Notch
Notch and neurogenesis: The PNS and bristle formation (part 2/2) Genetically, Hairless (H) acts as an antagonist of most neurogenic genes and may insulate neural
precursor cells from inhibition. H function is required for commitment to the bristle sensory organ
precursor (SOP) cell fate and for daughter cell fates. Using Notch gain-of-function alleles and
conditional expression of an activated Notch transgene, enhanced signaling in these cases produces
H-like loss-of-function phenotypes by suppressing bristle SOP cell specification or by causing an
H-like transformation of sensillum daughter cell fates. Furthermore, adults carrying Notch gain of
function and H alleles exhibit synergistic enhancement of mutant phenotypes. Over-expression of
an H+ transgene product suppresses virtually all phenotypes generated by Notch gain-of-function
genotypes. Phenotypes resulting from over-expression of the H+ transgene blocked by the
Notch gain-of-function products, indicating a balance between Notch and H activity. The results
suggest that H insulates SOP cells from inhibition and indicate that H activity is suppressed by
Notch signaling (Lyman, 1995).
To learn about the acquisition of neural fate by ectodermal cells, a very early sign of neural commitment in Drosophila has been analyzed, namely the specific accumulation of achaete-scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). An AS-C enhancer has been analyzed that directs expression specifically in SMCs. To delimit the sequences responsible for expression in SMCs, subfragments of a 3.7-kb fragment immediately upstream of scute were assayed for their ability to drive lacZ expression in wing discs. The necessary sequences are within a 356-bp fragment. This fragment specifically directed expression in SMCs. It also promotes expression in SMCs of other imaginal discs and of the embryonic PNS, but not in neuroectoderm neuroblasts. The SMC enhancer is shown to promote macrochaetae formation. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. The conserved sequences contain three E boxes: these are putative binding sites for bHLH proteins of the Achaete, Scute, and Daughterless type. The most proximal of the three is adjacent to an N box, a site that can be recognized by the E(spl)-C bHLH proteins. In addition, there are three copies of a motif reminiscent of a consensus binding site for the NF-kappaB family of transcription factors (named alpha1, alpha2, and alpha3), and three copies of a T-rich motif (termed beta1, beta2 and beta3) that does not fit with known protein-binding sequences. In spite of considerable effort, the NF-kappaB family member binding to the alpha motifs has not been identified (Culi, 1998).
In order to promote transcription, the SMC enhancer requires, besides proneural proteins, either additional activating factors or the removal of inhibitors. Activating factors might interact with the alpha and beta boxes necessary for efficient enhancer action. To identify the minimum number of different motifs sufficient to constitute an SMC-specific enhancer, the enhancer activity of a synthetic oligonucleotide containing two E1 boxes and one alpha2 box were examined. It promotes beta-galactosidase accumulation only in SMCs, although a weak one. A four tandem repeat of the same oligonucleotide drives much stronger lacZ activity and this also occurs exclusively in SMCs. In contrast, a four tandem repeat with E1 boxes, but without alpha2 boxes, drives strong expression in many cells of proneural clusters. A four tandem repeat of alpha2 boxes without E1 boxes fails to drive expression. Hence, both E and alpha boxes are sufficient, in the context of the minienhancer, to constitute an SMC-specific enhancer (Culi, 1998).
Promoters of other genes share similar enhancer motifs. The asense sequences that direct expression in SMCs contain several E boxes necessary for optimal expression in SMCs. The corresponding DNA from D. virilis was sequenced and compared with that of D. melanogaster. Similar to the sc SMC-enhancer, the stretches of D. virilis conserved DNA contain E boxes, one N box, two alpha boxes, and one beta box, supporting the relevance of these boxes for SMC enhancer function. Moreover, the neurogenic gene Bearded, which is expressed in proneural clusters and SMCs, contains in its regulatory region one E box, necessary for its expression, and one motif identical to the alpha2 box. An evolutionarily conserved alpha box is also found within the regulatory region of rough, a homeobox gene important for restricting photoreceptor R8 specification (Culi, 1998).
Notch signaling prevents more than one of a proneural cluster from becoming SMCs. When the N pathway is not operative, as for instance in Su(H) larvae or in larvae harboring a Nts allele raised at a nonpermissive temperature, Ac and Sc proteins accumulate in many cells of proneural clusters at levels higher than in the wild type. The extra accumulation of Ac and Sc might be mediated by the cluster-specific enhancers, by the SMC enhancer (which under insufficient N signaling may promote expression in many cells of the proneural cluster as they become SMCs), or by both. To distinguish among these alternatives, an examination was carried out of the activity promoted by each type of enhancer, in both wild-type and in Nts discs. N inactivation allows the SMC enhancer to drive expression in many cells of proneural clusters. Expression can occur in contiguous cells, indicating the failure of lateral inhibition. In contrast, N inactivation does not modify the activity of the enhancer that drives expression in the vein L3 and TSM (twin sensilla of the wing margin) proneural clusters, although the accumulation of Sc in these clusters is increased. Hence, the SMC enhancer is responsible for most of the increased levels of proneural protein that occur in proneural clusters under insufficient N function (Culi, 1998).
N signaling, triggered by Ac-Sc in the emitter cell, promotes in the receptor cell the accumulation of E(spl)-C proteins, the main effectors of this signal. E(spl)-C proteins are detectable in proneural cluster cells, except for the SMCs. This correlates with the SMCs being the cells that signal maximally and inhibit their neighbors from acquiring the neural fate, while the SMCs themselves are not inhibited. Ectopic accumulation of E(spl)-C protein prevents SMCs from emerging, as detected by a neuralized enhancer trap line and the consequent absence of SOs in the adult fly. Overexpression of UAS-E(spl)-m8 or UAS-E(spl)-m7 transgenes driven by da-GAL4 or the C-253 GAL4 lines block the activity of the SMC enhancer and the development of the corresponding SOs. In contrast, either of these overexpressions allowed normal accumulation of Ac and Sc in proneural clusters despite the high levels of ectopic E(spl)-m8 mRNA, which are severalfold higher than those in the wild type. However, overexpression with presumably stronger GAL4 drivers does interfere with ac-sc expression in proneural clusters. Taken together these results indicate that the function of the SMC enhancer is more sensitive to E(spl)-C inhibition than are the proneural cluster enhancers, and suggest that the SMC enhancer is the main target of lateral inhibition mediated by the N pathway (Culi, 1998).
Does E(spl)-m8 bind to the SMC enhancer, given the inhibition of SMC enhancer function by E(spl)-C? E(spl)-m8 binds to the N box and, unexpectedly, also protects a broad region of the enhancer (nucleotides 142-182), which does not contain sequences that fit the E(spl)-C consensus binding site. Binding to an enhancer with a mutated N box is weaker, and binding to an enhancer without the N box and the second E(spl)-m8-binding site is undetectable. Remarkably, the removal of one or both binding sites does not modify the SMC specificity of the enhancer, as might be expected if these binding sites mediated the repression of enhancer function in response to N signaling. E(spl)-m8 is unable to bind to the synthetic SMC-specific minienhancer. These results were extended to other E(spl)-C proteins by verifying that [similar to E(spl)-m8] E(spl)-m5 binds to an oligonucleotide with the E1-N sequence, but not to oligonucleotides containing only E2 or E3 boxes. Thus, it is concluded that the E(spl)-C proteins restrict enhancer function to SMCs by a mechanism that does not require direct interaction with enhancer DNA. Thus the Enhancer of split bHLH proteins block the proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-kappaB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment (Culi, 1998).
Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of asymmetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).
The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).
Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).
polychaetoid is required for cell fate specification in the eye. In pyd mutants a slight roughness of the eye is detected. This roughness is due to the presence of mispositioned and duplicated mechanosensory bristles and occasional enlarged, irregularly shaped facets. In addition, the rows of ommatidia are misaligned. In the midpupal stage eyes of pyd mutants, a small fraction of ommatidia are observed to have extra cone cells and/or primary pigment cells. Some ommatidia have too few cone cells. Ommatidia with extra cone cells also have extra photoreceptors. Reduction of Delta or Notch function in pyd mutants strongly enhances both the pyd extra bristle and rough eye phenotypes (Chen, 1996).
The Notch receptor is the central element in a cell signaling mechanism controlling a broad spectrum of
cell fate choices. Genetic modifier screens in Drosophila and subsequent molecular studies have
identified several Notch pathway components, but the biochemical nature of signaling is still elusive.
The results are described of a genetic modifier screen of the bristle phenotype of a gain-of-function
Notch allele, Abruptex16. Abruptex mutations interfere with lateral inhibition/specification events that
control the segregation of epidermal and sensory organ precursor lineages, thus inhibiting bristle
formation. Mutations that reduce Notch signaling suppress this phenotype. This screen of
approximately 50,000 flies led to the identification of a small number of dominant suppressors in seven
complementation groups. These include Notch, mastermind, Delta,
and Hairless, known components in the pathway, as well as two novel mutations: A122 and M285. A122, appears to interact with Notch only during bristle development. M285, displays extensive genetic interactions with the Notch pathway elements and appears, in general, capable of suppressing Notch gain-of-function phenotypes while enhancing Notch loss-of-function phenotypes, suggesting that it plays an important role in Notch signaling. The profile of the genetic interactions
documented with M285 is quite similar to that of mutations in other known components of the Notch
pathway. Three kismet alleles were isolated as weak suppressors of the Ax16 bristle phenotype. Interestingly,
mutations in kismet have been isolated independently as enhancers of the eye phenotype associated
with the expression of constitutively activated forms of the Notch receptor. kismet, which may encode a structural component of chromatin, does not display broad genetic interactions with Notch. It has therefore been suspected that the identification of these alleles through the eye screen may reflect its effect on the expression of the transgene by perturbing normal chromatin function rather than significant
interactions with Notch signaling. The fact that
such alleles were isolated in the bristle screen may be indicative of a link between Notch signaling and kismet function; however, further analysis is necessary before such a relationship can be established (Go, 1998b).
An early step in the development of the large mesothoracic
bristles (macrochaetae) of Drosophila is the expression of
the proneural genes of the achaete-scute complex (AS-C)
in small groups of cells (proneural clusters) of the wing
imaginal disc. This is followed by a much increased
accumulation of AS-C proneural proteins in the cell that
will give rise to the sensory organ, the SMC (sensory organ
mother cell). This accumulation is driven by cis-regulatory
sequences, SMC-specific enhancers, that permit self-stimulation
of the achaete, scute and asense proneural
genes. Negative interactions among the cells of the cluster,
triggered by the proneural proteins and mediated by the
Notch receptor (lateral inhibition), block this accumulation
in most cluster cells, thereby limiting the number of SMCs.
In
addition, proneural proteins trigger positive interactions among cells of the cluster
that are mediated by the Epidermal growth factor receptor
(Egfr) and the Ras/Raf pathway. These interactions,
which are termed 'lateral co-operation', are essential
for macrochaetae SMC emergence. Activation of the
Efgr/Ras pathway appears to promote proneural gene
self-stimulation mediated by the SMC-specific enhancers.
Excess Egfr signaling can overrule lateral inhibition and
allow adjacent cells to become SMCs and sensory organs.
Thus, the Egfr and Notch pathways act antagonistically
in notum macrochaetae determination (Culí, 2001).
Egfr-mediated lateral cooperation should tend to activate the
SMC-specific enhancers in many cells of the proneural clusters. This, however, is prevented by N signaling, which is
activated by Ac and Sc in the cells of the cluster. This signaling, by means of the bHLH proteins of the E(spl)-C, blocks the ac-sc-ase self-stimulatory loop promoted by the SMC-specific enhancers. However, within a proneural cluster the cells of the proneural field accumulate greater amounts of Ac-Sc proteins. As it has been
hypothesized that cells that signal the most are the least
inhibited by their neighbors, eventually, a cell of the proneural
field will be released from the inhibitory loop and its levels of
E(spl)-C bHLH protein will become minimal. This cell will turn on the ac-sc-ase self-stimulation and become an SMC. The SMC signals maximally to its
neighbors and prevents them from following the same fate
(lateral inhibition). These results add to this scenario the requirement for Egfr-mediated signaling for one cell of the proneural field to turn
on the ac-sc-ase self-stimulatory loops and become an SMC. According to this model, Ac-Sc activate both the N-and Egfr-mediated signaling pathways, with their SMC-suppressing
and SMC-promoting abilities, respectively, and
both signaling systems appear to act on the same SMC-specific
enhancers. Since an excess signaling by the N or the
Egfr pathway will either prevent SMC determination or
promote emergence of ectopic SMCs, the respective levels of
signaling should balance each other so that only one SMC is
determined at a time from each proneural cluster. How is this
balance accomplished? This is at present unclear. The large
enhancement of rho/ve mRNA in proneural clusters under
conditions of insufficient N signaling suggests that this
pathway may prevent the Rho/Ve-promoted activation of
Egfr from rising to excessively high levels. In contrast, the
insensitivity of the levels of E(spl)-m8 protein to the
overexpression of UAS-aos in proneural clusters suggests that
the Egfr pathway does not affect N signaling. Antagonistic
interactions between the N and the Egfr pathways are found
in other developing systems, as in the wing preveins and in the reiterative recruitment, from a long-lived atonal proneural cluster, of the precursors of the 70-80 scolopidia of the femoral chordotonal organs. In this later case, Egfr signaling promotes
commitment of neural precursors and the Dl-N interaction
prevents too many cells from being committed (Culí, 2001).
The requirement for WASp function in N-dependent cell fate decisions prompted a search for genetic interactions between WASp and N pathway elements, making use of the WASp adult bristle-loss phenotype. Although the N pathway is involved in a wide variety of cell fate decisions during fly development, use of conditional mutant alleles has been successful in demonstrating that loss-of-function mutations in N itself and in its ligands results in PNS neuronal preponderance and associated phenotypes, in both embryos and adults, including the pIIa-to-pIIb and sheath-to-neuron transformations suggested for WASp. A WASp;N double mutant was constructed using the temperature-sensitive Nts1 allele. At 25°C, Nts1 flies display a wild-type morphology, including a normal array of neurosensory bristles. Introducing this very mild N hypomorphic genotype into a WASp mutant background results in a strong enhancement of the WASp bristle-loss phenotype. Double mutant flies lack practically all bristles on regions of the cuticle such as the thorax, which is only partially affected by the WASp mutation alone (Ben-Yaacov, 2000).
In contrast to the enhancement achieved by reducing N function, significant suppression of the WASp bristle-loss phenotype can be observed when activity of the N pathway is even moderately elevated. The neurosensory bristle pattern of WASp mutant flies, which also lack one copy of the established N antagonist Hairless (H), is close to wild-type in appearance. These flies eclose normally. Similarly, a significant, if somewhat less dramatic rescue of the WASp phenotype is obtained using a gain-of-function allele of the N receptor itself. A transgenic construct (Nint.hs), in which the constitutively active, intracellular portion of N is expressed under the control of a heat-shock promoter, was introduced into a WASp mutant background. Mild (29°C) heat treatment of such flies, which has no noticeable effect on Nint.hs flies, when combined with a WASp mutation leads to significant restoration of the bristle pattern, particularly in abdominal segments. Sensitive genetic interactions can thus be demonstrated between WASp and elements of the N pathway, raising the possibility of a common functional framework (Ben-Yaacov, 2000).
Notch and neurogenesis: Chordotonal organs The selection of Drosophila sense organ
precursors (SOPs) for sensory bristles is a progressive
process: each neural equivalence group is transiently
defined by the expression of proneural genes (proneural
cluster), and neural fate is refined to single cells by Notch-Delta
lateral inhibitory signalling between the cells. Unlike
sensory bristles, SOPs of chordotonal (stretch receptor)
sense organs are tightly clustered. It has been shown that for
one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results
from the progressive accumulation of a large number of
SOPs from a persistent proneural cluster. This is achieved
by a novel interplay of inductive epidermal growth factor-
receptor (EGFR) and competitive Notch signals. EGFR
acts in opposition to Notch signaling in two ways: it
promotes continuous SOP recruitment despite lateral
inhibition, and it attenuates the effect of lateral inhibition
on the proneural cluster equivalence group, thus
maintaining the persistent proneural cluster. SOP
recruitment is reiterative because the inductive signal
comes from previously recruited SOPs (zur Lage, 1999).
The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental
analysis of Ato expression has revealed that these SOPs accumulate
over an extended period of time in the dorsal region of each leg
imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained
requirement for proneural function throughout the process of
SOP accumulation. Unusually, Ato is persistently expressed in
a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity
formed by the folding of the disc. This invagination later
becomes visible as a distinctive 2-cell wide intrusion, which is
referred to as the 'stalk'. Cells at the deepest end of the stalk
undergo shape changes to form an amorphous inner SOP mass.
Invaginating cells are characterised by upregulation of Ato
expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101
enhancer trap line) are not expressed in all
the stalk SOPs. Instead, these markers are only apparent in older
cells, particularly at the time when they become part of the inner
mass (which is therefore referred to as mature SOPs).
Despite this, entry into the stalk seems to mark SOP
commitment, since both the stalk and the mature SOPs are
absent in discs from ato mutant larvae. This
apparent intermediate stage may not have a counterpart in
external sense organ precursor formation, although there is
some evidence for multiple steps between the uncommitted cell
and the SOP (the so-called pre-sensory
mother cell state).
Initially, Ato remains activated in all invaginated SOPs. This
extended period of proneural gene expression is unusual since
AS-C proneural expression is typically switched off in SOPs
shortly after commitment. Later, at
approximately 6 hours before puparium formation (BPF), Ato expression is switched off
synchronously in the mature SOPs, although expression
remains in the stalk SOPs and the PNC. At this
point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).
The process of chordotonal SOP formation described above is
at odds in several respects with the well-known paradigm of
SOP selection for sensory bristles. In the latter, the solitary
SOP expresses Delta, which triggers expression in the PNC of
genes of the E(spl)-C, thereby
preventing further SOP commitment and forcing loss of AS-C
expression and neural competence. In the case of the femoral
chordotonal organ, newly committed cells from the PNC are
in contact with previously committed SOPs in the stalk, but are
apparently not receiving (or not responding to) lateral
inhibition signals from these to prevent their commitment.
Likewise, the presence of committed SOPs does not switch off
ato expression in the PNC. Nevertheless, components of the N-Dl
pathway are expressed in patterns consistent with lateral
inhibition. The newly formed SOPs express Dl, suggesting that
they send inhibitory signals, while the PNC expresses
mgamma, a member of the E(spl)-C, suggesting that these
cells are responding to the Notch-Delta signal.
Indeed, mgamma is coexpressed with ato in the PNC throughout the
development of the SOP cluster. Chordotonal SOP formation is
shown to be sensitive to N inhibitory signaling.
Strong activation of N signaling or its effectors can
inhibit chordotonal SOP formation.
Thus, N signaling has an important role to play: it acts to limit
the process of SOP selection from the PNC. Some mechanism,
however, must prevent N signaling from completely inhibiting
multiple SOP formation (zur Lage, 1999).
The progressive accumulation of chordotonal SOPs suggests
that a recruitment mechanism could explain the clustering of
SOPs. The Drosophila Egfr signaling pathway is involved
in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the
induction of embryonic chordotonal precursors (zur Lage,
1997). Although there appear to be significant differences in
the process of SOP formation in imaginal discs, as compared with
the embryo, it was asked whether Egfr signaling is also
involved in forming the femoral chordotonal cluster. To address
this question, the pathway was conditionally disrupted by
expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr
DN results in a dramatic loss of chordotonal SOPs in
late third instar imaginal leg discs (as judged by Ase protein
expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is
required for the process of femoral chordotonal SOP
formation. In contrast, the appearance of bristle SOPs is
unaffected, arguing against the possibility of a nonspecific
effect on SOPs in general (zur Lage, 1999).
To determine whether Egfr signaling controls SOP
number, expression of components of the Egfr
pathway that determine the level of signaling was forced, thus resulting
in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription
factor and is activated in cells responding to
Egfr signaling. Both rho and pnt
are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the
commitment of PNC cells or by stimulating proliferation of
SOPs. Both functions would be consistent with known Egfr
roles, but
the current investigations favour the former. Analysis of Ato
expression in leg discs in which rho has been misexpressed
reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced
extent of mgamma expression. These observations are
consistent with an increased rate of SOP commitment upon
Egfr hyperactivation. Moreover, this effect is reminiscent of
the effect of N loss of function on Ato expression, suggesting
that Egfr signaling supplies the mechanism that interferes
with lateral inhibition of SOP commitment (zur Lage, 1999).
The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand.
Reiterative recruitment alone cannot entirely explain the
accumulation of SOPs. Such an accumulation also relies on the persistence of the
competent pool of PNC cells from which SOPs can be
recruited. For AS-C PNCs, this does not occur, because the
mutual inhibition required for continued competence is
unstable and resolves quickly to a state of lateral inhibition
once the SOP emerges from the PNC.
This results in rapid shutdown of AS-C expression and hence
competence within the PNC. It is possible
that the members of E(spl)-C that are expressed in the PNC
(notably mgamma and mdelta) are less aggressive inhibitors of proneural
gene expression than the E(spl)-C members expressed in AS-C
PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that
Egfr has a role to play in maintaining the PNC by partially
attenuating lateral inhibition on a PNC-wide scale. Thus, the
PNC is not completely shut off by inhibition from SOPs, but
instead kept in check, allowing continued mutual inhibition and
maintenance of competence but not allowing general SOP
commitment. Since neither rho nor dp-ERK are detected in the
PNC as a whole, this function of Egfr could be indirect and
achieved through partial attenuation of Dl signaling from the
stalk SOPs themselves. The trans- or auto-activation of EGFR
signaling between the stalk SOPs (as suggested by the co-expression
of dp-ERK and rho) might be an indicator
of this function. It is also possible, however, that Egfr
signaling is direct and that the dp-ERK antibody is not
sensitive enough to detect expression in the PNC cells (zur Lage, 1999).
Notch and sense-organ differentiation
in the Drosophila antenna The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. Three broad categories of sense-organs are known to be specified at the level of progenitor choice. However, little is known about how cell
fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups
of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero, Elav,
and Seven-up shows that there are three secondary progenitor fates. The lineages of these cells have been established by clonal analysis
and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to
the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically
segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated
sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle
for the analysis of how olfactory neurons acquire distinct terminal fates (Sen, 2003).
In sibling cells, a bias in N signaling occurs because of
an asymmetric distribution of the membrane-associated protein
Numb (Nb), which binds the intracellular region of N
antagonizing its function. N signaling
can also be compromised by ectopic expression of a dominant
negative (DN-N) construct of the N receptor which
interferes with ligand-dependent signaling. The
sca-Gal4P309 insertion strain was used to drive UAS-mediated Nb or
DN-N activity in secondary progenitors. Expression of sca-Gal4P309-driven GFP is visualized in the proneural domains, the primary and secondary
progenitors, but is not detected in the majority of
sensory clusters after division (Sen, 2003).
Animals of sca-Gal4P309/UAS-Nb show a strong
reduction in external structures on the adult antenna concomitant with an increase in glial cell numbers. The defect was also observed although significantly
weaker in sca-Gal4P309/UAS-DN-N. In both genotypes, there appears to be an
increase in neuronal number since the fascicles appeared
thicker than in the controls. It is proposed that
these phenotypes are caused by a transformation of PIIa to
PIIb; in the coeloconic lineages, this would result in an
increase of glial cells (Sen, 2003).
To test this hypothesis, pros-Gal4 was used to drive expression
of DN-N in PIIb/PIIIb and PIIc but not PIIa. The numbers of external cells (sensillar shafts) from
all three sense-organ types was counted and was found to be comparable to
normal controls. This is consistent with the findings
that tormogen and trichogen cells arise from the PIIa,
which is not being manipulated in this genotype. There was
also no change in glial cell number, even though N activity
is being lowered in the PIIb progenitors. While it
is possible that ectopically expressed DN-N is not sufficient
to compromise N signaling, the favored explanation is that N
is not required in PIIb/PIIIb. This would imply a PIIa-to-PIIb switch, which in the coeloconic lineages, results in
excess of glial cells (in addition to neurons) (Sen, 2003).
If indeed N signaling plays a role in the binary choice
between secondary progenitor types, then gain of N activity
would be expected to increase the external cells (socket and
shaft) that arise from the PIIa lineage. The truncated cytoplasmic
domain of the N receptor (N-intra) is constitutively
active, and its misexpression creates a dominant gain-of-function
condition. Expression of Nintra early during sense organ
development interfers with olfactory progenitor
choice and subsequent recruitment of secondary progenitors. These effects of N could be avoided by exploiting the thermosensitive nature of the Gal4/UAS system (Sen, 2003).
sca-Gal4P309; UAS-Nintra animals were reared at 16°C
until 10 h APF and then shifted to 28°C to activate N
specifically in secondary progenitors. The adult antennae show
a variety of defects affecting the external structures of the
sensory units. There were cases of multiple
sockets and sensilla with two shafts arising from
a socket. While, in principle, such phenotypes
could be explained by a role of N in binary choice between
shaft and socket cells, the appearance of sensilla with four
sockets or two shafts with a single socket can only
be explained by invoking conversion of PIIb/PIIc to PIIa (Sen, 2003).
Notch and gliogenesis 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).
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).
The expression patterns of the enhancer-trap lines M84 and
P101 are indistinguishable in abdominal segments: both are expressed in the SPGs including the A- and B-SPG and LV-SPG cells produced from NB1-1A.
To investigate the relationship between the three glial cells
derived from NB1-1A, M84/P101 (a stock double
homozygous for both the M84 and P101 insertions), embryos were stained with anti-ß-gal. In stage 12/13
embryos, a single M84/P101+ cell appears in dorsal positions
of each hemi-neuromere. Embryos double labelled
with anti-Even-skipped (anti-Eve) indicates that this cell is
located posterior to the Eve+ cluster of NB1-1 (aCC/pCC)
and NB7-1 progeny (CQ-neurons). Slightly
later, a second M84/P101+ cell appears anterior to the first cell. According to the positioning within the developing
nerve cord, the posterior cell represents the A-SPG, whereas
the anterior cell is the B-SPG. A third glial cell, representing
the LV-SPG, can be detected in ventral
positions only after the A- and B-SPG are already present,
suggesting that this cell is the last born glia within the lineage (Udolph, 2001).
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).
Removing Notch function results in the near complete
abolishment of all M84/P101 expression, indicating a more
general function for Notch in SPG specification.
These findings prompted an investigation whether Notch is
required in the specification of CNS glia in general. Experiments were performed in which glia-specific markers, either
enhancer trap lines (gcm-lacZrA87 and pnt-lacZ) or an antibody (anti-Repo) were used. gcm-lacZrA87 is an
insertion into the gcm gene and expresses ß-gal in the pattern
of gcm. pointed (pnt) is specifically expressed in glial cells and has been reported to act downstream of gcm. Nts1embryos were shifted to the non-permissive temperature after 6 hours of
development, and subsequently stained with anti-Eve and anti-ß-gal at late stage 16; taking into account the missing SPGs, both enhancer traps were expressed in a pattern reminiscent of wild-type embryos. However, an increase in the
number of ß-gal-positive cells was observed, which could be
in part due to a mild neurogenic phenotype caused by Nts1.
Furthermore, it was found that the glial-specific protein Repo
was widely expressed in the CNS of N55e11 (an amorphic N
allele) embryos. Thus, general glial specific markers
like gcm, pnt and repo are expressed in embryos that lack or
have strongly reduced Notch function, indicating that although
Notch is required for the formation of SPGs, there is not a
global requirement for Notch in the specification of all CNS glia (Udolph, 2001).
Another neurogenic gene, 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 also 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).
Clonal analysis demonstrates that the loss of Notch
function results in the loss of SPGs and a concomitant gain of
neuronal cells within the NB1-1A lineage, consistent with the
notion that a sibling cell fate relationship exists between cluster
neurons and glial cells in this lineage and that Notch is required
for the asymmetric divisions that generate these postulated
neuron/glia sibling pairs. The data support a hypothesis that glial cells and cluster neuron s share a sibling relationship within the NB1-1A lineage. Taken together, the data favour a model in which a series of three GMCs produced from
NB1-1A can each divide asymmetrically to produce a neuron
and a glia, with Notch signaling required to specify the glia
fate, and Numb and the absence of Notch signaling required
for the neuronal fate (Udolph, 2001).
It is known that gcm acts as a master regulator in gliogenesis because it has been described as a binary switch between glia and neurons. As expected, gcm appears to be required for the formation of SPGs. P101 marker gene expression is abolished in embryos homozygous for gcm. The similarity of gcm and Notch phenotypes suggests the possibility that both genes share a common pathway required for SPG specification. It was reasoned that if Notch acts downstream of gcm, then overexpression of activated N in a gcm minus genetic background should result in additional SPGs; however, if Notch acts upstream of gcm, then in a gcm minus background the overexpression of activated N should not result in the production of additional P101+ cells. In a genetic background lacking gcm function, activated N expression is unable to induce SPG development as indicated by the loss of marker gene expression. These findings are consistent with the notion that Notch functions upstream of gcm in the context of SPG development (Udolph, 2001).
Two types of neuroectodermally derived glial progenitors in
the embryonic nervous system of Drosophila have been
described. Glioblasts (GB) generate only glial progeny, and
NGBs produce both neurons and glial cells within the same
lineage. A mechanism by which both neurons and glia
can arise within the lineage (NGB6-4T) of a thoracic NGB has
recently been described. NGB6-4T represents a thoracic-specific
NGB in which glial and neuronal sublineages bifurcate
from each other during the first division of the parental NGB. Only one
of the two daughters expresses Gcm, a master regulator of glial
cell fate that is involved in regulating the expression of other
glia-specific target genes. In the NGB6-4T lineage, the
asymmetric distribution of Gcm results in a cell that is
specified as a GB within a neuroglioblast lineage. The GB will
exclusively generate the glial components, whereas its sibling
will exclusively give rise to the neuronal components of the
lineage (Udolph, 2001).
This study reports a novel mechanism by which glia
can be generated during CNS development. The data suggest
that NB1-1A gives rise to a set of three glial cells through a
series of three GMC asymmetric divisions. Several lines of
evidence support the notion that within a NGB lineage
GMCs can produce both glial and neuronal cells. (1) Immunohistochemical analyses indicate that the A-, B- and LV-SPG
arise at different times in development, and their non-simultaneous
birth, in conjunction with the fact that their
formation can be differentially affected by inactivation of
Notch, suggests that they do not derive from a common
precursor; (2) mutations in genes involved
in specifying alternative sibling cell fates affect SPG
development in a fashion that suggests these cells are
siblings with non-glial components of the lineage; (3)
transplantation experiments indicate that the 3 SPGs share
sibling relationships with the neuronal components of the
NB1-1A lineage, the cluster neurons (cN). Finally, analysis
of two cell FLP-clones demonstrate that SPG glia and neurons
share a direct sibling relationship (Udolph, 2001).
This proposed mechanism for the genesis of SPGs
is fundamentally different from the ones described for GBs,
e.g. anterior glioblast, and for NGBs,
e.g. NGB6-4T and NGB5-6A. The first division of NB1-1A
produces a neurogenic GMC that gives rise to a pair of sibling
neuron s (aCC/pCC), and at the level of the first division of
the parental NB no GB sublineage is bifurcated. In addition,
the NB1-1A derived glial cells share a direct sibling
relationship with neurons and does not involve the generation
of a lineage internal GB at all. The asymmetric origin of glia
described here provides a novel mechanistic framework of glial
origin that might be used as a model system to gain further
insights into the regulatory networks involved in glial cell fate
specification in the CNS. It is concluded that the mechanisms
leading to glial cell fate specification are complex and that
multiple developmental mechanisms lead to glial cell fate
specification. These mechanisms will be likely to require
different molecular machinery that involve distinct sets of
genes or genetic hierarchies; for example, Notch being
required specifically for the SPGs but apparently not for other
types of glia. However, most of the glial cells (except midline
glia) strictly require the gcm gene. Thus, there seems to be a
common molecular basis that is context and cell specifically
regulated during development.
This study provides the first evidence that Notch is a crucial
component in the specification of a subclass of CNS glial
cells, the subperineurial glia (SPG). As revealed by the
transplantation experiments, Notch is not only required for the
expression of the M84/P101 marker, but loss of Notch function
leads to the loss of the SPG cell fate per se. In the case of NB1-1A, the data indicate that the loss of glia is accompanied by
the conversion of these cells into neurons. In contrast, Notch
gain of function results in the overproduction of SPG-like cells
at the expense of the cluster neurons. Hence, Notch signaling
is sufficient for the specification of these cells. A second gene,
gcm, is also crucially required for the specification of the
SPGs; in a gcm mutant background M84/P101 expression is
completely absent. If an activated form of the Notch protein is
ectopically expressed in the embryonic CNS of animals lacking
gcm function, SPGs do not form; in addition, M84/P101
expression cannot be detected in the CNS of these embryos.
This indicates that gcm function is strictly required for Notch-mediated
SPG specification, suggesting that Notch functions
upstream of gcm. It is interesting to note that Notch also
appears to play an instructive role in gliogenesis for
mammalian neural crest stem cells in culture (Udolph, 2001).
These results, in conjunction with what is already known about
the origin of the aCC and pCC neurons, as well as the terminal
lineage of NB1-1A, allow the following model to be proposed for the
NB1-1A lineage: in total, NB1-1A gives rise to five pairs of
sibling cells. The first pair comprises the aCC/pCC neurons
resulting from GMC1-1a. In addition, three later born GMCs
each give rise to one cluster neuron and one SPG-glia. A fifth
GMC gives birth to two additional cluster neurons. The order
of appearance and the position of the MP84/P101-labelled cells
would suggest that, of the three glia-producing GMCs, the
earliest born GMC gives rise to the A-SPG; the latest born
GMC gives rise to the LV-SPG; and the GMC born in the
middle gives rise to the B-SPG. The time of
birth of the fifth GMC in this lineage, which is postulated
to produce two cluster neurons, cannot be determined. Clearly, refinement and proof
for this proposal will require experiments that provide direct
temporal information (Udolph, 2001).
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).
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).
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).
The Drosophila central brain is composed of thousands of neurons that derive from approximately 100 neuroblasts per hemisphere. Functional circuits in the brain require precise neuronal wiring and tight control of neuronal numbers. How this accurate control of neuronal numbers is achieved during neural development is largely unclear. Specifically, the role of programmed cell death in control of cell numbers has not been studied in the central brain neuroblast lineages. This study focusses on four postembryonic neuroblast lineages in the central brain identified on the basis that they express the homeobox gene engrailed (en). For each lineage, the total number of adult-specific neurons generated was determined, as well as number and pattern of en-expressing cells. Programmed cell death has a pronounced effect on the number of cells in the four lineages; approximately half of the immature adult-specific neurons in three of the four lineages are eliminated by cell death during postembryonic development. Moreover, programmed cell death selectively affects en-positive versus en-negative cells in a lineage-specific manner and, thus, controls the relative number of en-expressing neurons in each lineage. Furthermore, evidence is provided that Notch signaling is involved in the regulation of en expression. Based on these findings, it is concluded that lineage-specific programmed cell death plays a prominent role in the generation of neuronal number and lineage diversity in the Drosophila brain (Kumar, 2009).
In postembryonic CNS development of holometabolous insects such as flies, a combination of programmed cell death and neuronal process re-innervation allows the larval nervous system to reorganize and innervate new body structures. During metamorphosis many adult-specific neurons in the ventral ganglia are targeted by programmed cell death, particularly in abdominal segments. Furthermore, extensive cell death occurs during postembryonic development in the insect visual system, where cells are overproduced and those that do not make the appropriate targets are eliminated by apoptosis. By contrast, very little is currently known about the prevalence and functional roles of programmed cell death in development of the insect adult central brain (Kumar, 2009).
This report identifies four neuroblast lineages in the postembryonic central brain and finds that programmed cell death occurs in all four lineages, albeit to different extents. Whereas cell death plays only a minor role in the medial cluster MC1 lineage, it has dramatic effects in anterior cluster (AC), posterior cluster (PC) and medial cluster MC2 lineages, in which nearly half of the adult-specific neuronal progeny are programmed to die during larval development. It is noteworthy that the adult-specific neurons targeted by cell death are generated during larval development and are eliminated before their respective neuroblasts stop proliferating (12-24 hours after pupal formation). Because the cell death reported here occurs before neuronal differentiation, it is probably not involved in events of brain reorganization that take place during metamorphosis (Kumar, 2009).
Another central feature of the cell death events demonstrated here is that none of the four lineages is completely eliminated by cell death; all four neuroblasts and a significant number of their neuronal progeny survive at the end of larval development, and these neuronal progeny are largely present in the adult. In this sense, the programmed cell death reported here is likely to be functionally different from the cell death observed in the ventral ganglia, where the neuroblast itself undergoes apoptosis, regulating neuronal numbers in the abdominal segments (Kumar, 2009)
These experiments indicate that programmed cell death plays a prominent role in determining lineage-specific features; if cell death is blocked the total neuronal number increases in all four lineages and the number of en-expressing neurons increases in AC, PC and MC2. Furthermore, the axonal projection pattern of H99 mutant (deleting rpr, hid and grim) and Notch mutant en-expressing lineages was examined, comparing them to wild type. Both cell death defective H99 and Notch mutant PC lineages showed an additional projection that was not present in the wild type, whereas the other three H99 lineages did not appear to change drastically in their projection patterns. In conclusion, programmed cell death appears to contribute to the cellular diversity of neuronal lineages in the central brain (Kumar, 2009).
Studies on neuroblast lineages in the developing ventral ganglia indicate that proliferating neuroblasts generate a largely invariant clone of neural cells. In general, each neuroblast division produces a distinctly fated GMC, and each GMC division produces two sibling progeny of different fates. There is some evidence that the fate of these progeny is controlled by the parental GMC; the two siblings are restricted to a pair of different cell fates, with one sibling adopting an 'A' fate and the other adopting a 'B' fate. This, in turn, has led to a model in which a neuroblast lineage can be thought of as composed of two hemilineages, with one hemilineage comprising 'A'-fate cells and the other hemilineage comprising 'B'-fate cells. It is thought that an interaction between Notch and Numb is responsible for generating distinct neural fates of the two GMC daughter cells, with a loss of Notch or Numb resulting in reciprocal cell-fate duplication. However, Notch signaling does not appear to confer a particular fate; rather, it acts generically as a mechanism to enable two siblings to acquire different fates, and other developmental control genes that are inherited from the specific parental GMC are thought to be instrumental in determining the final identity of each progeny (Kumar, 2009).
Findings on lineage-specific cell death support a comparable model in which all four brain neuroblasts can generate one en-positive hemilineage and one en-negative hemilineage. In this model, programmed cell death is then targeted in a lineage-specific manner to either the en-negative hemilineage (AC, PC), or the en-positive hemilineage (MC2), or neither hemilineage (MC1). Alternatively, en-positive and en-negative neurons in the lineages could be generated in a temporal fashion and subsequently en-positive or en-negative neurons could be eliminated in a lineage-specific manner. However, the results suggest that this is unlikely. In particular, in the PC lineage, more than 80% of the two-cell clones examined were composed of one en-positive and one en-negative cell. If the above did occur, a significant number of two-cell en-positive clones should have been obtained along with two-cell clones comprising one en-positive and one en-negative neuron. Similar analysis of single and two cell clones in the other three en lineages is further required to confirm the occurrence of hemilineage-specific programmed cell death (Kumar, 2009).
Based on these experimental results, it was postulate that Notch signaling is an important generic mechanism underlying generation of the two different hemilineages, as in the absence of Notch signaling, cell-fate duplication of GMC siblings occurs. Indeed, analysis of Notch loss-of-function neuroblast clones suggests that in the absence of Notch signaling most of the neurons in the four lineages acquire an en-positive cell fate. Alternatively, in the four lineages examined, being positive for en may be the default state of the cells, and Notch induces secondary fate by repressing en in subsets of cells in each lineage. These en-positive neurons then appear to survive or undergo programmed cell death depending on the lineage-specific context. However, it remains to be seen whether Notch itself acts on the apoptotic machinery, independent of en (Kumar, 2009).
This study, used en as a molecular marker to identify four lineages in the postembryonic central brain. Might en itself be functionally involved in regulating programmed cell death in these lineages? For the PC lineage, there is some indication that the total clone size is reduced by approximately half in en loss-of-function mutants, compared with wild type. Although this suggests that en may be involved in promoting survival of en-positive neurons in PC (and probably AC), it does not explain the role of en in the MC2 lineage, where it would have to play an opposing role, as en-positive neurons die in this lineage. Thus, en could act either as a pro-apoptotic or an anti-apoptotic factor, depending on the lineage-specific context. Moreover, a direct genetic interaction between en and the apoptotic machinery remains to be investigated. As en is known to have multiple interactions with other proteins, a complex regulatory network involving target proteins of en may be responsible for regulating apoptosis in a lineage-specific manner. Further analysis of interactions with such target proteins is necessary to reveal the full regulatory network in more detail (Kumar, 2009).
The lineage-specific effects of cell death and of Notch signaling in AC and PC are distinctly different from those observed in MC1 or MC2 lineages. However, when compared with each other, many aspects of AC and PC lineages are similar. In wild type, both lineages consist of similar numbers of adult-specific neurons, and the majority (approximately 80%) of these neurons are positive for en, whereas neuroblasts and GMCs are negative for en in both lineages. Blocking cell death results in a substantial (approximately double) increase in total cell number in both lineages, and this increase is almost exclusively due to an increase in the number of surviving en-negative neurons in both lineages. Moreover, loss of Notch function causes a marked increase in the number of surviving en-positive neurons without affecting the number of en-negative neurons in both lineages. The only significant difference between AC and PC lineages observed in this study is that the AC lineage is located in the protocerebrum, whereas the PC lineage is located in the deutocerebrum (Kumar, 2009).
What might be responsible for these similarities in the AC and PC neuroblast lineages? There is some evidence for the existence of serially homologous neuroblasts in the fly brain and VNC. In the VNC, serially homologous neuroblasts, defined by comparable time of formation, similar positions in the neuromeric progenitor array and similar expression of developmental control genes, such as segment polarity genes, dorsoventral patterning genes and other molecular markers, can give rise to almost identical cell lineages. This suggests that similar regulatory interactions take place during development of serially homologous neuroblasts and their neural lineages. A comparison of molecular expression patterns in neuroblasts from different neuromeres of the brain and ventral ganglia suggests that several of them might be serial homologs of each other. For example, neuroblasts NB5-6 in the abdominal, thoracic and subesophageal ganglia have been proposed to be homologous to NBDd7 in the deutocerebrum and NBTd4 in the tritocerebrum (Kumar, 2009).
Given the remarkable similarities in AC and PC neuroblast lineages, it is possible that the protocerebral AC lineage and the deutocerebral PC lineages represent serial homologs. If this is the case, then investigations of the cellular and molecular mechanisms that control their lineage-specific development should be useful for understanding of how regionalized neural diversity in the brain evolves from a basic metameric ground state. However, as neither the combination of developmental control genes expressed in AC and PC neuroblasts nor the position of the two brain neuroblasts in their neuromeres of origin are currently known in sufficient detail, further experiments are needed before the issue of serial homology can be resolved for these brain neuroblast lineages (Kumar, 2009).
Drosophila larval neurogenesis is an excellent system for studying the balance between self-renewal and differentiation of a somatic stem cell (neuroblast). Neuroblasts (NBs) give rise to differentiated neurons and glia via intermediate precursors called GMCs or INPs. E(spl)mγ, E(spl)mβ, E(spl)m8 and Deadpan (Dpn), members of the basic helix-loop-helix-Orange protein family, are expressed in NBs but not in differentiated cells. Double mutation for the E(spl) complex and dpn severely affects the ability of NBs to self-renew, causing premature termination of proliferation. Single mutations produce only minor defects, which points to functional redundancy between E(spl) proteins and Dpn. Expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling from the GMC/INP daughter to the NB. When Notch is abnormally activated in NB progeny cells, overproliferation defects are seen. This depends on the abnormal induction of E(spl) genes. In fact E(spl) overexpression can partly mimic Notch-induced overproliferation. Therefore, E(spl) and Dpn act together to maintain the NB in a self-renewing state, a process in which they are assisted by Notch, which sustains expression of the E(spl) subset (Zacharioudaki, 2012).
This paper presents an analysis of the expression and function of two types of bHLH-O proteins expressed in Drosophila neuroblasts, Dpn and the E(spl) family. The main conclusions are that (1) these two types of factors have distinct expression modalities: E(spl)mγ and m8 are targets of Notch signalling, whereas Dpn is not; (2) these factors have redundant functions to maintain NBs in a self-renewing state in normal development, yet (3) in a pathological NB hyperproliferation context, Dpn and E(spl) have distinct functions (Zacharioudaki, 2012).
It was heretofore thought that embryonic NBs are cells that escape from Notch signalling, which is only perceived in the surrounding neuroectoderm. Only in the later post-embryonic period, were the NBs thought to respond to Notch. This study has shown that this happens much earlier, already in the embryonic NBs. Soon after the NB delaminates, a time when it sends, but does not receive, a Notch signal, it starts asymmetrically dividing. The results are consistent with the daughter GMCs sending a Delta signal back to their sister NBs, thereby initiating E(spl) expression. E(spl)mγ expression ceases when the NB enters quiescence, only to restart when proliferation resumes (Zacharioudaki, 2012).
Dpn, another bHLH-O protein, is also expressed in NBs, but much less dynamically. Its expression initiates upon NB delamination from the neuroepithelium and persists throughout its life. dpn does display some degree of dynamic expression, as it is rapidly turned off in the immature intermediate progenitors (iINPs), only to be reactivated upon maturation. Loss-of-function data clearly indicate that it is not a target of Notch in the NB, in contrast to E(spl). Paradoxically, dpn is induced upon Notch hyperactivation. This could be an indirect effect mediated through E(spl). Indeed, E(spl)mγ overexpression can induce ectopic dpn expression. Still, dpn does harbour a Notch-responsive enhancer that drives expression in larval NBs. This same region scored positively for Su(H) binding in a ChIP-chip approach in a cell line of mesodermal origin . How this enhancer contributes to the overall expression pattern of dpn will be a matter of future analysis (Zacharioudaki, 2012).
Despite their different expression modalities, Dpn and E(spl) have redundant functions in the larval NBs, as only double mutant clones show proliferation defects. These mutant NBs do not stop proliferating immediately, rather gradually terminate their cycling within a few days following homozygosing of the mutant alleles. It is proposed that Dpn/E(spl) keep the NB in an undifferentiated state and proliferation is a consequence of the ability of these cells to respond to mitogens. Upon Dpn/E(spl) loss, this state becomes unstable and prone to switch to a terminally differentiated state. This transition takes a few days, probably reflecting the time needed to accumulate pro-differentiation factors. A redundant role of Dpn/E(spl) in maintaining the undifferentiated state also during quiescence transpired from the genetic analysis of NB re-activation after embryogenesis. Whereas dpn–/– NBs quite successfully re-entered the cell cycle, dpn–/–;E(spl)+/– NBs were unable to do so, despite trophic growth factor stimulation (Zacharioudaki, 2012).
E(spl) expression has been associated with the less differentiated of two alternative outcomes in other instances. For example, during NB formation, E(spl) genes are expressed in the undifferentiated embryonic neuroectoderm and not in the NBs. The same happens in the optic lobe neuroepithelium. This work has presented evidence for a similar role for Dpn/E(spl) in the NB. Excessive Dpn/E(spl) activity in GMCs/INPs can revert these partially differentiated cells back to a NB-like fate. For this reason, NB asymmetric divisions must ensure that Dpn and E(spl) are never expressed in the GMC or iINP. Regarding E(spl), it is proposed that this is ensured by the directionality of Notch signalling (GMC to NB). Dpn is also never seen to accumulate in the GMCs/iINPs, suggesting a repression mechanism at work in these cells, e.g., via Pros. These modes of transcriptional control are probably combined with active protein clearance by degradation (Zacharioudaki, 2012).
An anti-differentiation role has also been proposed for vertebrate homologues of Dpn/E(spl), the Hes proteins. Hes1, Hes5 and Hes3 are all expressed in proliferating neural stem cells of the embryonic CNS. Upon Hes knockout, neural stem cells prematurely differentiate resulting in a hypoplastic nervous system, with increasing severity as more Hes genes are lost. In an interesting analogy, only Hes1 and Hes5 are direct targets of Notch signalling. Another example where anti-differentiation during quiescence is mediated by high Hes1 expression are cultured fibroblasts and rhabdomyosarcoma cells. Similar to what was observed with Dpn/E(spl)-mutant embryos, a quiescence trigger, like serum depletion, can result in irreversible cell-cycle withdrawal, if Hes1 activity is compromised (Zacharioudaki, 2012).
The results have shed light on the paradox of why Notch loss of function has only minor effects in larval neurogenesis, whereas its hyperactivation causes significant overproliferation. Notch loss of function decreases E(spl) expression, leaving Dpn levels unaffected. Furthermore, Notch pathway disruption does not seem to directly affect NB proliferation, as Cyclin E expression is not eliminated. Therefore, Notch signalling from the GMC/iINP to the NB acts to ensure robustness in NB maintenance, in collaboration with Dpn (Zacharioudaki, 2012).
When Notch signalling is aberrantly activated in the GMCs/iINPs, both type I and type II lineages overproliferate, although the former do so with lower penetrance (fewer lineages) and expressivity (smaller clones). Yet, for both types of lineages, E(spl) genes are necessary to implement overproliferation. This is consistent with the hypothesis that ectopic E(spl)/Dpn activity in the GMCs/iINPs inhibits their differentiation and makes them competent to respond to mitogenic stimuli (Zacharioudaki, 2012).
Why are Type II lineages more sensitive than type I lineages to Notch gain of function? A crucial difference between these NBs is the lack of expression of Ase in type II, as its artificial reinstatement can revert the latter to type I-like behaviour. It was recently demonstrated that Ase downregulates E(spl) expression. It is even possible that Ase antagonizes E(spl) proteins post-transcriptionally, as the two can interact and extensive antagonistic interactions. Thus, N hyperactivation will probably cause a smaller increase in E(spl) levels/activity in type I cells, compared with type II. If resistance to differentiation stimuli depends on the level of E(spl)/Dpn activity, this would account for the relative resilience of type I lineages to Notch-induced overproliferation (Zacharioudaki, 2012).
In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1 --> RP2/sib lineage, it was found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. It was further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. These results tie Numb and Notch-signaling through a single player, Neur, thus giving a more complete picture of the events surrounding asymmetric division of precursor cells. It was also shown that Neur and Numb are interdependent for their asymmetric-localizations (Bhat, 2011).
The results in this paper tie the localization of Numb and the signaling-processing of Notch through a single upstream player, Neur. This gives a more complete picture of the events that surround asymmetric division of neural precursor cells. The E3 Ubiquitin ligase protein Neur regulates asymmetric division of Numb and Notch-sensitive neural precursor cells in the CNS via two distinct, sequential mechanisms: first, by promoting the asymmetric localization of Insc and Numb in GMCs and second, via non-cell autonomously potentiating or enhancing the activation of Notch signaling in the Numb-negative daughter cell. While Neur is known to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch, an earlier role for it in asymmetric division via Insc and Numb localization has not been discovered. In fact, these results show that this is the primary role for Neur in generating asymmetry in the CNS. That Neur plays a secondary role or a role which is not absolute in the potentiation or enhancement of Notch signaling is indicated by the finding that in neur; numb double mutants, both sibling cells often but not always adopt a mixed fate as opposed to an RP2 fate seen in Notch; numb double mutants. If the role of Neur in Notch potentiation in this lineage is an absolute one, the same result would have been seen in neur; numb as N; numb mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch, however, the penetrance of this effect is weak (Bhat, 2011).
Previous studies had shown that the RP2-sib binary fate decision is regulated by unequal segregation of the Notch regulator Numb. The simplest interpretation of the current results would suggest that Neur is required for sib fate specification via Notch. However, the results indicate that the requirement of Neur for sib-specification to a daughter cell of a GMC-1 via regulating Notch is preceded by its requirement in GMC-1 for Numb localization, where Neur itself is expressed and becomes asymmetrically localized to the basal Numb-domain. Thus, the loss of sib identity in neur mutants appears to be mainly due to the non-asymmetric localization of Insc and Numb in GMC-1. Moreover, the levels of Pdm1 are responsive to both loss of function neur (Pdm1 level is up-regulated) and gain of function neur (the Pdm1 level is down-regulated), which are more likely a consequence of Neur function within GMC-1. This regulation of Insc and Numb localization appears to be via regulation of Pdm1 levels inside GMC-1, whereas regulating Notch processing is later and the source of Neur is from outside. By regulating asymmetric localization of Numb, Neur ensures that one of the two daughters is free of Numb, thus, later on the activation of Notch-signaling in that cell can occur. The source of Neur for this Notch processing appears to be from outside of the lineage since a division-arrested GMC-1 in numb; cyc A double mutant can still adopt a sib fate. Thus, the two roles of Neur in this lineage are distinct and separable. But then is it possible Notch has a role in the asymmetric localization of Numb and this activity of Notch is regulated by Neur? It certainly is possible but then one would have to disregard the presence of asymmetrically localized Neur in a GMC-1 as anything but of no consequence to the asymmetric division of GMC-1. It should also be pointed out that the identity of GMC-1 per se in neur is not altered, if it did, two neurons of some other identities would have been seen, not RP2s (or sibs) (Bhat, 2011).
A previous study in the sensory system of the PNS indicated that Neur protein localizes asymmetrically in the pI cell of SOP. It then segregates to pIIb, where it is thought to enhance the endocytosis of Dl to promote N activation in the pIIa cell. This represents a trans-differentiation mechanism to specify different cell fates. The results confirm the findings in SOP lineage but at the same time extends the data on SOP lineage in that this trans-determination process is a potentiation step to mediate a more efficient Notch-signaling-processing, but it is not necessarily a deterministic one. What is new and different from the SOP lineage is that Neur controls not only the asymmetric localization of Numb during mitosis, but also controls the localization of Insc, an apical cue that controls spindle orientation and participates in Numb basal localization. In neur mutant cells, Insc is no longer asymmetric indicating that Neur is somehow needed to localize Insc. The fact that Neur is somehow needed for Insc localization is also consistent with the finding that genetically insc is epistatic to neur, therefore that it is downstream of neur (Bhat, 2011).
Finally, while insc is epistatic to neur in the RP2 lineage defect in insc; neur double mutants, as for the neurogenic phenotype, neur is epistatic. This is not surprising since epistasis relationships are lineage/cell-type/tissue specific, depending upon whether or not the two genes in question are expressed in the same lineage and if the two single mutants give the same (or opposing) phenotype. Insc has no role during the neural versus ectodermal fate decisions and loss of function for insc does not cause a neurogenic phenotype, hence, the neurogenic phenotype of neur mutants is not expected to be present (epistatic) in the double mutant (Bhat, 2011).
It is clear from the results that Neur regulates asymmetric division of GMCs in the CNS. This was examined in at least two different GMCs, the GMC of the RP2/sib lineage (GMC-1 or GMC4-2a of NB4-2) and the GMC of the aCC/pCC lineage (GMC-1 or GMC1-1a of NB1-1). In neur, these GMCs symmetrically divide to generate two of the same cells, RP2 neurons in the case of GMC-1 and aCC neurons in the case of GMC1-1a. It is thought that many more GMC lineages are affected by loss of function for neur. Being a neurogenic protein, Neur is also involved in selecting neural versus ectodermal fates for the neuroectodermal cells. Due to its neurogenic property, the mutant will generate extra copies of many of the NBs in the nerve cord, which in turn, will generate more of the GMCs and neurons. Several lines of evidence indicate that symmetric division of a GMC indeed occurs at a high frequency in the CNS in neur mutants. For example, GMC-1 normally generates an RP2 and a sib, RP2 is larger than the sib and the two have distinct gene expression profiles and patterns. This is also the case for aCC/pCC pairs—they also have distinct gene expression profiles. These specific criteria were used to separate the ones that are generated by the symmetric division from those generated due to a neurogenic effect of neur mutation (Bhat, 2011).
Several additional pieces of evidence indicate a role for Neur in generating asymmetry. These include the asymmetric localization of Neur in GMCs, non-asymmetric localization of Numb in GMC-1 in neur mutants, non-asymmetric localization of Neur in numb mutants, genetic interaction results and effect on downstream players such as Pdm and Numb. All these results point to a specific role for Neur in regulating asymmetric mitosis of precursor cells (Bhat, 2011).
The results show that Neur itself is asymmetrically localized in GMC-1 to the Numb-domain and opposite to that of the Insc-domain (Neur is also localized to the basal end of several NBs, the significance of which is not known). In neur mutants, both Insc and Numb are not localized but found uniformly distributed along the cell cortex. This suggests that Neur is upstream of Insc and Numb localization but not their expression per se. The levels of Numb or Insc are also not affected in neur mutants indicating that Neur does not participate in Numb degradation (via ubiquitination, or otherwise). There is no evidence that Neur has a direct role in the localization of Numb. Do these results therefore mean Neur basically regulates the identity or the fate (i.e. gene expression program) of the GMC-1 prior to its division and therefore that Neur has only one function, which is potentiating Notch signaling? The GMC-1 was examined in neur mutants with several different GMC-1 markers (Eve, Pdm1, Zfh-1, Spectrin, etc.) and with the exception of a higher than normal Pdm1 in a late GMC-1, none of these markers were affected. A higher than normal levels of Pdm1 does not change the identity of a GMC-1. Indeed, several studies have shown that high levels of Pdm1 or its sister protein Pdm2 will induce a GMC-1 to undergo symmetric division to produce two GMC-1s and then two RP2s and two sibs. In order for a GMC-1 to change its identity, many of its genes should be turned off and a new set of genes has to be initiated. Such a drastic change does not occur in GMC-1 of neur mutants. Similarly, an identity change should result in this GMC-1 in neur mutants to produce different sets of neurons, which it does not. Instead, it produces two RP2s. Given these results and that Neur is necessary for the normal localization of Numb, whether this is directly mediated or indirectly mediated, the conclusion that Neur regulates asymmetric division at two different levels during the lineage development is based on firm grounds (Bhat, 2011).
The main question is how might Neur regulate Insc and Numb localization. A clue to this question comes from previous studies. It was shown that over-expression of Pdm POU transcription factors (Pdm1 or Pdm2) in GMC-1 causes non-localization of Insc and Numb and their segregation to both daughter cells of GMC-1; these cells then adopt an RP2 fate, with Numb blocking the N-signaling from specifying a sib fate. Pdm1 was up-regulated in GMC-1 in neur mutants and down-regulated with over-expression of Neur. This shows that the localization of Insc and Numb is altered in neur mutants indirectly via the up-regulation of Pdm protein. At the moment, it is not clear how an up-regulation of Pdm alters Insc or Numb localization. A most likely possibility is that Pdm proteins, being transcription factors, their over-expression may cause changes in the expression of genes that are needed for the proper localization of Insc and Numb but without altering the cell-identity itself (since this cell still produces RP2 neurons and not some other neurons). These conclusions are all consistent with the overall expression pattern and mutant effects of pdm genes: Pdm proteins are down-regulated in GMC-1 prior to its division, loss of function for Pdm causes loss of GMC-1 identity (Bhat, 2011).
The gain of function for these pdm genes indicates that the GMC-1 division is quite sensitive to varying levels and timings of expression of these POU proteins. For example, a high level of pdm gene expression in a GMC-1 from pdm transgenes causes a symmetric division of GMC-1 into two GMC-1s and then each of these GMC-1s generates an RP2 and a sib. In contrast, a symmetric division of GMC-1 into two RP2s can also be observed in these embryos. In this case, the cells from the GMC-1 express Zfh1; a GMC-1 does not continually express (a late GMC-1 about to divide does express Zfh1 at a very low level), a sib transiently expresses Zhf-1, and an RP2 stably expresses Zfh-1. Moreover, both these cells inherit Insc and Numb. No more cells are produced from these two cells, and each of these cells generates a projection as that of an RP2. When these genes are over-expressed for a prolonged period of time, a GMC-1 divides multiple times producing a GMC-1 and a differentiated progeny: First two unequal sized cells are observed. Only one of the two (the smaller cell) expresses markers such as Zfh1. Later on, three cells, and then five cells, etc., are sequentially seen, all in a tight cluster; from these clusters, as many as 5 RP2s are formed. Indeed, with this prolonged over-expression of pdm genes for 90 min from a heat shock promoter causes hemisegments with all the above types of divisions depending upon the time of over-expression. In contrast, it is not clear what the sensitivity range of GMC-1 is to varying concentrations in terms of the kind of division pattern generated. One clue to this comes from an earlier study, that GMC-1 in embryos carrying a duplication chromosome for the chromosomal region containing the two POU genes undergo a single self-renewing asymmetric division of GMC-1. This suggests that when the copy numbers for these genes are doubled, this presumably results in producing twice the amount of these proteins (from their own promoters), and causes a single self-renewing division. Having said that, it was also found that in neur mutants a GMC-1 rarely divides symmetrically into two GMC-1s and then each produces a sib and an RP2, or a GMC-1 dividing more than once with self-renewing asymmetric division as in pdm-GOF situations (Bhat, 2011).
Based on these results with gain of function for pdm genes, a loss of function for pdm genes should suppress the neur defects. However, this experiment is not possible to do since loss of function for the pdm genes causes loss of GMC-1 identity (GMC-1 becomes some other GMC) and therefore GMC-1 is undetectable with GMC-1 markers (Bhat, 2011).
While the exact mechanism as to how the level of Pdm1 is up-regulated in GMC-1 of neur mutants or down-regulated when Neur is over-expressed in GMC-1, is not known, one possibility is that Neur is involved in the degradation of Pdm1 in GMC-1. This scenario is most likely since Neur has the RING domain, one of the signature domains for E3 Ubiquitin-ligase proteins involved in protein degradation. Neur has also been shown to ubiquitinate proteins in vitro. One indication that Neur might be involved in the degradation of Pdm1 is the result that while ectopic or over-expression of full length neur from a transgene down-regulated Pdm1 and resulted in the same phenotype as loss of function for pdm genes, a similar ectopic or over-expression of a neur transgene missing the RING domain (Hs-neurΔRF) did not result in a down-regulation of Pdm1 or resulted in any phenotypes. Pdm1 appears to be specifically affected in GMC-1 of the RP2/sib lineage and not in other cells where Pdm proteins are present. Even if the up-regulation of Pdm proteins in neur mutants is via an indirect mechanism, say via factor X or Y, the results define a major role for Neur in regulating asymmetric division prior to the Notch-potentiation role of Neur: regulating Numb localization via down-regulating (directly or indirectly) Pdm proteins (Bhat, 2011).
Results from the analysis of neur, neur; numb double mutant embryos and neur gain-of-function embryos show that Neur functions to increase the efficiency of Notch-signaling but not essential for it. None of the previous studies have made this important distinction. Previous results have indicated that Neur activates Notch-signaling via endocytosis of Delta and the Delta-bound extracellular domain of Notch. However, in neur null mutants (embryos homozygous for a deficiency that removes neur completely), sib specification still occurs in ~ 10% of the hemisegments. While this may arguably be due to a partial redundancy for neur, there is another line of evidence that suggests a role for Neur in enhancing the efficiency of Notch signaling. That is, while in Notch; numb double mutants both daughter cells of a GMC-1 adopt an RP2 fate (note that for the specification of an RP2 fate Numb is needed only when there is an intact Notch-signaling), in neur; numb double mutants the daughters often adopt a mixed identity. This result indicates that Notch is still able to specify some features of a sib identity (i.e., reduced levels of Eve expression) in the absence of Neur activity. If Neur is absolutely needed for Notch signaling, the double mutant results would have been exactly the same as Notch; numb double mutants where both daughters adopt an unambiguous RP2 fate (Bhat, 2011).
In contrast, the results from Neur over-expression experiments indicate that when present at high levels Neur is able to overcome the Numb block and induce both the progeny of GMC-1 to adopt a sib fate. This phenotype is strikingly similar to the phenotype observed with the over-expression of the intracellular domain of Notch or the phenotype in numb mutants. These results suggest that over-expression of Neur leads to processing of Notch in the cell that has Numb. It is also pointed out that the source of Neur for the trans-effect on Notch-signaling need not be only from the “RP2” cell, but may also be from the neighboring cells. This is indicated by the previous result that while the GMC-1 in embryos mutant for cyclin A adopts an RP2 fate, the same GMC-1 in cyclin A; numb double mutants adopts a sib fate (Bhat, 2011).
These results show that the asymmetric basal localization of Numb in neur mutants and Neur in numb mutants is affected. This shows the interdependence of localization of these two proteins. Whether there is any initial localization of Numb or Neur in the two mutants was examined to determine if the localization of the one protein falls apart in the absence of localization of the other. However, no such initial localization was observed for either of the two proteins. It is possible that both Neur and Numb control the same pathway(s) that directly or indirectly mediates localization of the other. Perhaps Neur and Numb interact physically with each other in the cytoplasm prior to any localization and it is this Neur-Numb complex that gets localized to the basal pole; in the absence of either of the two proteins, no such complex is formed, and no localization occurs. This model has not been tested due to lack of appropriate reagents. In contrast, loss of Numb-localization in neur could be due to loss of Insc localization; loss of Neur localization in numb mutants could be more direct where Neur is downstream of Numb and Numb mediates directly or indirectly the localization of Neur. The function of Neur in GMC-1, however, appears to be required for the down-regulation of Pdm and allow localization of such proteins as Insc. Thus, Neur is both upstream and downstream of Numb in GMC-1. Another important distinction between Neur and Numb is that while non-asymmetric localization of Numb in GMC-1 will lead to both daughters of GMC-1 inheriting Numb and adopting RP2 fates, a non-asymmetric localization of Neur and inheritance of Neur by both daughters will not make them adopt an RP2 fate, but a sib fate (Bhat, 2011).
In numb mutants, the localization of Neur is affected in such a way that both daughters inherit Neur. Does this have a consequence? The results argue that unlike Numb there is no consequence to the non-asymmetric localization and segregation of Neur to both daughters. For instance, in wild type the sib cell does not inherit Neur, thus, the potentiation of Notch in this cell by Neur occurs in a cell non-autonomous mechanism (removing the extracellular domain of Notch bound by Delta) and there is no role for Neur in the sib itself. Thus, in numb mutants although both daughters inherit Neur, they still adopt a sib fate (Bhat, 2011).
The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. This paper reconstructs the complex morphogenesis of the optic lobe during the larval period, and establishes a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. These findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions (Ngo, 2010).
The structure of the optic lobe primordium of the larva is highly dynamic and, towards the later stages, very complex. As a result, only a rudimentary understanding is available of how the different neuropiles and cell types of the adult optic ganglia map onto the larval optic lobe primordium. Moreover, the dynamic changes in shape that characterize the optic lobe at the different larval stages make it very difficult to interpret mutant phenotypes of genes controlling optic lobe development. This study depicts the normal development of the larval optic lobe, focusing on the outer optic anlage and its derivatives, the distal (outer) medulla and the lamina (see Topology of the developing optic lobe in the larva; Ngo, 2010).
The OOA of the early larva starts out as an expanding rectangular sheet of epithelial cells, formed dorso-ventrally oriented columns of cells. Starting at the late first instar and continuing throughout larval life, the OOA epithelium bends along the dorso-ventral-axis. As a result, cells are aligned in C-shaped curves. What this spatial transformation means when looking at optic lobes sectioned along the 'standard' frontal plane (Ngo, 2010).
During the second larval instar, the OOA becomes subdivided into two domains, visibly separated by a furrow called lamina furrow. Cells lateral of this furrow (OOAl) give rise to the lamina; the much larger medial domain (OOAm) form the distal medulla. At around the time when the lamina furrow divides the OOA into a lateral and medial domain, epithelial cells along the edges of these domains convert into asymmetrically dividing medulla neuroblasts (Mnb). This transition can be followed effectively by labelling optic lobes with anti-Crumbs (Crumbs being expressed at the apical membrane of all ectodermally derived tissues). Once cells have converted to neuroblasts, they 'bud off' progeny in the direction perpendicular to the plane defining the OOA. Because of this directed proliferation, neurons born first come to lie at ever increasing distances from the neuroblast/OOA. At the same time as the medulla neuroblasts divide, new rows of neuroblasts appear as, one by one, rows of epithelial cells along the medio-lateral-axis convert into neuroblasts. In the late larva, medulla neuroblasts start to disappear. Thus, the lineages at the medial edge of the optic lobe, which had been the first to appear, are no longer capped by a neuroblast. The fate of the medulla neuroblasts after they cease to divide has not yet been followed in detail. Similar to neuroblasts of the central brain, they are likely to undergo programmed cell death (Ngo, 2010 and references therein).
The correlation between neuron position and birth date can be visualized by BrdU pulse-chase experiments. Early pulses (24 h) result in faint labelling of medulla neurons located deep. In this experiment, BrdU is taken up by all cells of the epithelial OOA which, around 24 h, divide symmetrically. As the epithelium converts into neuroblasts, all neuroblasts 'inherit' the (by then already diluted) label. When neuroblasts start their rapid asymmetric division, only the first born neurons receive enough BrdU to maintain detectable label; these are the neurons located deeply. Pulses administered at mid-larval stages (72 h) result in strong labelling of neurons located in the medial medulla at deep and intermediate levels. In this experiment, the BrdU pulse reaches the OOA at a stage when the epithelial cells have all but ceased to divide, and the medial cells have converted into rapidly dividing neuroblasts. These are the cells that incorporate BrdU. Due to the rapid division/dilution of the label, only early born neurons, located deeply, receive enough label. Late pulses, followed by immediate fixation, result in labelling of most neuroblasts and superficially located neurons. At this stage, the most medial lineages no longer proliferate (Ngo, 2010).
The description of OOA development above indicates that two spatio-temporal gradients are in the OOAm. One gradient, directed along the medio-lateral axis of the OOAm ('ml-gradient'), describes the sequence in which rows of neuroblasts are formed; the second gradient, directed from the surface inward, perpendicular to the plane of the OOA ('z-axis'), underlies the order in which each neuroblast produces neurons ('z-gradient'). The ml-gradient correlates with the anterior-posterior axis of the retina. Thus, axons that grow towards the first-born OOAm neurons, derived from the most medial row of neuroblasts, are the R7/8 axons originating from posterior retina, as well as L- neurons from the posterior lamina. The next set of axons, arriving later, captures neurons of the next (more lateral) row of OOAm neuroblasts, etc. What this implies is that the ordered progression of NE-NB conversion may match the progression of ingrowing axons, and that this matching may be important for the formation of an ordered medulla neuropile. The significance of the z-gradient has not yet been investigated. It seems likely that, similar to what is known for lineages of the central brain and ventral nerve cord, it accounts for the sequential generation of different neuronal cell types (Ngo, 2010).
The inner optic anlage (IOA) also undergoes a NE-NB conversion, bending along the dv-axis, similar to what has been described above for the OOA. Briefly, in the late larva, the IOA consists of a C-shaped epithelial component (IOAep). Further laterally, neuroblasts derived from the IOA (IOAnb) form a mass of cells that is also bent, and therefore seen twice in a frontal section. The IOA neuroblasts produce two populations of neurons. Neurons pushed anteriorly (or outward, taking into account the curvature of the IOA) become the proximal medulla (Mp); those pushed interiorly, or centrally, become the lobula and lobula plate (Lo) (Ngo, 2010).
Neural progenitors give rise to the diversity of cell types seen in the central nervous system (CNS). Intrinsic factors expressed in progenitors, as well as extrinsic cues from neighboring cells specify cell fate. In both vertebrates and invertebrates, the specification of cell types follows a highly invariant spatio-temporal pattern. Typically, one can distinguish an early phase where the pool of progenitors (e.g., the neuroepithelium of the neural tube in vertebrates) expands by symmetric cell division. Subsequently, progenitors start leaving the pool of expanding cells and either directly differentiate into specific cell types, or undergo asymmetric divisions where one daughter cell keeps the properties of a progenitor, whereas the other differentiates (Ngo, 2010).
Neurogenesis in the Drosophila optic lobe follows a similar pattern. Segregating from the embryonic neuroectoderm as a small epithelial placode, the optic lobe anlagen undergo a phase of growth by symmetric cell division in the early larva, followed by a highly ordered transition into asymmetrically dividing neuroblasts. The medio-lateral gradient that characterizes this transition in the OOA is correlated with the posterior-anterior gradient of eye development: photoreceptor axons of the earliest developing (posterior) row of ommatidia arrive first and capture the first born neurons, formed (in case of the medulla) from the medial edge of the OOA. Later born axons occupy medulla neurons forming later, at increasingly lateral levels. It is to be assumed that this temporal match between target neuronal development and afferent axonal development plays an important role for correctly wiring the optic lobe; this hypothesis, though, requires rigorous testing (Ngo, 2010).
This study shows that the Notch pathway is critically involved in the ordered NE-NB conversion. The most significant effect resulting from decreasing Notch function in the larval brain was the reduction in size of the epithelial optic anlagen, as shown by the loss of the epithelial marker, Crb. It is therefore likely that the function of Notch in the optic anlagen is to maintain its undifferentiated, neuro-epithelial state. Clonal analysis has led to the same conclusion. This would match a similar function of Notch in the embryonic neuroectoderm, where Notch activity is also required for cells to stay epithelial. The only difference is the topology of the neuroblast (i.e., cell that moves out of the epithelium): in the embryonic neuroectoderm, neuroblasts are mostly scattered cells, surrounded on all sides by epithelial cells. In the optic anlagen, there is a continuous front where all epithelial cells convert to neuroblasts. However, this difference aside, the way in which Notch signaling acts and is controlled during the NE/NB conversion could be quite similar in the embryonic neurectoderm and the late larval optic anlagen (Ngo, 2010).
Surprisingly, Notch activity, despite of its continued expression throughout development, appears to be dispensable during the earlier phase of optic lobe development during which the epithelial optic anlagen grow by symmetric mitosis. Neither early temperature shift experiments with Nts, nor temporally restricted optic lobe expression of Su(H)DN resulted in premature neuroblast formation. Also the active lifespan of the optic lobe neuroblasts appear to be independent of Notch activity. Optic lobes of late Nts larvae raised at the restrictive temperature were mostly devoid of (Dpn-positive) neuroblasts, and lineages or overall volume of the medulla primordium were not noticeably enlarged (Ngo, 2010).
Taken together, these findings suggest the following model of Notch signaling in the larval optic lobe. Moderate levels of the Notch ligand Dl, as well as Notch activity, are present in the entire optic lobe anlage of the early larva. Starting during the mid larval stage, the proneural gene l'sc is expressed at the medial margin of the OOA. This expression sets in motion a cascade of events that result in the ordered NE/NB conversion. L'sc locally upregulates Delta and other proneural genes (ase) that promote first the conversion of OOA epithelium to neuroblasts, followed by rapid asymmetric division and neuronal differentiation. At the same time, once cells have converted to neuroblasts, L'sc and Delta are downregulated, even though N stays on in a dynamic manner in neuroblasts and neurons. L'sc remains high in a laterally moving band of cells at the medial OOA margin. A second mechanism that may act on the localized Dl upregulation, where it appears that that the cell cycle arrest in the OOAm, caused by activation of the Fat/Hippo pathway, is prerequisite for the accumulation of Dl. Throughout the third larval instar, a continued, interdependent expression of L'sc and Delta in the OOA could be the mechanism that accounts for the slow, gradual release of neuroblasts from the OOA margin. Thus, L'sc is known to upregulate Delta in other neural precursors, and this could be the case also in the OOA. The L'sc induced peak in Delta levels at the medial OOA margin would then signal to its neighbors laterally, increasing Notch activity, and thereby preventing a premature advance of L'sc towards lateral (Ngo, 2010).
How initiation and maintenance of L'sc is controlled is still unclear. Wingless (Wg), a known activator of proneural genes in other tissues, is expressed in a fairly restricted pattern in the apices of the OOA. It is possible that a long range effect of Wg could be responsible for L'sc activation along the OOA margin (Ngo, 2010).
A large number of studies in vertebrate and invertebrate systems alike suggest that the fundamental role of N in the developing nervous system is to maintain cells in an undifferentiated (neuroepithelial) state at any given moment. Cells released from N activity enter a differentiative pathway (typically accompanied by structurally visible changes, such as a switch from epithelial cell to neuroblast, and/or a switch in mitotic behavior (symmetric vs. asymmetric). The temporally controlled release/birth of neurons from the neuroepithelium is often tied to different cell fates. This has been shown very convincingly in the retina of vertebrates and Drosophila. For example, in the vertebrate retina, the first wave of differentiation results in ganglion cells, the second wave of differentiation at a later point includes photoreceptors, followed by bipolar cells, and others. If N activity is reduced at an early time point, the number of ganglion cells produced increases massively, at the expense of later born cell types. In Drosophila, the first retinal cell type to be born behind the morphogenetic furrow is the R8 photoreceptor. If at the time of R8 specification, N function is decreased, the number of R8 cells is increased, and other cell types born later are decreased. With later pulses of N depletion, one gets different phenotypes; what they all have in common is that the cell types born at the time of the pulse are increased in number, the ones born later decreased (Ngo, 2010).
In the Drosophila OOA investigated in this paper, the temporal progression of neuroblast formation that is controlled by N activity is linked to the coordinated growth between eye (and lamina) and medulla, derived from the OOA. However, it is well possible that the temporal progression is also tied into the control of different cell fates. The way in which the multitude of different medulla cell types map onto the larval optic lobe is not clear. It is most likely that (as in the lineages of the ventral nerve cord) most cell types are specified along the z-axis, which would imply that each part of the OOA (in the medio-lateral and dorso-ventral dimension) would produce the same cell types. However, it is well possible that some cell types which are actually not found in all medulla columns, such as wide field tangential neurons, are produced by different parts of the OOA. Such cell types then might be affected by premature or delayed conversion of the OOA into neuroblasts; identifying specific markers that label cell types at early stages, and using such markers in the background of Notch loss or overactivity, will help clarifying this question (Ngo, 2010).
A role of the Notch pathway has been described for later stages in neural development, that is, the specification of neurons from ganglion mother cells (GMCs). Asymmetric neuroblast proliferation in the ventral nerve cord and brain produces a series of GMCs which each divides one more time into two, often different, neurons/glial cells. It has been shown that this fate choice between sibling pairs depends on Notch activity, both during embryonic and post-embryonic stages. Such may also be the case for the neuroblasts emerging from the OOA. The relatively high level of the Notch reporter, E(spl)m8-lacZ maintained in the OOA-derived lineages would speak for a continued role of Notch in these cells; however, detailed investigations of the neurogenesis of these lineages need to be carried out in order to address the potential later Notch function (Ngo, 2010).
This study found that reduction in Stat activity causes a premature loss of the epithelial state of the OOA. This is accompanied by accelerated proliferation and gross abnormalities in the architecture of the optic lobe neuropile, as also seen in Notch mutant brains. Furthermore, continued activity of Stat in the epithelial OOA is dependent on Notch, and vice versa. The mutual interaction between both signaling pathways is most likely indirect, mediated via a number of intermediate steps. Thus, Delta levels are normal in optic lobes of Statts mutant brains up to 48 h after hatching. It is only during later stages, when the structurally visible premature change of OOA epithelium to neuroblasts occurs in the Statts mutant, that Delta expression is reduced. It remains to be seen what are the intermediate genetic events that interconnect the signaling activities of the Notch and Stat pathway (Ngo, 2010).
The larval optic lobe represents but one of many scenarios in which interdependency between Notch and Stat have been reported. The types of genetic interactions between these pathways appear to be as diverse as the developmental events or cell fates which they control. For example, in the Drosophila ovary, mutual inhibition Notch and Stat set up the boundary between the stalk (Jak/Stat dependent) and the main-body follicle cells (Notch-dependent. In the adult midgut, Notch is necessary for the differentiation of cells derived from the intestinal stem cells (ISCs) into enteroblasts and enterocytes. Thus, high Notch activity in one of the daughter cells derived from an ISC division prompts these cells to become an enteroblast (EB), which then either differentiates into enterocyte (EC) or enteroendocrine cell (EE). High Notch activity in the EB promotes the EC fate; low Notch activity allows for the formation of EEs. Jak/Stat signaling intersects with the Notch pathway at multiple steps: for example, it acts upstream in an activating manner. Thus, under stressful conditions (e.g. bacterial infection or JNK-induced stress), Stat functions to induce Notch to allow for self-renewal and proliferation. In addition, Jak/Stat is required during the differentiation of different ISC-derived cell types. The same kind of dynamic and complex relationship between the two signaling pathways can be seen in the eye, where Stat can function both upstream and downstream of Notch. It has even been suggested that Jak/Stat can function to inhibit Notch as well. From all these studies, it is clear that many of the intermediates which link Notch and Jak/Stat signaling are still unknown. One can envision scenarios where subtle differences in the spatial distribution and timing of signals may contribute to how Stat and Notch interact during development. The Drosophila optic lobe is likely to present a highly favorable system to address these complexities which impact the role of the two signaling pathways (Ngo, 2010).
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).
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