Notch
Hartenstein (1992) has extensively documented the complex effects of Notch mutation on different aspects of morphogenesis. A summary of these findings is found in the Notch pathway site.
Notch and neurogenesis: The neuroectoderm and the CNS The loss of dynamin activity, which is encoded by the gene shibire and is required for endocytosis, results in a neurogenic phenotype similar to that caused by mutation in Notch. The requirement of shibire function for Notch signaling during the segregation of sensory bristles on the notum of the fly has been investigated. Overexpression of different constitutively active forms of Notch in shibire mutant flies indicates that shibire function is not necessary for transduction of the signal downstream of Notch, even when the receptor is integrated in the plasma membrane. However, when wild-type Notch is activated by its ligand Delta, dynamin is required in both signaling and receiving cells for the normal singling out of precursors. shibire is epistatic over the Abruptex coded gain of function form of Notch, which results from a point mutation in the extracellular domain. These results suggests an active role on the part of the signaling cell for ligand-mediated receptor endocytosis in the case of transmembrane ligands. It is suggested that Dynamin requirement in the signaling cells reflects Dynamin function in an active pinching off of the macrovillar process of the signaling cells, allowing internalization of the membrane-bound ligand into the receiving cell (Seugnet, 1997b).
Loss of any one of several neurogenic genes of Drosophila results in overproduction of embryonic
neuroblasts at the expense of epidermoblasts. Dominant lethal, antineurogenic phenotypes were produced by
expression of three classes of mutant proteins: (1) a protein comprised of the cytoplasmic domain of
Notch and devoid of sequences permitting membrane association; (2) a transmembrane protein
lacking the extracellular, lin12/Notch repeats; and (3) transmembrane proteins carrying amino acid
substitutions replacing one or both extracellular cysteines thought to be involved in Notch
dimerization. These Notch proteins not only suppress the neural hypertrophy observed in Notch-
embryos, but also generate a phenotype in which elements of the embryonic nervous system are
underproduced. Action of the intracellular cdc10 repeats appears to be essential for wild-type
Notch function or for the antineurogenic activity of these proteins. The activities of the dominant,
gain-of-function proteins indicate that Notch functions as a signal transducing receptor during
ectoderm development. Production of antineurogenic Notch proteins in embryos deficient for the
other neurogenic genes allowed functional dependencies to be established. Delta, mastermind,
bigbrain, and neuralized appear to function in elaboration of a signal upstream of Notch. Genes of
the Enhancer of split complex act after Notch. The cytoplasmic domain of Notch contains nuclear
localization sequences that function in cultured cells, and one of the Notch antineurogenic proteins,
the cytoplasmic domain, accumulates in nuclei in vivo (Lieber, 1993).
The Notch gene encodes a receptor protein that is involved in many processes during development. Its best understood role occurs
during neurogenesis, in a process called "lateral inhibition." However, it has been proposed that Notch also has a role in first defining
the proneural clusters. This raises the possibility that the Notch protein is acting as a multifunctional receptor.
To test this hypothesis, a genetic analysis was carried out of molecularly characterized Notch alleles to identify alleles that
affect only one of the two proposed functions. Notch alleles can be identified that appear to
affect the function of Notch during either lateral inhibition or the definition of proneural clusters. Hypermorphic mutations increase bristle number in comparison to wild type while hypermorphic mutations decrease bristle number. It is concluded that there are at least two functions for Notch: the hypermorphic mutations functioning in the initiation of bristle development and the hypomorphic mutations functioning during lateral inhibition. These results indicate
that there may be discrete regions of the Notch protein required for each function. Lateral inhibition requires the extracellular EGF repeats 10-12 that bind Delta, as well as an intracellular region, the RAM-23 domain and the cdc10 repeats, which are involved in the implementation of the Delta signal. The second functional module contains a region, centered around EGF repeats 24-26: this might be involved in binding an extracellular ligand. Another region, which probably lies in the area C-terminal to the cdc10 repeats, is involved in linking the Notch receptor to an intracellular signaling cascade other than Suppressor of Hairless (Brennan, 1997).
Transplantation of single homozygous Notch minus cells into the ventral neuroectoderm of Drosophila wild-type embryos reveals the non-autonomous behaviour of these cells. Notch mutant cells in proneural territories do not express the neural phenotype autonomously, as they should if these cells were behaving autonomously. However, mitotic recombination events induced in heterozygous cells in imaginal discs lead to the generation of homozygous Notch- cells, which do differentiate cell-autonomously. Various possible explanations have been tested for the non-autonomous behaviour of Notch mutant cells following transplantation. Previous results have been confirmed using different Notch alleles. Increasing the number of wild-type Notch copies in a cell increases the probability that it will take on an epidermal fate. However, single Notch mutant cells behave differently, depending on whether they are placed in the ventral neuroectoderm, the procephalic neuroectoderm or the proctodeal anlage. Following transplantation into host embryos devoid of mesoderm, almost all single Notch mutant cells behave autonomously. Thus, the presence of Notch + cells in the mesoderm can influence cell fate in the ectoderm. Absence of a mesodermal layer correlates with a higher proportion of neural mutant Notch clones, that is to say, with a cell-autonomous expression of the Notch phenotype. These results suggest an influence of the mesoderm on ectodermal development. Further evidence strongly suggests that Delta acts as the signal source in lateral inhibition (Stüttem, 1997).
Mutations in Notch and wingless have been used to study the process of neurogenesis in the neuroectoderm. Patterns of delamination and mitosis are closely correlated: delamination occurs
either immediately after a cell has divided (in the case of microchaete precursors) or shortly before the
division (in the case of neuroblasts). In addition, cytoskeletal changes similar to those occurring
during mitosis can be seen in delaminating neuronal precursors. Thus, during both mitosis and
delamination, the discrete apicobasally oriented microfilament-tubulin bundles break down.
Microfilaments form a dense, diffuse cortical layer surrounding the entire cell body. Microtubules
are concentrated at the apically located centrosome. The relationship between mitosis and
delamination is supported by the finding that the neurogenic gene Notch and segment polarity gene
wingless (wg) affect both proliferation and delamination in the ventral neurectoderm. Thus, in
embryos expressing the truncated cytoplasmic domain of the neurogenic gene Notch under
heat-shock control, all ventral neurectodermal cells go into mitosis
prematurely, followed by the absence of neuroblast delamination. In wg loss-of-function mutants,
mitosis in the ventral neuroblast is irregular and generally postponed, accompanied by irregularities in the timing of
neuroblast delamination in general and the absence of a subset of neuroblasts (Hartenstein, 1994).
The CNS midline of Drosophila should not be considered as an isolated autonomous entity but as an organizing center for the rest of the CNS. Cells located at the midline of the developing central nervous system perform a number of conserved
functions during the establishment of the lateral CNS (the rest of the CNS as distinguished from the midline). The midline cells of the Drosophila CNS are required for correct pattern formation in the ventral ectoderm (which gives rise to the rest of the CNS) and for
induction of specific mesodermal cells. The midline cells are also required for
the correct development of lateral CNS cells. Embryos that lack midline cells through genetic
ablation show a 15% reduction in the number of cortical CNS cells. A similar thinning of the ventral
nerve cord can be observed following mechanical ablation of the midline cells. A
number of specific neuronal and glial cell markers have been identifed that are reduced in CNS midline-less embryos, as for example in
single-minded embryos, in early heat-shocked Notch(ts1) embryos or in embryos where
the midline cells have been mechanically ablated. Genetic data suggest that both neuronal and glial midline cell lineages are required for differentiation of lateral CNS cells. One marker, the rR226 enhancer trap insertion, reveals a reduction in the number of marker positive cells in midline ablated embryos. It is thus concluded that
the CNS midline plays an important role in the differentiation or maintenance of the lateral CNS cortex (Menne, 1997).
strawberry notch synergistically affects Notch phenotypes in a tissue-specific manner. For example, split alleles of Notch affect the eye, while notchoid alleles affect the wing. In double mutant combinations with sno, the phenotypes in these respective tissues are enhanced. This suggests a role for sno in many independent Notch-related pathways (Coyle-Thompson, 1993)
Previous studies of big brain (bib) genetic interactions and expression agree that bib acts as a channel protein in proneural cluster cells that adopt the epidermal cell fate and serves a necessary function in the response of these cells to the lateral inhibition signal. These prior studies had not revealed any interaction between big brain and the other neurogenic genes. The neural hypertrophy in big brain mutant embryos is less severe than that in embryos mutant for
other neurogenic genes. This paper shows that bib cannot rescue the phenotype in
embryos mutant for other neurogenic genes. Ectopic bib
expression does not rescue the cuticle-defect of embryos mutant for Dl, N, E(spl), mam or neu. In reciprocal experiments, ectopic Dl and neu do not rescue the neurogenic
phenotype in bib mutant embryos. In contrast, ectopically activated
N still has an antineurogenic effect in bib mutant embryos. These results
indicate that bib, Dl and neu cannot functionally replace one another and that bib functions upstream of or parallel to activated
N. Using mosaic analysis in the adult, it has been demonstrated that big brain activity is required autonomously in epidermal precursors to prevent neural development. Ectopically
expressed big brain acts synergistically with ectopically expressed Delta and Notch,
providing the first evidence that big brain may function by augmenting the activity of
the Delta-Notch pathway (Doherty, 1997).
Sanpodo regulates Notch-mediated sibling cell fate decisions but
is not involved in Notch-mediated lateral inhibition. Notch functions in the neurogenic ectoderm to limit the number of cells adopting a neural fate. spdo mutation does not alter the number of neuroblasts that delaminate from the ectoderm, but instead is involved only in regulating sibling cell fate in the progeny of neuroblasts. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation
characteristic of Notch mutations. Mutations in two other genes, Delta (10 alleles)
and mastermind (1 allele) have been identified that yield similar equalization of sibling neuron fates. Because both Delta and mastermind are in the well-characterized Notch signaling pathway, null and hypomorphic alleles of several 'Notch pathway'
genes have been tested: Delta, Notch, mam, neuralized and E(spl). Mutations in all these genes result in an excess of neuroblasts due to failure of lateral inhibition within the neuroectoderm.
However, mutations in neuralized and E(spl) have no effect on the
identity of the sibling neurons that were assayed, despite strong
defects in the earlier process of neuroblast formation. In contrast, Delta, Notch and mam mutations all yield similar sibling neuron phenotypes, in addition to excessive neuroblast
formation. These results can be illustrated using embryos homozygous for a hypomorphic mam allele in which neuroblast formation is essentially normal but sibling neuron fates are equalized. Loss of mam does not affect eve expression in GMCs, but leads to the duplication of RP2, Usib, aCC and dMP2 fates at the expense of the RP2sib, U, pCC and vMP2 fates, respectively. Thus, mutations in three genes (Delta, Notch and mam) have precisely the same sibling neuron phenotype as spdo mutations, suggesting that spdo, Delta, Notch and mam act together to specify asymmetric sibling neuron fate (Skeath, 1998).
Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the
Drosophila embryo. In one of the well-studied neuronal lineages in
the ventral nerve cord (the NB4-2->GMC-1->RP2/sib lineage), Notch (N) signaling interacts with asymmetrically localized Numb (Nb) to
specify sibling neuronal fates to daughter cells of GMC-1. The NB4-
2 is delaminated in the second wave of NB delamination during
mid stage 9 (~4.5 hours) of embryogenesis and is located in the 4th row along the
anteroposterior axis and 2nd column along the mediolateral
axis within a hemisegment. The NB4-2 generates its first GMC
(GMC-1, also known as GMC4-2a) ~1.5 hours after formation.
The GMC-1 divides ~1.5 hours later to generate two cells, the
RP2 and the sib. The RP2 cell eventually occupies its position
in the anterior commissure along with the other RP neurons
(RP1, RP2, RP3 and RP4) and projects its anteroipsilateral
axon to the intersegmental nerve bundle (ISN) and innervates
muscle #2 on the dorsal musculature. The sib cell migrates to
a position posterior and more dorsal to RP2. The DiI tracing of the NB4-2
lineage indicates that the sib has no axonal projection at mid
stage 17 of embryogenesis; thus, its
ultimate fate has not been determined. In this study, loss-of-function
mutations in N and nb, cell division mutants cyclinA (cycA), Regulator of cyclin A1
(rca1) and string/cdc25
phosphatase (stg/cdc25
phosphatase), and the microtubule destabilizing agent, nocodazole, were all used to investigate
asymmetric cell fate specifications by N and Nb in the context of cell cycle. Mutation in rca1 gene was initially identified as a dominant
suppressor of roughex (rux) eye phenotype. In rux, the cells enter
S-phase precociously due to ectopic activation of a CycA/Cdk
complex in early G1 (Dong, 1997). In embryos lacking the
rca1 activity, the cells appear to arrest in G2 of the cell cycle (at stages 15-
16) similar to cycA mutants (Wai, 1999 and references).
The loss of cycA, rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1
exclusively adopts an RP2 identity. The requirement of cycA or rca1 for cell division in the
CNS is lineage specific.
Anti-Eve staining of cycA or rca1 mutant embryos indicates
that loss of these gene products does not affect all the Eve-positive
lineages in the ventral nerve cord. Eve is expressed in
other neuronal lineages such as the CQs, the Us and the ELs. The CQs are formed from NB7-1, an S1 neuroblast. The GMC for these
neurons are formed at the same time as the GMC for the
aCC/pCC neurons (generated from another S1 neuroblast,
NB1-1) and divide at the same time as GMC for the aCC/pCC
lineage. The NB7-1 in cycA or rca1 mutants
does not divide to generate an Eve-positive GMC for the CQs.
However, the effect on CQs is partially penetrant in both
the mutants. Thus, ~75% of the hemisegments had missing CQs in cycA mutants; in rca1
mutants, this figure is ~50%. The effect on the generation of U
neurons is as follows: in cycA mutants, the effect is fully
penetrant; whereas, in rca1 mutants, 65% of the hemisegments
were missing the Us. It must be pointed out that in those
hemisegments where these neurons (Us and CQs) are formed,
the number of these neurons is fewer than normal. Finally, the
effect of the loss of cycA or rca1 on another Eve-positive lineage,
the EL neurons, is minimal. The EL neurons are formed from
NB3-3, an S4
neuroblast (the formation of this neuroblast extends between
S3-S5). None of the hemisegments have
missing EL neurons, in either the cycA mutants or the rca1 mutants.
The above result indicates that the loss of rca1 or cycA does
not affect the division of all neuroblasts. One possibility for this result is that
the maternal deposition of these gene products is masking the
zygotic loss of these gene products in these lineages. However,
this seems unlikely since the GMCs for the
aCC/pCC or the RP2/sib lineages are generated earlier than the
GMCs for the EL neurons. Moreover, the maternal deposition
of CycA, for example, is completely exhausted before stage 7 and none of the
neuroblasts have delaminated from the neuroectoderm at this
stage of development. Thus, these results
indicate that the effect of loss of cycA or rca1 is lineage specific
and every neuronal lineage is not sensitive to the loss of these
cell division genes. It is most likely that some other cyclins
(i.e., Cyclin B) complement the loss of CycA in these lineages (Wai, 1999).
While the loss of N leads to the specification of RP2 fates to both
progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1
in the double mutant between nb and cycA assumes a sib fate.
While the GMC-1 fails to divide to generate two
cells in these double mutants, the GMC-1 assumed a sib fate.
About ~35% of the hemisegments show this
phenotype. This penetrance of the phenotype is slightly higher
than the phenotype observed in nb single mutants alone. This
suggests that cycA mutation has an enhancing effect on the nb
phenotype. This would argue that normally a small amount of
the Nb protein segregates into a sib cell and that, in the absence
of cell division, all of Nb is accumulated in one cell, and therefore, is much
more effective in blocking the N signaling. Moreover, since the
nb phenotype is epistatic to the cell division mutant phenotype,
Nb must be acting downstream of these genes. This result is
consistent with the finding that Nb becomes localized during
metaphase and is not localized in stg mutants. Thus, in rca1 or cycA mutants, the absence of a
localized Nb prevents the N signaling from specifying sib fate
and, as a result, the GMC-1 assumes an RP2 fate.
These epistasis results indicate that both N
and nb function downstream of cell division genes and that progression through cell cycle is required for the
asymmetric localization of Nb. In the absence of entry into metaphase, the Nb protein prevents the N signaling
from specifying sib fate to the RP2/sib precursor. These results are also consistent with the finding that the
sib cell is specified as RP2 in N;nb double mutants. Finally, these results show that nocodazole-arrested
GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that
microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib
fate specification (Wai, 1999).
During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, Ed has been shown to have a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed it has been shown that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. This work establishes a role for Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway (Ahmed, 2003).
Notch signaling and the mesectoderm Notch signal transduction appears to involve the ligand-induced
intracellular processing of Notch, and the formation of a processed
Notch-Suppressor of Hairless complex that binds DNA and activates the
transcription of Notch target genes. This suggests that loss of either
Notch or Su(H) activities should lead to similar cell
fate changes. However, previous data indicate that, in the Drosophila blastoderm embryo, mesectoderm specification
requires Notch but not Su(H) activity. The
determination of the mesectodermal fate is specified by Single-minded
(Sim), a transcription factor expressed in a single row of cells
abutting the mesoderm. The molecular mechanisms by which the
dorsoventral gradient of nuclear Dorsal establishes the single-cell
wide territory of sim expression are not fully understood. Notch activity is required for sim
expression in cellularizing embryos. In contrast, at this stage,
Su(H) has a dual function. Su(H) activity is required to up-regulate sim expression in the mesectoderm, and to
prevent the ectopic expression of sim dorsally in the
neuroectoderm. Repression of sim
transcription by Su(H) is direct and independent of Notch
activity. Conversely, activation of sim transcription by Notch
requires the Su(H)-binding sites. Thus, Notch signaling appears to
relieve the repression exerted by Su(H) and to up-regulate sim
transcription in the mesectoderm. A model is proposed in which
repression by Su(H) and derepression by Notch are essential to allow
for the definition of a single row of mesectodermal cells in the
blastoderm embryo (Morel, 2000).
To gain insight into the molecular mechanisms by which Su(H) and
Notch regulate sim expression, an examination was carried out to see whether Su(H) regulates sim expression in a direct manner. The regulatory elements necessary for mesectodermal expression of sim are contained within a 2.8-kb genomic DNA region. Sequence analysis has
identified 10 putative Su(H)-binding sites, with 6 of these exactly
matching the GTGRGAA consensus binding sites (Su4, Su5, Su7, Su8, Su9, and Su10). In gel shift experiments, Su(H)
binds strongly to oligonucleotides corresponding to each of these sites. Two additional sites, Su2 and Su6, match the consensus RTGRGAR that accomodates
nearly all sites that have been shown to bind Su(H) in vitro. These two sites bind weakly to Su(H), both
in direct binding assays and in competition
experiments. The ability of two noncanonical sites, Su1 and Su3, to bind Su(H) in vitro was also examined. Both Su1 and Su3 bind weakly to Su(H). Other sequences that differ
from the RTGRGAR at a single position are not known to bind Su(H) in
vitro. Thus, the sim regulatory sequences contain at least 10 binding sites for Su(H). Eight of these sites are clustered in a 500-bp
region that contains functional binding sites
for Dorsal, Twist, and Snail. Moreover, the organization of this regulatory region has been
conserved throughout evolution between D. melanogaster and
D. virilis. Together, these data
strongly suggest that Su(H) regulates sim transcription
directly (Morel, 2000).
Su(H) not only mediates the Notch-dependent activation of sim transcription, but also acts as a transcriptional repressor. This latter conclusion is supported by
the following two findings: (1) a complete loss of Su(H) activity leads to weak ectopic expression of sim in the
neuroectoderm; (2) the deletion of all of the Su(H)-binding sites
from the sim regulatory region also results in ectopic
activation of the sim promoter in the ventral neuroectoderm.
In Notch mutant embryos, repression by Su(H) is observed not
only in the neuroectoderm, but also in the mesectoderm. Because
Su(H) is expressed maternally,
it is speculated that uniformly localized Su(H) might repress the
activation of sim transcription in all of the cells in which
Notch is not activated (Morel, 2000).
This study provides the first evidence that Su(H) can act as a
transcriptional repressor in Drosophila, and that its
repression activity is inhibited by the activation of the Notch
receptor. In mammals it has been suggested that the binding of
processed Notch to CBF1 competes with the binding of corepressors to
CBF1 to promote the formation of an activation complex. The results presented here suggest that Su(H) might mediate such a transcriptional switch at the sim promoter in mesectodermal cells (Morel, 2000).
This regulatory mechanism, in which transcriptional repression is
inhibited by a signaling input, may be a general feature of
Notch-mediated gene regulation. Consistent with this view, repression
by Su(H) might contribute to the difference seen between Notch
and Su(H) mutant cuticular phenotypes. The finding that Su(H) can repress a Notch target gene
indicates that phenotypic differences between Notch and
Su(H) mutations do not necessarily imply that Notch signals in
a Su(H)-independent manner (Morel, 2000).
How is a single-cell wide territory of sim expression
established on the basis of the nuclear gradient of Dorsal? The data presented here, together with previous studies, suggest the following model. In the mesoderm, transcriptional activation of sim by Dorsal and Twist is inhibited by Snail. Whether Su(H)
and/or Notch play any role in these cells is not known.
In more dorsal cells that do not accumulate Snail, it is proposed that
positive regulation of sim by low levels of Dorsal and Twist
is antagonized by Su(H). However, in cells bordering the mesoderm,
negative regulation by Su(H) would be relieved locally by Notch
signaling. This would lead to the specific expression of sim
in these cells, which will then form the mesectoderm (Morel, 2000).
An important feature of this model is that Notch signaling overcomes
repression by Su(H) only in the single row of cells abutting the
mesoderm. One possible explanation for this is that Notch participates
in the contact-dependent reception of a mesodermal signal. Results from
nuclear transplantation experiments support the existence of a
mesodermal signal. When transplanted into
snail/twist double mutant embryos that do not
express sim, wild-type nuclei can induce the expression of
sim in neighboring mutant cells. This
result suggests that, in wild-type embryos, mesodermal cells may
produce an inductive signal that activates sim transcription
in the mesectoderm. Although the molecular nature of this signal is not
known, it is speculated that this mesodermal signal might participate in
the activation of Notch (Morel, 2000).
Consistent with the view that Notch is specifically activated in
ventral cells, changes in the subcellular distribution of both Notch
and Delta have been observed ventrally in stage 5 embryos. (1) Lower
levels of Notch are found in ventral cells as the ventral furrow forms. (2) In cellularized embryos, Delta is found at
the cell membrane, except in ventral cells, in which it predominantly
accumulates in vesicles. Both down-regulation of
Notch and vesicular accumulation of Delta are consistent with Delta
activating Notch in ventral cells in stage 5 embryos (because Snail
represses sim transcription, activation of Notch in the
mesoderm may have no effect on sim transcription). It will
thus be of interest to determine whether these changes in the
subcellular distribution of Notch and Delta can be observed in both
mesodermal and mesectodermal cells, but not in the more dorsal
neuroectodermal cells (Morel, 2000).
In conclusion, repression by Su(H) can be viewed as a refining
mechanism ensuring that Notch target genes are expressed only in cells
reaching a high threshold of Notch activation. In the early embryo,
repression of sim expression allows for the definition of a
single row of mesectodermal cells. In these cells, a high level of
Notch activity might be induced by a juxtacrine (contact-dependent) inductive signal produced by the mesoderm. In view of this hypothesis, the sharp mesodermal boundary defined by snail expression
would be shifted dorsally by one cell, thereby defining a single row of
mesectodermal cells (Morel, 2000).
The activity of Notch is required
for the transcriptional activation of the sim gene in the
mesectoderm, and Su(H) directly regulates sim expression.
However, both the sim gene and the
simmut-lacZ construct that does not respond to activated
Su(H) are expressed in mesectodermal cells in the complete absence of
Su(H) activity. These results
might suggest that Notch signals, at least in part, in a
Su(H)-independent manner to activate sim expression in the
mesectoderm. Alternatively, the observation that Su(H) acts to repress
sim expression raises the possibility that Notch might be
required to antagonize repression by Su(H). To distinguish between
these two possibilities, the expression of
simmut-lacZ was examined in Notch mutant embryos. simmut-lacZ is expressed at a low level
both in the mesectoderm and ectopically in the dorsal neuroectoderm. This pattern is very similar to that observed for
simmut-lacZ in wild-type embryos, and dramatically
differs from the complete loss of sim-lacZ expression seen in
Notch mutant embryos. This shows that the Su(H)-binding sites
are required to repress sim transcription in the mesectoderm
as well as in the neuroectoderm in the absence of Notch signaling.
Furthermore, this demonstrates that repression of sim
expression by Su(H), both in ventral neuroectodermal and mesectodermal
cells, does not require Notch activity. It is concluded that Su(H)
acts as a Notch-independent repressor. Thus, no evidence has been found for a
Su(H)-independent function of Notch in the regulation of sim expression (Morel, 2000).
Notch and gut morphogenesis Early in development the Drosophila endoderm segregates into three non-neural cell types: the principle midgut epithelial
cells, the adult midgut precursors, and the interstitial cell precursors. This process requires proneural and neurogenic genes. In
neurogenic mutants the principle midgut epithelial cells are missing and the other two cell types
develop to great excess. Consequently, the midgut epithelium does not form. In achaete-scute
complex and daughterless mutants the interstitial cell precursors do not develop and the number of
adult midgut precursors is strongly reduced. Development of the principle midgut epithelial cells and
formation of the midgut epithelium is restored in neurogenic proneural double mutants. The
neurogenic/proneural genes, in contrast to the neuroectoderm, are not expressed in small clusters of
cells but initially homogeneously in the endoderm, suggesting that no prepattern exists which
determines the position of the segregating cells (Tepass, 1995).
Notch and salivary gland development To identify X chromosomal genes required for salivary gland development in the Drosophila embryo, embryos hemizygous for EMS-induced lethal
mutations were screened to find mutations causing gross morphological
defects in salivary gland development. The parental
strain carried a lac Z transgene on the second
chromosome, which was specifically expressed in the
salivary glands so the mutations could be unambiguously
identified. Embryos from 3,383 lines were tested for salivary
gland abnormalities following lacZ staining. From
63 lines exhibiting aberrant salivary gland phenotypes,
52 stable lines were established containing mutations affecting
salivary gland development. From these, 39 lines could be assigned to nine complementation groups: armadillo, brinker, folded gastrulation, giant, hindsight, Notch, runt, stardust and twisted gastrulation (Lammel, 2000).
The identified X chromosomal
genes with respect to their possible contributions
to salivary gland development is discussed here. For mutations in giant
and Notch, severe defects or the absence of the salivary
glands have been described previously. In giant mutant larvae, the gnathocephalic
structures are affected, which correspond to the labial
segment. This is the region where most of the salivary gland anlage originates. giant is required for the expression of Sex combs reduced (Scr), the master regulator for salivary gland development.
Scr expression is absent in the labial lobe in giant mutations. The small glands seen in the absence of giant may form from remaining
cells of PS 2 still expressing Scr protein. Notch is involved in the formation of epidermal cells, and in its absence neural precursor cells are formed
instead. As a result, in Notch mutants a
coherent sheet of epidermal cells is found only in the
most dorsal position, outside the neurogenic ectoderm.
Only a few ventrolateral epidermal cells are left, which
may fail to form a salivary gland anlage. Similar explanations
may hold for mutations such as stardust, which
affect other general aspects of epithelial development,
since in the presence of these mutations the maintenance
of coherent epidermal sheets is disrupted (Lammel, 2000 and references therein).
Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).
A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).
The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).
Activated Notch cooperates with scrib-, resulting in neoplastic overgrowth, and although no anti-apoptotic role for Notch signaling in the eye has been described previously, NACT exerts hyperproliferative effects in flies, and Notch signaling is required for proliferation of eye disc cells. Although it is not known if NACT induces the same critical downstream targets as RasACT to cause overgrowth of scrib- tissue, removing ras function in scrib- cells overexpressing NACT rescues the overgrowth phenotype, suggesting that the effects of NACT are at least partially dependent on Ras (Brumby, 2003).
Initially it seemed likely that the cooperative effects of RasACT or NACT on scrib- tissue could be explained by the ability of these oncogenes to promote cell proliferation while blocking apoptosis. However, the expression of neither cyclin E nor E2F1/DP, in combination with the apoptosis inhibitor p35 (or with the inhibitor of JNK pathway activity, BskDN), was capable of phenocopying the effect of RasACT or NACT in scrib- clones. It is therefore suggested that other downstream effectors, apart from anti-apoptotic and cell cycle regulators, must be important in mediating the oncogenic effects of RasACT or NACT. In fact, in Drosophila, Ras has also been shown to be a potent inducer of cellular growth, while cyclin E and E2F1 mainly promote cell cycle progression. Whether NACT also promotes cell growth in Drosophila has not been examined in detail. If growth promotion targets downstream of RasACT or NACT are critical in promoting the overgrowth of scrib- tumors, these are likely to be independent of the PI3 kinase pathway since ectopic PI3 kinase signaling in scrib- clones does not induce synergistic overgrowth, and RafACT is able to induce overgrowth as equally extensive as RasACT (Brumby, 2003).
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