numb mutants have lost most of the peripheral nervous system. Neural cells are transformed into support cells (Uemura, 1989). The IIb lineage is transformed into a IIa lineage, that is, into a hair and socket cell progenitor. Thus numb is required for the neuron and sheath fate. Induction of numb inappropriately in the IIa lineage creates a IIb phenotype, that is, a neuron and sheath fate (Rhyu, 1994).
The embryonic peripheral nervous system of Drosophila contains two main types of sensory neurons: type I neurons, which innervate external sense organs and chordotonal organs, and type II multidendritic neurons. Type I neurons are characterized by their single dendrite whose distal part is a modified cilium. In contrast, type II neurons possess several dendrites lacking ciliated structures. Type I neurons are associated with accessory cells (socket and shaft cells, known respectively as tormogen and trichogen cells) that form the non-neuronal part of the sense organ. Type II neurons are not associated with accessory cells. In Notch mutant embryos, the type I neurons are missing while type II neurons are produced in excess, indicating that the type I/type II choice relies on Notch-mediated cell communication. It is proposed that a protoprecursor cell exists called p0, having both external sense organ and multidendritic cell potentiality. In the absence of Notch the two daughters of the protoprecursor will adopt the same fate, the multidendritic fate (Vervoort, 1997).
Both type I and type II neurons are absent in numb mutant embryos and also after the ubiquitous expression of tramtrack. This indicates that the activity of numb and the absence of tramtrack are required to produce both external sense organ and multidendritic neural fates. Numb is thought to repress tramtrack, a gene that promotes non-neuronal verses neuronal fate. The analysis of string mutant embryos reveals that when the precursors are unable to divide they differentiate mostly into type II neurons, indicating that the type II is the default neuronal fate. A new mutant phenotype has been described, called X1. It prevents the acquisition of external sense cell fate. In these mutants, ASC-dependent neurons are converted into type II neurons, providing evidence for the existence of one or more genes required for maintaining the alternative (type I) fate (Vervoort, 1997).
A new hypomorphic numb mutant not only displays a double-socket phenotype, due to a hair cell to socket cell transformation, but also a double-sheath phenotype, due to a neuron to sheath cell transformation. This provides direct evidence that numb functions in the neuron/sheath cell lineage as well. These results, together with the observation from immunofluorescence analysis that Numb forms a crescent in the dividing IIa and IIb cells suggest that asymmetric localization of Numb is important for the cell fate determination in all three asymmetric cell divisions in the sensory organ lineage. In the hair/socket cell lineage, but not the neuron/sheath cell lineage, a Suppressor of Hairless mutation acts as a dominant suppressor of numb mutations whereas Hairless mutations act as enhancers of numb. Therefore Su(H) and Hairless are required for determining the hair/socket cell lineage but not the neuron/sheath cell lineage. The neuron/sheath cell lineage is not affected by the loss or increase of Su(H) activity. Epistasis analysis indicates that Suppressor of Hairless acts downstream of numb. Results from in vitro binding analysis show a physical interaction between Numb and Hairless and suggest that the genetic interaction between numb and Hairless may occur through direct protein-protein interaction. These studies reveal that Suppressor of Hairless is required for only a subset of the asymmetric divisions that depend on the function of numb and Notch. Since numb and Notch are involved in an antagonistic manner in all three asymmetric divisions of the sensory organ precursor lineage, and Su(H) is involved in only a subset of these, there must exist a Su(H)-idependent Notch signaling that has not yet been well characterized (Wang, 1997).
The dorsal bipolar dendritic (dbd) lineage is the most simple and well-characterized asymmetric cell division in the PNS: the precursor cell divides asymmetrically to give rise to an md neuron and a glial cell. The dbd neuron is often duplicated in sanpodo embryos. Glial markers show no glia associated with the duplicated dbd neurons. Hence the sibling cell is transformed into an md neuron. This transformation is the opposite of the numb phenotype, which consists of two glial cells instead of two dbd neurons. This suggests that in spdo mutants, for those md neurons that have a lineage-related sibling, the non-neuronal cells as well as the neuronal cell adopt the neural fate. Staining with an md-specific marker reveals another feature of spdo mutants. Es neurons are programmed to become md neurons in spdo mutants. This observation is particularly interesting because the same phenotype occurs in Notch mutants, suggesting that Notch signaling is impaired in spdo mutants. These observations suggest that spdo is a neurogenic gene and when mutated results in too many neurons, the hallmark of the neurogenic phenotype (Dye, 1998).
Spdo functions downstream of Numb, a protein known to be involved in the Notch pathway. Within the es lineage, numb function is required in both the SOPIIb and its daughter cells to correctly specify cell fate. spdo function is also required within the es lineage, but in a manner just the opposite of that in numb. To place spdo in the Numb/Notch pathway, an analysis of numb;spdo double mutants was undertaken. spdo function is likely downstream of numb, because numb;spdo embryos have many more neurons than those that only lack numb. Alternatively, Spdo functions as an antagonist of Numb. Based on the epistatic interaction studies, it is proposed that the transformation of SOPIIb into SOPIIa brought about by loss of numb is often blocked in the absence of spdo. Because loss of Numb in the SOPIIb leads to increased Notch signaling, Notch activation and/or signaling may require Spdo function. A role for Spdo in Notch signaling is supported by the finding that reducing Notch protein function at the time of SOPIIa division decreases the number of es glial cells and increases the number of es neurons. The es neurons express md-specific markers in spdo mutants, as is observed in Notch mutants. It is therefore unlikely that Spdo functions in an independent pathway, although at the present time this possibility can not be formally excluded (Dye, 1998). Numb is known to bind to the intracellular domain of Notch and antagonize Notch signaling but, with the exception of the dMP2/vMP2 neurons, it has not been reported to play a role in sibling neuron development in the CNS. However, due to the widespread role of Notch in specifying asymmetric sibling neuron identity, the CNS function of numb was re-investigated. Sibling neuron development was assayed using four different numb alleles and a deficiency that uncovered the numb locus. In embryos homozygous for the strongest numb allele (nb 2 ), an equalization of sibling neuron phenotype is observed for all siblings tested, with the exception of aCC/pCC. RP2 is transformed into RP2sib approximately 50% of the time; three Usibs are transformed into three U neurons; and dMP2 is transformed into vMP2. The numb phenotypes for RP2, Usib and dMP2 neurons are reciprocal to those observed in spdo, Delta, Notch or mam embryos. This is consistent with studies showing that Numb antagonizes Notch function and extends this interaction to a diverse array of CNS sibling neurons. In addition, a strong decrease in the number of Eve + EL neurons is observed in numb mutant embryos. There is clear evidence of maternal numb function during CNS development, which may account for the lack of a fully penetrant numb sibling neuron phenotype. Changing the dose of maternal numb product directly affects CNS development and suggests that numb may have earlier CNS functions in addition to sibling neuron specification (Skeath, 1998).
spdo and numb have opposite sibling neuron phenotypes and so the epistatic relationship between the two genes was determined by examining the phenotype of a numb;spdo double mutant. The numb;spdo double mutant phenotype is identical to embryos lacking spdo alone. Thus, spdo is genetically downstream of numb, just as has been observed for Notch pathway mutations in other lineages. sanpodo and numb exhibit dosage-sensitive interactions, as gene products that act in the same biochemical pathway often do. The sibling neuron phenotype in numb embryos is sensitive to the level of spdo. For example, homozygous nb 2 embryos show a loss of EL and RP2 neurons, but reducing the dosage of spdo by one-half in nb 2 embryos leads to a recovery of Eve + EL and RP2 neurons. Thus, halving the dosage of spdo strongly suppresses the numb CNS phenotype. These results show that the numb phenotype is extremely sensitive to the dosage of spdo, consistent with the two proteins acting in the same biochemical pathway (Skeath, 1998).
In numb embryos, there is a striking decrease in the number of Eve + EL neurons. Notch, Delta, mam and spdo single mutants do not alter the number of Eve + EL neurons. Importantly, numb;spdo double mutant embryos show a complete rescue of Eve + EL neurons , suggesting that Numb acts to prevent Spdo-mediated downregulation of eve expression (i.e. in the absence of Spdo, the loss of Numb is irrelevant). These data are consistent with a model in which Numb blocks Notch/Spdo-mediated downregulation of eve in the neurons of the EL lineage (Skeath, 1998).
WASp mutant phenotypes generally resemble those described for positive mediators of N signaling, whereas mutations in the N antagonist numb are distinct and opposite in character. Thus, adult sensory organ development in the absence of numb function leads to formation of multiple sockets, since both progeny of the pI division in this case assume a pIIa fate, and the subsequent division is characterized by shaft-to-socket transformations. The opposite effects on cell fate provide an opportunity to determine an epistatic relationship between WASp and numb. This issue was examined by producing clones of numb cells in a WASp mutant background. A powerful system for producing mutant clones in derivatives of the eye imaginal disc that includes the cuticle of the adult head capsule, has recently been described. This system has been successfully adapted for the study of numb and other regulators of sensory organ formation. Using this adaptation, large numb head clones, in which the multiple socket phenotype characteristic of numb was observed throughout the head cuticle, were consistently produced. When such clones are made in animals hemizygous for WASp alleles, multiple sockets are rarely observed, while the WASp smooth head cuticle phenotype predominates. These observations demonstrate that WASp is epistatic to numb, i.e., a requirement for WASp during adult sensory organ formation persists even in the absence of numb gene function. This finding is consistent with the normal segregation of Numb and Pon-GFP in WASp mutants, with both observations suggesting that WASp is not involved in localization of asymmetrically localized components, but rather provides a function further downstream (Ben-Yaacov, 2000).
During asymmetric cell division, protein determinants are segregated into one of the two daughter cells. The Numb protein acts as a segregating determinant during both mouse and Drosophila development. In flies, Numb localizes asymmetrically and is required for cell-fate specification in the central and peripheral nervous systems, as well as during muscle and heart development. Whether its asymmetric segregation is important to the performance of these functions is not firmly established. This study demonstrates that Numb acts both in a localization-dependent and in a localization-independent manner. numb mutants were generated that affect only the asymmetric localization of the protein during mitosis. Asymmetric segregation of Numb into one of the two daughter cells is absolutely essential for cell-fate specification in the Drosophila peripheral nervous system. Numb localization is also essential in MP2 neuroblasts in the central nervous system and during muscle development. Surprisingly, in dividing ganglion mother cells or during heart development, Numb function is independent of its ability to segregate asymmetrically in mitosis. These results suggest that two classes of asymmetric cell division exist, each with different requirements for asymmetric inheritance of cell-fate determinants (Bhalerao, 2005).
The mutant numbS52F is defective in asymmetric localization of Numb. Previously, mutations in bazooka and inscuteable or numb overexpression have been used to analyze the importance of Numb localization during cell division. In all cases, the phenotypes observed are different from the numbS52F phenotype. In these mutants, localization of other cell-fate determinants is also affected, causing phenotypes not solely as a result of Numb mislocalization. Other biological processes like epithelial polarity, spindle positioning, and cell size are affected in bazooka and inscuteable mutants and could be responsible for the observed phenotypes in these mutants. Numb localization is also inhibited upon overexpression of the protein, presumably as a result of saturation of the localization machinery. Upon overexpression, Numb is segregated into both daughter cells that then adopt the fate of the daughter that normally inherits numb. In numbS52F mutants, however, loss-of-function phenotypes were observed in most of the lineages analyzed. Either numbS52F is a hypomorph that only partially retains its ability to suppress Notch, or, alternatively, asymmetric localization is crucial for Numb to act as a cell-fate determinant. The second possibility is favored for several reasons: (1) NumbS52F completely retains its ability to bind Sanpodo and α-Adaptin; (2) numbS52F is as potent as the wild-type protein in inducing cell-fate transformations upon overexpression; (3) NumbS52F is fully functional in inhibiting Notch when cell division is blocked in Cyclin A mutants, and (4) numbS52F cannot be consistently placed into an allelic series. In the P2 lineage, for example, numbS52F mutants are completely wild-type, whereas in the SOP lineage, they behave like null alleles. It is therefore concluded that NumbS52F is specific for asymmetric localization and that asymmetric localization is essential for Numb to act as a cell-fate determinant during one class of asymmetric divisions that occur in the SOP cells, MP2 neuroblasts, and muscle precursors (Bhalerao, 2005).
Two classes of asymmetric cell divisions can be distinguished. In class I divisions, Numb localization is essential for the two daughter cells to assume different fates. In these divisions, one cell takes on a different fate only when Numb is concentrated in this cell. During class II divisions, however, Numb acts independently of its asymmetric segregation. In such divisions, which occur in GMCs and during heart development, one possibility would be that Numb functions via downstream effectors other than those involved in class I divisions. However, the Notch/Delta pathway is the downstream target of Numb function in these lineages as well. Sanpodo, another gene acting downstream of Numb, is involved not only in divisions of the SOP, MP2, neuroblast, and muscle precursors but also during divisions of the P2 and GMC4-2a (Bhalerao, 2005).
What could be the reason for the asymmetric outcome of these divisions? Other segregating determinants could exist that act redundantly with Numb during class II divisions but may not be present during class I divisions. Neuralized would be a candidate because it is the only other segregating determinant known to act on the Notch/Delta system. However, Neuralized has been shown to be essential only for asymmetric cell division in ES organs. Because these divisions belong to class I, Neuralized is unlikely to be responsible for the asymmetric outcome of class II divisions. Alternatively, feedback loops in the Notch/Delta pathway could amplify small, random differences in Notch activity to establish distinct fates even when Numb concentrations are the same. Finally, Numb could act redundantly with polarized extracellular signals that act differently on the two daughter cells. (Bhalerao, 2005).
It is proposed that Numb can act in both a localization-dependent and -independent way. The results are of particular importance for vertebrates, where both localizing and nonlocalizing homologs of Numb are involved in nervous-system development. It is conceivable that localization-dependent and localization-independent functions have separated into two distinct homologs during evolution. The existing data do not allow distinguishing whether vertebrate neural-precursor divisions are of class I or class II. However, Serine 52 is conserved in mouse Numb, and its targeted mutation could be used to address the relevance of Numb localization in vertebrates (Bhalerao, 2005).
Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical
Notch pathway in the process of lateral inhibition. The subsequent cell type specifications rely on distinct subsets of Notch
signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the
Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act
upon Su(H), influences strongly the entire cell lineage, demonstrating that it functions through targets other than Su(H) within the inner
lineage. Thereby, Hairless and Numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications
involve different sets of mediators depending on the cell type considered (Nagel, 2000).
Earlier work has identified numb as an important factor
during binary cell fate decisions within the sensory organ
lineage, where it is partitioned into those cells in which it
acts as intrinsic inhibitor of the Notch signal. Both loss and gain of numb activity causes cell transformations very similar to H except for
the selection of the SOP during lateral inhibition, which is
controlled by H and not by numb. It is noted, however, that phenotypes
are always much more penetrant when H is involved in
comparison to numb. H mutations act as dominant enhancers of numb mutations, and the two proteins might physically interact with each other. Both
activities are required for normal bristle development
because loss of function of either gene gives a similar
phenotype regarding cell type specification. To what degree
do these activities overlap? Close inspection of the phenotypes shows that numb has a very strong influence on the
pIIa/pIIb fate selection but less on the subsequent binary
decisions because cell type transformations are always
partial. Although this might be a quantitative difference, it is noted
that H activity is strictly required to guard both shaft cell and
neuron from a faulty Notch signal and that
complete cell type transformations are observed as a consequence of H activity loss. Furthermore, H activity is also
required during the process of lateral inhibition, suggesting
that H is the more general antagonist in Notch-dependent
processes (Nagel, 2000).
Both in loss of function and gain of
function combinations numb is epistatic to
Su(H) within the pII cells, indicating that Su(H) acts downstream of numb. This is in agreement with a model, whereby
numb antagonizes Notch signaling through direct interference with the Notch receptor. Within the
pIIa progeny, however, Su(H) can override the inhibiting
activity of ectopic numb protein. This interference might again be direct, because preliminary evidence from yeast interaction trap experiments shows that
Numb and Su(H) physically interact.
Furthermore, by inhibiting Notch signaling, numb might
indirectly modulate the levels of Su(H) transcriptional activity. Thus, the conflicting epistasis data
might reveal once more different sensitivities of the sensory
organ cell lineage regarding Notch signaling, especially the
preference of the pIIb over the pIIa fate (Nagel, 2000).
During the development of mechanosensory organs,
Notch is required at two distinct steps: the singling out of
the sensory organ precursor, SOP, and the correct specification of cell fates within the sensory organ lineage, SOL.
Apparently, different subsets of Notch signaling components are used for these two processes. Whereas SOP selection in the process of lateral
inhibition requires the 'classical' battery of Notch signaling
components, namely Su(H), dx, mam, E(spl) bHLH and H,
the subsequent asymmetric cell divisions require only
certain Notch components plus the intrinsic activity of
numb. Numb plays a major role in the distinction between
the pII siblings. In the pIIa progeny, socket cell fate is
enforced with the help of Su(H) and, to a lesser degree,
dx, and the role of H is to protect the shaft from this fate. In
the sub-epidermal progeny, Notch signaling determines
sheath cell fate, promoted to some degree by dx and
Su(H). However, since neither of the components, Su(H), dx nor E(spl) bHLH are strictly required for the selection of
thecogen fate, the Notch signal is transduced by other
factor(s), X. The role of mam in this process is as yet undecided, since the mutant cell clones are rather uninformative and
appropriate overexpression constructs are unavailable. The
neuron has to be protected from the Notch signal, and both
numb and H play a pivotal role in this process. Apparently,
the target of numb is the Notch receptor itself. It is not clear whether H acts at the same level, or whether it acts on a different target, maybe
directly involving the presumptive signal transducer X. Although both mam and dx might be targets of H, no physical interactions were observed in the yeast interaction trap
assay.
Overall, H represents a key player in antagonizing the Notch
signal and thus assures, that in the end all four different cell
types of the mechano-sensory organ arise (Nagel, 2000).
A summary of Notch signaling during mechano-sensory organ development is presented. Notch signaling is required in the entire cell lineage, as is the
antagonist Hairless. Whereas the lateral inhibition process uses the classical
battery of Notch signaling components, the subsequent binary cell fate
specifications rely only on a subset of these components and involve in
addition the intrinsic antagonist Numb. In a first step SOP is singled out by lateral
inhibition from a proneural cluster, protected through the activity of H. The surrounding cells are forced by the SOP into epidermal
fate through the activation of the Notch receptor, implemented with the help
of Su(H), dx, mam and E(spl) bHLH proteins (classical pathway). In a second step,
Notch signal, promoted by Su(H) and dx, forces one SOP daughter cell into pIIa fate from which the pIIb cell is protected by the antagonists
Numb and H. The pIIb gives rise to the pIIIb and a glial cell. In a third step, the
progeny of pIIa are socket and shaft cell. The socket cell receives the Notch
signal via Su(H) and dx, the effector genes are unknown. The
shaft cell is protected by H and numb from the Notch signal. In a fourth step, the
progeny of the pIIIb are sheath cell and neuron. The sheath cell receives
a Notch signal promoted by unknown factor X, whereas the neuron is
protected by H and numb (Nagel, 2000).
By using gain-of-function mutations it has been proposed
that vertebrate Notch promotes the glial fate. In
vivo glial cells are produced at the expense of neurons
in the peripheral nervous system of flies lacking Notch and
that constitutively activated Notch produces the opposite
phenotype. Notch acts as a genetic switch between neuronal
and glial fates by negatively regulating
glial cells missing, the gene required in the glial precursor
to induce gliogenesis. Moreover, Notch represses
neurogenesis or gliogenesis, depending on the sensory
organ type. Numb, which is asymmetrically localized in the
multipotent cell that activates the glial cell fate, inducing
glial cells at the expense of neurons. Thus, a cell-autonomous
mechanism inhibits Notch signaling (Van De Bor, 2001).
Strikingly, N seems to act in opposite directions
in fly and some vertebrate peripheral glial cells. Indeed,
gain-of-function N mutations promote differentiation of
Müller, radial and Schwann glial cells. Two possible
explanations can account for these results: (1) the genetic
switch between neuronal and glial fates has different
requirements in flies and vertebrates; (2) the role of N
depends on the subtype of glial cell. The analysis of other
classes of fly glial cells will help elucidate this point.
Preliminary analyses on the embryonic CNS
suggest that the response of central glial cells to N depends
on the subtype. The observation that
oligodendrocyte differentiation, like fly peripheral glial cells,
is repressed by N, also argues in favor of the second
hypothesis (Van De Bor, 2001).
One of the most striking results is that repression of the N
pathway throughout the development of the sensory organ
(obtained by N loss-of-function mutations or by Numb
overexpression) leads to sensory organs composed of six glial
cells. The competence to adopt
the glial fate is restricted to some cells of the sensory organ
lineage; the strongest phenotype observed upon overexpression
of gcm throughout the lineage is the differentiation of a
sensory organ composed of five cells, three of which are Repo-positive. Thus, gcm is not sufficient
to induce a IIa into IIb transformation. This indicates that the
pathway mediated by N and Numb affects the competence of
sensory organ cells to adopt the glial fate. In molecular terms,
this implies the control of expression of gcm regulators,
positive co-factors and/or repressors (Van De Bor, 2001).
An essential feature of the organization and function of the vertebrate and insect olfactory systems is the generation of a variety of olfactory receptor neurons (ORNs) that have different specificities in regard to both odorant receptor expression and axonal targeting. Yet the underlying mechanisms that generate this neuronal diversity remain elusive. This study demonstrates that the Notch signal is involved in the diversification of ORNs in Drosophila. A systematic clonal analysis showed that a cluster of ORNs housed in each sensillum were differentiated into two classes, depending on the level of Notch activity in their sibling precursors. Notably, ORNs of different classes segregated their axonal projections into distinct domains in the antennal lobes. In addition, both the odorant receptor expression and the axonal targeting of ORNs were specified according to their Notch-mediated identities. Thus, Notch signaling contributes to the diversification of ORNs, thereby regulating multiple developmental events that establish the olfactory map in Drosophila (Endo, 2007).
In the Drosophila olfactory system there are about 50 different types of ORNs, each of which is characterized by the expression of a specific odorant receptor. Yet little is known about the molecular mechanisms underlying the generation of this diverse array of ORNs. By analyzing the projection patterns of ORNs and the cell lineage of the olfactory organ in mastermind (mam) and numb (nb) clones, it was shown that differential Notch activity in the two sibling ORN precursors leads to ORN diversification and thereby regulates multiple developmental events in the organization of the olfactory system (Endo, 2007).
There are several types of sensory organs in Drosophila, and the component cells in each organ are generally derived from a single SOP. All of the neuronal and non-neuronal cells that constitute the olfactory sensilla are derived from three precursors that arise before early pupal stages. MARCM-clone analyses was used to examine developmental events in the olfactory sensory lineage that precede these stages, and it was found that all the cells in the olfactory sensillum are generated from a single SOP that arises as early as 30 h BPF. Therefore, the olfactory sensilla seem to adopt developmental mechanisms similar to those seen in other sensory organs. However, although only one or two neurons are produced in the canonical, or ancestral, peripheral nervous system lineage, 1 to 4 ORNs are generated in the lineages of the olfactory sensilla. These findings reveal that this difference in the number of generated neurons is a consequence of the generation of more inner-cell precursors in the olfactory sensilla lineage. Interestingly, the gustatory sensillum is also innervated by 2 to 4 gustatory receptor neurons. It is proposed that the gustatory and olfactory systems share similar developmental mechanisms for producing a variety of neurons (Endo, 2007).
Although Notch signaling asymmetrically differentiates the ORN precursors, lineage analysis of nb clones in the antenna suggests that Notch does not diversify the Notch-ON-class ORN siblings (referring to a class with activate Notch signaling) on their generation from a common precursor. This notion is consistent with the results of systematic mam- and nb-clone analyses in the antennal lobe, where most ORNs are categorized into either Notch-ON or Notch-OFF classes. Therefore, the Notch-ON-class ORN siblings seem to be diversified by Notch-independent mechanisms. The asymmetric distribution of other proteins may mediate this process in ORN siblings. Thus, the olfactory sensory lineages use both Notch-dependent and Notch-independent mechanisms to generate a variety of neurons as well as non-neuronal cells (Endo, 2007).
The data in this study indicate that the genetic information that specifies the characteristics of the olfactory organ, such as sensilla and ORN type, is derived from SOPs in the developing antennal disc. Part of the information in SOPs is possibly encoded by the proneural genes atonal and amos, because they are essential for the differentiation of SOPs that produce different types of sensilla. It is proposed that the identities of SOPs arise, in part, from positional information allocated along the axes of the disc. This hypothesis is supported by the observation that ORNs expressing different types of odorant receptors are roughly segregated into distinct domains that are distributed along the proximo-distal axis. Positional information along other axes is also likely to refine genetic information in SOPs, as evidenced by a correlation between the regional localization of sensilla types on the antenna and the patterned projections of the corresponding ORNs in the antennal lobe. The data of lineage analysis indicate that Notch signaling further diversifies the genetic information of SOPs to produce the outer and inner cell lineages, and subsequently differentiates ORN precursors to generate two Notch-mediated classes of ORNs. Importantly, Notch signaling diversifies not only the genetic information specific to each SOP, but also the information shared by a subset of SOPs, as evidenced by the observation that a subset of ORNs of a given Notch-mediated class share their axonal pathways, target domains or both in the antennal lobe. Thus, Notch signaling controls multiple aspects of ORN development that contribute to the organization and function of the olfactory circuit (Endo, 2007).
In the adult olfactory system, odor information is first represented as a spatial map of activated glomeruli in the antennal lobes. Recent optical recording studies in vertebrates have shown that distinct chemical classes activate distinct regions of glomeruli, forming a chemotopic map. In Drosophila, although the larval olfactory system has a glomerular organization, in which two distinct domains represent specific classes of chemicals, the chemotopic organization of glomeruli in the adult brain is less obvious than that in the fly larvae and vertebrates. The glomerular domains identified in this study may be correlated with the chemical properties of some odorants, but no specific chemical classes have been assigned to these domains, and there are no significant sequence similarities among odorant receptors expressed in ORNs that project to the same glomerular domains compared with those expressed in ORNs that project to distinct domains. One possible implication of this domain organization is that glomeruli are physically segregated, as is typically found in the posterior group, and the sensory information from ORNs is independently processed in distinct domains. Alternatively, these domains may evoke different behavioral responses. In the Drosophila gustatory system, the axonal targets of the gustatory receptor neurons are segregated by taste category: neurons that recognize sugars project to a different region from those that recognize noxious substances. Currently, little is known about the relationship between the olfactory circuitry and behavior. Further functional studies would be necessary to assess whether Notch-mediated glomerular domains function in olfactory coding and behavior (Endo, 2007).
A prominent feature of glial cells is their ability to migrate along axons to finally wrap and insulate them. In the embryonic Drosophila PNS, most glial cells are born in the CNS and have to migrate to reach their final destinations. To understand how migration of the peripheral glia is regulated, a genetic screen was conducted, looking for mutants that disrupt the normal glial pattern. Analysis of two of these mutants is described: Notch and numb. Complete loss of Notch function leads to an increase in the number of glial cells. Embryos hemizygous for the weak NotchB-8X allele display an irregular migration phenotype and mutant glial cells show an increased formation of filopodia-like structures. A similar phenotype occurs in embryos carrying the Notchts1 allele when shifted to the restrictive temperature during the glial cell migration phase, suggesting that Notch must be activated during glial migration. This is corroborated by the fact that cell-specific reduction of Notch activity in glial cells by directed numb expression also results in similar migration phenotypes. Since the glial migration phenotypes of Notch and numb mutants resemble each other, these data support a model where the precise temporal and quantitative regulation of Numb and Notch activity is not only required during fate decisions but also later during glial differentiation and migration (Edenfeld, 2007).
Within the peripheral nervous system of Drosophila axons are insulated from the surrounding hemolymph by only a few identified glial cells. Most of the peripheral glial cells are born in the CNS and migrate to stereotypic positions along the segmental nerves. To understand the genetic programs that govern the selection of migrating cells, the control of the normal migratory path and the final conversion into sessile cells, a genetic screen was conducted. Most of the mutants identified affected the fidelity of glial cell migration along the segmental nerves. Among this class of alleles Notch and numb were identified as being required for normal PNS glia migration (Edenfeld, 2007).
Notch was first extensively studied in Drosophila where it was found to be required for mediating lateral inhibition during the selection of neural progenitor cells. Since then, Notch has been shown to be involved in a large variety of cellular processes controlling cell proliferation, fate, survival and differentiation in organisms ranging from worms to man. Regulation of Notch activity appears to be an example for an archetype of signaling system. Upon binding of one of its most important ligands, Delta or Serrate (Jagged in vertebrates), the intracellular domain of Notch (Notchintra) is liberated by the large Presenilin protein complex that is equipped with transmembrane protease activity. Subsequently, Notchintra is able to access the nucleus where it interacts with Su(H) to activate the transcription of target genes. However, genetic evidence supports the notion that Notch can also activate signaling pathways independent of Su(H) (Edenfeld, 2007).
Notch signaling has frequently been implicated in different aspects of gliogenesis not only in Drosophila but also in different vertebrate glial lineages including Müller glia, radial glia, astrocytes, Schwann cells, and oligodendrocytes. Developing oligodendrocytes express the Notch1 receptor that is activated by neuronally expressed Jagged. Jagged-induced Notch activation conveys an inhibitory function in regulating the timing of axonal myelination. In contrast, the GPI-linked neuronal adhesion protein F3/contactin is capable of promoting Notch-dependent oligodendrocyte differentiation. Both receptor-ligand interactions lead to the generation of Notchintra but nevertheless cause different cellular responses, suggesting that the tight temporal and spatial regulation of Notch activity is of extreme importance (Edenfeld, 2007).
In vertebrates, axonally derived Notch ligands such as Jagged or F3/contactin appear in an excellent position to regulate glial growth and myelination. This study shows that in Drosophila Notch becomes activated in migrating peripheral glial cells. Numb generally inhibits Notch function. This study found Numb expression at early stages among the glial cells close to the nerve exits of the CNS. These cells represent the prospective subpopulation of glial cells that subsequently will migrate along the growing nerves out into the periphery. During the migration (from stage 12 onwards) Numb expression becomes down-regulated and thus correlates with the activation of Notch. Loss- or gain-of-function of numb leads to a disruption of peripheral glial cell migration comparable to the Notch mutant phenotype. This suggests that regulation of Notch activity in migrating glial cells depends on the precise control of the amounts and timing of Numb expression. Thus, this cell-autonomous mechanism appears to be responsible for the temporal control of glial migration (Edenfeld, 2007).
In previous reports, Notch expression has been found on neuronal membranes. However, since Notch is generally involved in direct cell-cell contact, it may be difficult to resolve Notch expression to axonal or glial cell membranes by standard light microscopic techniques. A function of Notch during cell migration was first implicated in the analysis of Delta-1 mutant mice, where neural crest migration phenotypes where found. In Drosophila, a disruption of Notch function impairs morphogenetic movements that lead to the formation of the proventriculus. This phenotype, however, may be due to fate changes, but the finding that the expression of short stop, which encodes a cytoskeletal linker protein of the Spectraplakin family, is regulated by Notch activity provides some evidence that Notch activity can influence cell structure and motility (Edenfeld, 2007 and references therein).
The Notch mutant phenotype is characterized by an increase in the number of filopodia on glial cells. This suggests that Notch activity can - directly or indirectly - influence the dynamics of the cytoskeleton. In the context of the dorsal closure of the Drosophila embryo, it has been shown that Notch may regulate JNK and RhoA signaling, which act on controlling actin dynamics. A direct function of Notch in influencing actin dynamics has also recently been described for a NIH 3T3 tissue culture model, where soluble forms of Delta and Jagged antagonize Notch signaling and in addition affect different cellular functions. Whereas soluble Jagged expression leads to an impaired cell motility, reduced F-actin stress fibers and focal adhesion site formation, the expression of soluble Delta does not interfere with the normal motility but reduces the amount of contactin phosphorylation (Edenfeld, 2007 and references therein).
Taken together, these data indicate that Notch acts in a cell-autonomous manner to instruct peripheral glial cell migration along the peripheral nerves. The precise temporal control and the quantitative adjustment of Notch activity is crucial for this migration. Neuronally expressed Delta presumably initiates Notch activation, however, the fine-tuning of Notch activity is mediated through regulating the levels or localization of Numb expression (Edenfeld, 2007).
A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition
within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of
equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the
other, the sibling cell, achaete expression is lost. In this paper
the mechanisms are charted by which achaete expression is differentially maintained in the tip cell lineage to stabilize cell fate. Initially,
wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells.
Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Then,
Notch signaling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete
expression is maintained only in the tip cell. In embryos mutant for Notch, tip cells segregate at the expense of sibling cells,
whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when
numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the
developing tubules of embryos lacking Wingless, after the critical period for tip cell allocation, reveals an additional
requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect
on the final tubule cell number (Wan, 2000).
The tip cell progenitor is selected from a group of competent
cells by lateral inhibition and is demarcated by the
continued expression of ac. Further extrinsic and intrinsic cues (Wg signaling and
the asymmetric distribution of Nb) operate to ensure the
continued expression of ac and so confirm tip cell potential.
The selection of cell fate from an equivalence group by
lateral inhibition alone relies on chance fluctuations in the
equilibrium of signaling between cells and therefore may
not be completely reliable. The activity of other genes, by
biasing lateral inhibition, serves to make the selection of
cells to specific fates more robust. Such mechanisms have
been shown to confirm cell fate in the PNS and
of the anchor cell in the nematode gonad.
The results presented here indicate that wg and nb are required for the
specification of the tip cell and sibling cell fate in the
Malpighian tubules. The activity of these two genes biases
the outcome of intercellular signaling at separate stages in
this process, resulting in the reliable allocation of tip and
sibling cell fates, suggesting that this distinction is important
to the development of the tubules. However, it is clear
that continued cell division in the tubules relies only on the
allocation of the tip cell progenitor and not on the differentiation
of fate between the tip cell's daughter cells, in which nb plays
an important role (Wan, 2000).
This result is surprising, since Nb is active where sister
cells of specific lineages are allocated to separate cell fates,
for example, in the PNS, in the CNS, and in myogenesis. Separation between sister cell
fates involves the maintenance of gene expression in one
sibling and its repression in the other, for example, of Kr,
eve, and S59 in sibling muscle founder cells.
This pattern is also seen in the tubules: ac, Kr, and Dl
continue to be expressed in the tip cell but are repressed in
the tip cell's sibling. In the neural and myogenic lineages the correct
allocation of sibling cell fates underpins normal tissue
differentiation. In the tubules, the
separate roles of the tip cells and their siblings are not yet known; they both
appear to be active in regulating cell proliferation but later
only the tip cell expresses genes characteristic of neuronal
cells. The later function of both cell
types has yet to be elucidated. By manipulating nb, Malpighian tubules that lack sibling cells can be generated, but these have two
tip cells or have two sibling cells but lack tip cells, thus
providing an important tool for this analysis (Wan, 2000).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.