC and IPC. E(spl)m8 protein and possibly additional members of the E(spl)-C may suppress the expression of proneural genes in the optic lobe. The proneural genes of the achaete-scute complex (as-c) comprise four members, achaete, scute, L'sc, and asense. achaete is not expressed in the optic lobe, but scute is expressed in both the neuroepithelial cells and neuroblasts in the OPC implying that scute expression in the neuroepithelial cells is not suppressed by Notch signaling activity. By contrast, asense is only expressed in the neuroblast and GMCs and L'sc is transiently detected in an advancing stripe of neuroepithelial cells of 1-2 cells wide that are just ahead of newly formed medulla neuroblasts. Thus, E(spl)-C proteins may suppress L'sc and/or ase expression, the release of this suppression may allow the neuroepithelial cells to begin to differentiate into medulla neuroblasts. It should be noted, however, that the removal of the E(spl)-C activity does not seem to be sufficient to allow full differentiation of neuroepithelial cells into medulla neuroblasts, suggesting that additional factors downstream of Notch signaling may be involved in the suppression of medulla neuroblast formation (Wang, 2011).

The phenotypes of Notch pathway mutants are reminiscent of those of JAK/STAT mutants. For example, inactivation of either pathway led to early depletion of the OPC neuroepithelium; either pathway inhibits neuroblast formation, and ectopic activation of either pathway promotes the growth of the OPC neuroepithelium. The remarkable phenotypic similarities in Notch and JAK signaling mutant brains suggest that these pathways may act in a linear relationship such that activation of one pathway is relayed to the second, perhaps by inducing the expression of a ligand. Alternatively, these pathways may act in parallel and converge onto some key downstream effectors or target genes. Further experiments will be needed to test whether Notch interacts with JAK/STAT and if it does, to find out where the interaction occurs during the development of the optic lobe (Wang, 2011).

The roles of Notch signaling in mammalian brain development have been studied intensely. Many Notch pathway components have been examined in knockout mice, which showed defects in brain development. Mice deficient for Notch1 or Cbf all display precocious neurogenesis during early stages of nervous system development. This has led to the view that the role of Notch signaling in the mouse brain is to maintain the progenitor state and inhibit neurogenesis. However, it is not clear from these studies whether the premature neurogenesis in Notch signaling mutant mice was caused by premature differentiation of neuroepithelial stem cells into neurons or by premature differentiation of neuroepithelial stem cells into progenitor cells, which then generated neurons. In fact, it has been proposed that Notch activation can promote the differentiation of neuroepithelial stem cells into radial glial cells, the progenitor cells that generate the majority of neurons in the cerebral cortex. This is based on the observation that ectopic Notch activation using activated forms of Notch1 and Notch3 (NICD) caused an increase in radial glial cells as compared to control. The radial glial cells resemble medulla neuroblasts in the Drosophila optic lobe in that they are both derived from neuroepithelial stem cells and undergo asymmetric division to self-renew and generate neurons, although morphologically radial glial cells are still polarized while medulla neuroblasts have lost epithelial characters and are rounded in shape. Based on the current results, it is suggested that Notch signaling maintains the pool of neuroepithelial stem cells and promotes their expansion in both Drosophila and mammals and that the precocious neurogenesis in Notch signaling mutant brains arise due to premature differentiation of the neuroepithelial stem cells into the progenitor cells (Wang, 2011).

However, ectopic Notch activation may indeed promote progenitor cell proliferation in the brain. Ectopic neuroblasts were observed in the medulla cortex when NACT was ectopically expressed by the neuroblast/GMC driver insc-Gal4, by ubiquitous expression using hs-Gal4, or when numb15 mosaic clones were induced at later larval stages when neuroblasts normally begin to form. Since the results have shown that the Notch pathway is not essential for medulla neuroblast formation or self-renewal, the ectopic neuroblasts are a novel phenotype solely induced by ectopic Notch signaling activity. This is consistent with Notch activation promoting ectopic neuroblast formation in the central brain and VNC without being required for neuroblast self-renewal in these regions of the CNS; and Notch has been shown to be an oncogene in mammals. Since the sizes of the ectopic neuroblasts were in the range of GMC or neurons, they may resemble the transit-amplifying (TA) neuroblasts that are found in the dorsal-medial region of the central brain. The origin of these ectopic neuroblasts in the medulla cortex is not clear, but it is unlikely that they are derived from differentiated medulla neurons as ectopic expression of NACT using elav-Gal4, which is active in medulla neurons, did not result in ectopic neuroblasts and by the fact that ectopic neuroblasts can be induced in numb15 mosaic clones, which could only arise from mitotically active cells that include neuroepithelial cells, medulla neuroblasts, and ganglion mother cells (GMCs), but not neurons. The ectopic neuroblasts could be generated by a transformation of GMCs into a neuroblast identity as suggested for ectopic neuroblasts in brat mutant central brains. Ectopic Notch signaling activity may even directly promote the expansion of neuroblasts after they have differentiated from the neuroepithelial cells in the OPC. In either case, ectopic Notch signaling activity may block the normal path of neuronal differentiation and lock the cells in a proliferative state. This is indeed what was observed in numb15 mosaic clones in which numerous ectopic neuroblasts were induced in the medulla cortex without generating medulla neurons. Perhaps ectopic Notch signaling activity may also promote the proliferation of neural progenitors in vertebrates, such as the radial glial cells in the mouse brain (Wang, 2011).

The regulation of apoptosis by Numb/Notch signaling in the CNS serotonin lineage

Apoptosis is prevalent during development of the central nervous system, yet very little is known about the signals that specify an apoptotic cell fate. The role of Numb/Notch signaling in the development of the serotonin lineage of Drosophila has been studied; it is necessary for regulating apoptosis. When Numb inhibits Notch signaling, cells undergo neuronal differentiation, whereas cells that maintain Notch signaling initiate apoptosis. The apoptosis inhibitor p35 can counteract Notch-mediated apoptosis and rescue cells within the serotonin lineage that normally undergo apoptosis. Furthermore, tumor-like overproliferation of cells is observed in the CNS when Notch signaling is reduced. These data suggest that the distribution of Numb during terminal mitotic divisions of the CNS can distinguish between a neuronal cell fate and programmed cell death (Lundell, 2003).

The segmented Drosophila nerve cord develops from stereotyped division of 30 neuroblasts (NB) in each hemisegment. A pair of serotonergic neurons in each hemisegment arises from NB7-3. The divisions of the NB7-3 lineage have recently been determined using a combination of molecular markers and clonal analysis. NB7-3 produces three GMCs. GMC-1 produces two neurons: GW, a motoneuron, and EW1, the more medial serotonergic neuron. GMC-2 produces EW2, the more lateral serotonergic neuron. GMC-3 produces EW3, a neuron that synthesizes the neuropeptide corazonin. The GW neuron projects an axon ipsilateral and posteriorly, and the three EW interneurons all project axons anterior to the posterior commissure (Lundell, 2003 and references therein).

Several genes have been shown to be essential in the differentiation of the NB7-3 lineage. Sequential expression of the segmentation transcription factors, Hunchback->Krüppel->Pdm1, within neuroblasts has been shown to be important in the development of several lineages, including NB7-3. The subsequent GMCs and neuronal progeny maintain the expression of the transcription factors that are present in the NB at their birth. In the case of NB7-3, Hunchback (Hb) is expressed only in GMC-1 and its progeny, and is both necessary and sufficient to define the fates of these cells. Krüppel (Kr) is expressed in both GMC-1 and GMC-2 and is necessary and sufficient to establish the fate of the EW2 serotonergic neuron. Pdm1 is expressed primarily in EW2. Differentiation of the NB7-3 lineage is also affected by mutations in wingless (wg) and other members of the Wingless signaling pathway such as, engrailed (en), hedgehog (hh) and patched (ptc). In addition, mutations in the transcription factors eagle (eg) and huckebein (hkb) also disrupt NB7-3 differentiation. eg has been shown to suppress a rough eye phenotype caused by the overexpression of Ras1, suggesting that eg may be involved in Ras signaling. hkb is regulated by both en and hh in the NB7-3 lineage. The exact relationship between these genes and signal transduction pathways within the NB7-3 lineage remains to be determined (Lundell, 2003 and references therein).

The results of this study demonstrate that the intercellular Notch signaling pathway can be modulated during terminal divisions of the CNS to direct a choice between neuronal development and programmed cell death. The division of GMC-1 produces two distinct neuronal cell fates: the EW1 interneuron and the GW motoneuron. In this division, genetic alteration in the expression of Notch leads to switching between these two cell fates. A loss of Notch activity in spdo mutants leads to two Ddc/Hb-expressing EW1 cells and the overexpression of Notch leads to two Zfh-1 expressing GW cells. Therefore, Notch signaling must be inactivated during development of the EW1 neuron. Numb appears to have a minor role in this inactivation. In a numb1 mutant, 7% of the hemisegments do not develop an EW1 neuron, and a similar number of numb1 hemisegments show two Zfh-1-expressing GW cells. This transformation from an EW1 cell fate to a GW cell fate is what one would expect if Numb were inhibiting Notch. However, most EW1 neurons develop normally in a numb1 mutant and do not convert to the GW cell fate. Therefore, inactivation of Notch signaling in EW1 is mostly independent of Numb function. One possible explanation is that EW1 has a factor that is redundant for Numb function, which can inhibit Notch signaling and is capable of masking the effect of a numb1 mutation in most hemisegments. The unique expression of Hb in GMC-1 progeny could be responsible for establishing this redundancy. However, if a redundant Numb-like factor does exist, it is insufficient to protect EW1 during expression of the UAS-NotchACT transgene (Lundell, 2003).

The A8 segment is unique in that it has only a single serotonergic neuron instead of the pair of serotonergic neurons found in the more anterior segments. In a wild-type fly, this cell appears to be a derivative of GMC-2, because it expresses Zfh-2, but in a numb1 mutant, this cell appears to be a derivative of GMC-1, because it expresses Hb. A single Hb/Ddc-expressing cell in A8 is identical to the phenotype in the more anterior segments of a numb1 mutant. This suggests that in a numb1 mutant an EW1 cell is the default developmental pathway for this lineage. One possibility is that the redundant Notch-inactivating mechanism proposed for EW1 is induced only in the presence of a numb mutation. This would explain why the A8 EW1 cell is seen only in the numb mutant and not in a wild-type animal. If this were true, then the preservation of EW1 cells in a numb mutant would be due to the mutation itself. Until a putative redundant factor is identified it is impossible to determine whether it is expressed normally in wild-type animals or is expressed only in numb mutant animals (Lundell, 2003).

Like GMC-1, most GMCs divide, producing two progeny cells. However, GMC-2 and GMC-3 of the NB7-3 lineage produce only one neuron. It has been suggested that the mitotic sisters of EW2 and EW3 may undergo apoptosis. This idea is supported by the detection of apoptotic cells with TUNEL in the wild-type NB7-3 lineage and experiments with the apoptosis inhibitor p35, which can produce ectopic Ddc and corazonin-containing cells. The origin of the ectopic cells within NB 7-3 has not been formally determined by lineage tracing; however, the hypothesis that they are mitotic sisters of EW2 and EW3 is supported by the observations that GMC-2 and GMC-3 progeny often appear as mitotic pairs and that ectopic NB7-3 cells are immunoreactive for Zfh-2 (Lundell, 2003).

During the divisions of GMC-2 and GMC-3, genetic alterations in the expression of Notch lead to a switching between a neuronal cell fate and apoptosis. A reduction of Notch signaling with either spdoG104 or UAS-Numb embryos produces ectopic NB7-3 cells that express Zfh-2. Conversely, the overexpression of Notch in either UAS-NotchACT or numb1 embryos led to an increase in TUNEL labeling of GMC-2 and GMC-3 progeny. Additionally, inhibiting apoptosis with UAS-p35 or reducing Notch activity with spdoG104 can rescue the numb1 phenotype. It is hypothesized that during the divisions of GMC-2 and GMC-3, Numb partitions asymmetrically into EW2 and EW3 where it inactivates Notch signaling and leads to neuronal development. The mitotic sisters of EW2 and EW3 do not receive Numb, maintain Notch signaling and undergo apoptosis. The difficulty in detecting wild-type hemisegments that have more than four immunoreactive Eg cells, suggests that any other cells produced during divisions of the NB7-3 lineage quickly undergo apoptosis (Lundell, 2003).

Ectopic Eg cells in the NB7-3 lineage can be induced at stage 15 by H99, UAS-Numb, spdoG104 and UAS-p35. However, the ability of these alleles to produce ectopic Ddc and corazonin-containing neurons at later stages is variable. No significant ectopic Ddc or corazonin-containing cells were detected in either H99 or UAS-Numb CNS. For UAS-Numb it was shown that the ectopic Eg cells detected at stage 15 can undergo apoptosis. spdoG104 mutants produce only ectopic Ddc cells, but the reduction in the number of corazonin-containing cells in general suggests that either GMC-3 does not consistently form in these mutants or that GMC-3 progeny may convert from a corazonin-containing cell fate to a serotonergic cell fate. UAS-p35 mutants produce both ectopic Ddc and corazonin-containing cells at low frequency, but the allele is much more efficient at rescuing the EW neurons in numb1 and UAS-Notch mutants. Therefore, apoptosis is harder to reverse in cells that normally undergo apoptosis, than in the cells genetically induced to undergo apoptosis. The ability of these various alleles to produce ectopic Ddc- and corazonin-containing cells could be influenced by mutant effects they cause outside the NB7-3 lineage or may reflect different roles they have in the apoptotic pathway. The mechanism by which Notch induces apoptosis in the NB7-3 lineage remains to be determined, but the apoptotic genes reaper, grim and hid may be involved because all three of these genes are deleted in the H99 allele (Lundell, 2003).

Notch-induced apoptosis in the NB7-3 lineage will probably be regulated by other factors in addition to Numb. The Ras signaling pathway has been shown to inhibit Notch-induced apoptosis in the Drosophila pupal retina. Wingless has been shown to mediate Notch signaling and mutations in the Wingless pathway can lead to ectopic serotonergic cells. It will be a challenge to determine how these different signaling pathways interact to specify apoptosis within the NB7-3 lineage (Lundell, 2003).

The tumor-like expansion of Ddc-expressing cells observed in heterozygous spdoG104 larvae suggests that Notch-induced apoptosis may be essential for regulating cell proliferation. This spdo phenotype is reminiscent of three tumor-suppressor genes; discs large (dlg), lethal giant larvae (lgl) and scribble (scrib), which produce tumors in the CNS and imaginal disks. Interestingly, these three genes work in a common pathway that regulates cell polarity, and lgl and dlg have been shown to be essential for the distribution of Numb and other asymmetric determinants. Further investigation will be necessary to determine if spdo is part of this same mechanism and exactly how spdo mutants inhibit Notch signaling. Spdo expression is ubiquitous throughout embryogenesis and persists through the larval stages and into adults. If a spdo mutation can alter the response of the Notch receptor to environmental cues that induce apoptosis, one would expect to see overproliferation in additional tissues (Lundell, 2003).

Numb/Notch signaling is also known to affect development of the midline dopaminergic cells. The expression of Ddc is essential to the biosynthesis of both serotonin and dopamine. Anti-Ddc antibody detects not only the serotonergic neurons, but also midline dopamine neurons. As a consequence of using Ddc as a marker for the serotonin lineage, a number of observations were made about the development of midline dopamine cells. In a numb1 mutant very few midline dopamine cells are detectable with Ddc. spdoG104 mutants produce ectopic dopamine cells and can rescue dopamine cells in the numb1 mutant phenotype. Thus, Numb/Notch signaling also has a role in the development of midline dopamine cells, but further investigation into the significance and whether apoptosis is involved in this lineage will require lineage analysis to determine the origin of the midline dopamine cells (Lundell, 2003).

Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster

Loss of cell polarity and cancer are tightly correlated, but proof for a causative relationship has remained elusive. In stem cells, loss of polarity and impairment of asymmetric cell division could alter cell fates and thereby render daughter cells unable to respond to the mechanisms that control proliferation. To test this hypothesis, Drosophila larval neuroblasts were generated containing mutations in various genes that control asymmetric cell division and then their proliferative potential was assayed after transplantation into adult hosts. It was found that larval brain tissue carrying neuroblasts with mutations in raps (also called pins), mira, numb or pros grew to more than 100 times their initial size, invading other tissues and killing the hosts in 2 weeks. These tumors became immortal and can be retransplanted into new hosts for years. Six weeks after the first implantation, genome instability and centrosome alterations, two traits of malignant carcinomas, appeared in these tumors. Increasing evidence suggests that some tumors may be of stem cell origin. These results show that loss of function of any of several genes that control the fate of a stem cell's daughters may result in hyperproliferation, triggering a chain of events that subverts cell homeostasis in a general sense and leads to cancer (Caussinus, 2005).

Malignant transformation and loss of cell polarity are tightly correlated in human carcinomas. Likewise, Drosophila larval tissues with mutations in dlg1, l(2)gl or scrib have impaired apicobasal polarity and neoplastic growth in the imaginal epithelia and nervous system. There are several hypotheses to explain how loss of polarity contributes to neoplastic transformation. Most of them involve models in which changes in cellular architecture impinge directly on the cell cycle either by inhibiting signals that restrain cell proliferation or by enhancing mitogenic pathways. An alternative hypothesis is that loss of polarity in stem cells that divide asymmetrically impairs the mechanisms that specify the fate of the resulting daughter cells. If these daughter cells are unable to follow their normal developmental program, they may not respond to the mechanisms that control proliferation in the wild-type lineage (Caussinus, 2005).

Drosophila neuroblasts are stem cells whose asymmetric cell-division machinery is fairly well characterized and thus provide a good model to test this hypothesis. In the embryo, Insc integrates into the apical cortex of two neuroblast protein complexes, Baz-DmPar6-aPKC and Gialpha-Raps, by associating with Baz and Raps. These two complexes mediate the basal localization of Mira and Pon and their interacting proteins, Pros and Numb, whose segregation into the ganglion mother cell (GMC) is required for the unequal fate of the two neuroblast daughter cells. The basal localization of Mira and Pros, as well as the spindle orientation and asymmetry of daughter-cell sizes, require the functions provided by dlg1, l(2)gl and scrib. Larval neuroblasts originate from quiescent embryonic neuroblasts, and their asymmetric division seems to be controlled by the same molecular complexes, although minor differences have been reported (Caussinus, 2005).

To assess the effect of disrupted stem-cell asymmetric division on cell proliferation, larval neuroblasts were generated with mutations in aPKC, raps, mira, pros or numb and their proliferation potential was assayed after transplantation into adult hosts. No substantial growth of 101 pieces of wild-type larval brains were observed 2 weeks after transplantation. Similar results were observed for 109 implants that carried homozygous aPKCk06403 clones, none of which grew to any noticeable extent. In contrast, pieces of brains from rapsP89/raps P62 larvae or from larvae carrying homozygous numb03235, miraZZ176 or pros 17 clones grew to more than 100 times their original size, severely damaging and displacing the host's organs in the abdomen. Of the 103 flies studied in detail, 92% had one or more small tumor colonies derived from the implanted tissue but located at a long distance from the point of injection. The efficiency of tumor development ranged from 8% for numb03235 clones to 20% for rapsP89/rapsP62 tissue (Caussinus, 2005).

To assess further the growth potential of these tumors, they were cut into pieces and reimplanted into new hosts. More than 90% of these flies developed a tumor, even when they were implanted with numb 03235 tissue that had initially developed tumors in only 8% of implanted adults. This result suggests that the growing tumor mass adapts itself very rapidly to its new environment. Pieces of brain lobes from 9- to 12-d-old homozygous brat k06028 and l(3)mbt ts1 larvae, in which overgrowth was already apparent, developed tumors in 91% and 58%, respectively, of the implanted hosts (Caussinus, 2005).

All the tumors described here have been maintained in the laboratory, some for more than 2 years. This shows that the transformed cells became immortal and can proliferate without end, in contrast to cells of wild-type imaginal discs implanted into adult hosts, which remain alive after years of culture but very rarely proliferate. Among the established cell lines, substantial differences were observed in speed of growth, host lifespan or frequency or average number of additional tumor colonies, that could be attributed to the mutant background from which the tumors originated. Using the same criteria, these tumors were indistinguishable from dlg1, l(2)gl and scrib neuroblastomas (Caussinus, 2005).

Attempts were made to determine the kinds of cells that could be found in these tumors. Using green fluorescent protein as a clonal marker, it was observed that in tumors derived from tissue carrying numb 03235, miraZZ176 or pros17 clones induced by mitotic recombination, neither the wild-type twin nor the heterozygous background cells were able to proliferate upon implantation and were lost within 2 weeks. These cells accounted for most of the implanted mass, and so their inability to hyperproliferate provided a valuable internal control to substantiate the conclusion that tumor growth in this assay required the loss of the genes under study and was not just the result of dissection and transplantation into adult hosts. It also showed that the tumor growth induced by the loss of function of these genes was cell-autonomous (Caussinus, 2005).

Immunofluorescence staining for cell-specific markers identified the neuroblasts as relatively large cells, 8-12 microm in diameter, that expressed Mira. In miraZZ176 tumors, neuroblasts were identified by the expression of Wor. Ganglion cells were identified as small cells, 4-6 microm in diameter, that did not express Mira but did express Pros or, in pros 17-derived tumors, Numb. The intermediately sized cells that did not express Pros, some of which showed weak Mira staining, might be GMCs. Neuroblasts accounted for most of the mitotic activity observed in these tumors (86%). Daughter-cell size and Mira segregation during mitosis were symmetric in neuroblasts derived from rapsP89/rapsP62 tumors but asymmetric in those derived from numb03235 and pros 17 tumors. Daughter-cell size was also asymmetric in neuroblasts from miraZZ176 tumors (Caussinus, 2005).

Neither neuroblasts nor ganglion cells were markedly diluted or over-represented as the tumors aged from host to host. Therefore, like l(2)gl and dlg1 tumors, the tumors derived from numb03235, miraZZ176, pros17 and raps P89/rapsP62 were neuroblastomas that resulted from the uncontrolled division of neuroblast stem cells and were largely composed of the undifferentiated cell types that belong to this lineage. The mechanism by which these tumors grew is not understood, but it must account for the observed continuous expansion of both the neuroblast and the ganglion cell populations. One plausible mechanism could be a low frequency of neuroblast divisions that generate two neuroblast daughters. Real-time analysis of cell proliferation in these tumors may provide an answer to this issue (Caussinus, 2005).

In most solid human tumors, malignancy is very often correlated with genome instability, which is thought to contribute to multistage carcinogenesis. As in most animal cells, the frequency of natural cases of genome instability in wild-type Drosophila neuroblasts and GMCs is low (less than 10-3). This is also the case in numb03235, miraZZ176, pros 17 and rapsP89/raps P62 tumors shortly after transplantation. In 40-d-old tumors, however, 10%-15% of the cells presented different kinds of karyotype defects. Of the 340 karyotypes obtained from numb, mira, pros and raps tumors, 62% included segmental aneuploid; 9% were monosomic, trisomic or tetrasomic with respect to one or more chromosomes; 6% were triploid or tetraploid; and the remaining 23% included cells that could not be karyotyped owing to very high levels of ploidy, chromosome fragmentation or chromosome condensation (Caussinus, 2005).

The karyotypes obtained from cells in a single tumor were as different from one another as they were from the karyotypes of cells from other tumors, and none of the tumor lines that were established presented a distinct set of chromosome aberrations. Therefore, no substantial differences were observed attributable to the mutant condition that originated the tumor. In most tumor lines, the frequency of cells that contained abnormal karyotypes did not change noticeably over time, with one exception: 3 months after the first implantation, genome instability affected more than 95% of the cells in mirTF, a tumor line derived from miraZZ176. The absence or very low incidence of genome instability during the first round of implantation suggests that genome instability did not cause tumor formation in these tumor lines. But the onset of genome instability correlates well with a marked increase in the frequency of hosts that developed a tumor in later transplantations. Therefore, the possible contribution of genome instability to the evolution of these tumors remains to be assessed. Genome instability has also been reported in l(2)gl neuroblastomas (Caussinus, 2005).

In mammalian carcinomas, genome instability is tightly correlated with severe alterations of the centrosome cycle that affect the number of centrosomes per cell as well as centrosome size and shape. Supernumerary centrosomes can result in multipolar spindles and contribute to the generation of aneuploidy. Like the DNA cycle, the centrosome cycle is tightly controlled in wild-type neuroblasts, so that cells that have an abnormal number of centrosomes are exceptionally rare in wild-type tissue. This was not the case in numb03235, mira ZZ176, pros17 or raps P89/rapsP62 tumors: forty days after the first implantation, 15%-20% of those cells had more than two centrosomes. Some of these centrosomes were irregularly shaped, and their size range was much wider than that of control cells. A fraction of these could be centriole-less aggregates of pericentriolar material. The cells that had supernumerary centrosomes seemed to be hyperploid (Caussinus, 2005).

None of the mutant conditions from which these tumors originated has been reported to affect chromosome segregation or the centrosome cycle, which were both unaffected in early tumors. In addition, the cells of wild-type imaginal discs that have been kept for years in adult hosts maintain a stable genome and can differentiate into adult structures. Therefore, genome instability and impaired centrosome cycles in numb 03235, miraZZ176, pros17 and rapsP89/rapsP62 tumors cannot be considered a consequence of the mutant background or long-term exposure to the adult abdomen environment. Rather, the onset of genome instability and centrosome alterations suggests that once the mechanisms that control cell proliferation have been over-ridden, hyperproliferation triggers a chain of events that subverts cell homeostasis in a very general sense, including the DNA and centrosome cycles (Caussinus, 2005).

In summary, neoplastic transformation of Drosophila larval neuroblasts can be triggered by perturbing several of the functions that mediate asymmetric stem-cell division. In terms of growth rate, cell types, metastatic activity and extent of genome and centrosome instability, the resulting tumors are essentially indistinguishable from one another, regardless of the mutant from which they derive. The main conclusion that can be drawn from these data is that these tumors might have a common etiology: perturbation of neuroblast polarity and the resulting impairment of cell-fate determination. This argument is strengthened by the case of the homeobox-containing transcription factor Pros, which lies downstream of the other genes required for neuroblast asymmetric division (Caussinus, 2005).

The tumors in this study are practically indistinguishable from the neuroblastomas that arise in adults implanted with pieces of dlg1, l(2)gl or scrib mutant larval brains. Because these three neoplastic tumor suppressors are required for multiple aspects of neuroblast asymmetric cell division, including the basal localization of Mira, Numb and Pros, mislocalization of these proteins might explain, at least partially, the uncontrolled cell proliferation produced by loss of dlg1, l(2)gl or scrib function in larval neuroblasts (Caussinus, 2005).

The unequal segregation of cell-fate determinants resulting from asymmetric cell division is a fundamental mechanism for generating cellular diversity during development, organ homeostasis and repair. If impaired segregation of cell-fate determinants can cause the hyperproliferation of larval neuroblasts of Drosophila, it may similarly affect tissue stem cells in other species. At the moment, however, any parallel to stem-cell models of human cancer remains purely speculative. Consistent with this hypothesis, the inactivation of both Numb and Numb-like in the mouse dorsal forebrain leads to impaired neuronal differentiation, hyperproliferation of neural progenitors and delayed cell-cycle exit. In addition, loss of Lgl1 (also called Mlgl or Hugl), one of the two L(2)gl homologs in the mouse, results in a failure to asymmetrically localize Numb and leads to severe brain dysplasia (Caussinus, 2005).

In most human tumors, the identity of the first carcinogenic cell remains elusive. Indirect but growing evidence suggests that in some cases, the founders may be stem cells. Stem cells are self-renewing, have limitless replicative potential and produce differentiating cells, three features found in many cancers. Carcinomas occur in tissues that are maintained by a continuous supply of differentiating daughter cells originating from stem-cell division. Moreover, some of the signaling pathways that control stem-cell self-renewal, like the Notch, Wnt-ß-catenin and Hedgehog pathways, are known to have a role in carcinogenesis in these tissues. The results show that inactivation of any of several molecular mechanisms that control the asymmetry of the segregation of cell-fate determinants during stem-cell division may result in hyperproliferation of the stem-cell compartment and could contribute to cancer (Caussinus, 2005).

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

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