Effects of Mutation or Deletion

Table of contents

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 (StĀŸttem, 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 midline development

Mastermind mutations generate a unique constellation of midline cells within the Drosophila CNS

The Notch pathway functions repeatedly during the development of the central nervous system in metazoan organisms to control cell fate and regulate cell proliferation and asymmetric cell divisions. Within the Drosophila midline cell lineage, which bisects the two symmetrical halves of the central nervous system, Notch is required for initial cell specification and subsequent differentiation of many midline lineages. This study provides the first description of the role of the Notch co-factor, mastermind, in the central nervous system midline of Drosophila. Overall, zygotic mastermind mutations cause an increase in midline cell number and decrease in midline cell diversity. Compared to mutations in other components of the Notch signaling pathway, such as Notch itself and Delta, zygotic mutations in mastermind cause the production of a unique constellation of midline cell types. The major difference is that midline glia form normally in zygotic mastermind mutants, but not in Notch and Delta mutants. Moreover, during late embryogenesis, extra anterior midline glia survive in zygotic mastermind mutants compared to wild type embryos. This is an example of a mutation in a signaling pathway cofactor producing a distinct central nervous system phenotype compared to mutations in major components of the pathway (Zhang, 2011).

Notch has been shown to play multiple developmental roles in the CNS of several organisms. The Drosophila midline, with its easy to identify neural and glial lineages, has provided examples of multiple and reiterative roles of the Notch pathway within a single CNS lineage. In this study, the characterization of mamΔC mutants indicates how a co-factor within a signaling pathway contributes to the development of different midline cell types and adds to understanding of Notch signaling complexity (Zhang, 2011).

Initial activation of sim in the mesectoderm depends on maternal Notch expression, as N55e11 germline clones lack most sim expression and therefore, contain few midline cells. Likewise, mamΔC germline clones also show a reduction in sim expression. Thus, maternal contributions of both mam and Notch appear to act in the same pathway to activate sim early in development. Similarly, many midline neural phenotypes in zygotic mamΔC mutant embryos are largely consistent with those of N55e11 and Dl3, suggesting mam and Notch act together during the development of these neurons. Notch is required for formation of neurons expressing en and may be needed to maintain en expression in midline cells that develop in the posterior compartment of each CNS segment. The results described in this study suggest mam is also required for the formation of the midline neurons that express en and develop into the iVUMs, the MNB and its progeny. While these cells of the posterior compartment were absent, the H cell and mVUM midline neurons were expanded in mamΔC mutants, similar to N55e11 and Dl3 mutants, suggesting that mam function is needed within the Notch signaling pathway to obtain the variety of midline neurons found in wild type embryos (Zhang, 2011).

The major difference observed between zygotic mamΔC and N55e11 mutants was the presence of midline glia in mamΔC, but not N55e11 mutant embryos during mid to late embryogenesis. Not only were AMG present, but additional AMG survived in the mature CNS midline in mamΔC mutants compared to wild type embryos (and N55e11 mutants). The presence of AMG in mamΔC mutants suggests either (1) the mamΔC mutation is hypomorphic, (2) mam is not required within the Notch pathway for midline glial differentiation or (3) maternally deposited mam transcripts are stable and functional during the Notch signaling event needed for midline glial formation. Results with mam deficiency embryos indicated that midline glia formed and persisted in the complete absence of zygotic mam activity, suggesting it is not the hypomorphic nature of the mamΔC allele that allows the midline glia to form. Currently, it is not possible to distinguish between the other two possibilities, although the last hypothesis is favored due to the timing of midline cell divisions. At gastrulation, each segment contains 8 mesectodermal cells, which each divide, resulting in 16 MPs per segment at stage 10. Cells that give rise to AMG and PMG do not divide again, whereas MPs that develop into neurons each divide once at stage 11. Because MPs that give rise to glia undergo their last division earlier than MPs that give rise to neurons, the Notch signaling event needed for midline glial differentiation may occur prior to Notch events that dictate midline neural fates at stage 11. Maternal Mam protein may linger just long enough to allow midline glia to form, but not long enough to function when MPs divide to give rise to midline neurons slightly later. It is thought that this is the reason N55e11 mutants contain more midline cells per segment than wild type (and mamΔC). In N55e11 mutants, MPs that would normally form glia and not divide, instead take on neural fates and do divide. The data are consistent with this hypothesis, but future, additional experiments are required to properly test it (Zhang, 2011).

In addition to this temporal sensitivity, mam may also be sensitive to spatially restricted events within the midline. Existing evidence suggests the 16 MPs fall into 3 equivalence groups at stage 10: the MP1s, MP3s and MP4s. MP1s are in the anterior, MP3s in the middle and MP4s in the posterior of each CNS segment and effects of mamΔC vary according to these positions. The results indicate that neurons derived from the anterior MP1s are sensitive to N55e11, but not mamΔC; the middle MP3s are more sensitive to N55e11 than mamΔC; while the posterior MP4s are equally sensitive to N55e11 and mamΔC. In other words, mamΔC mutants 1) differ with N55e11 mutants in neurons derived from MP1s (MP1 neurons), 2) have similar, less severe effects compared to N55e11 mutants in cells derived from the MP3s (the H cell and H cell sib) and 3) the same effects as N55e11 mutants in cells derived from the posterior MP4s (mVUMs, iVUMS and MNB). These differences may be due to region specific differences in expression of other midline regulators that combine with Notch and/or Mam to control cell fate specification during embryogenesis. Possible candidates include hedgehog and wingless, which are expressed in the midline, affect cell fate and both interact with mam in a Notch-independent manner in other tissues. In any case, clear differences in zygotic mam and Notch mutations within the midline exist and demonstrate that variations in different Notch signaling components can alter the cellular composition of the CNS in unique ways (Zhang, 2011).

Close examination of mamΔC and N55e11 mutants during mid embryogenesis indicates they also differ in sim expression. After stage 10, sim diminishes in N55e11 mutants, but persists in mamΔC mutants. Likewise, midline glia, which are known to require sim expression to differentiate, do not develop in N55e11 mutants, but do develop in mamΔC mutants. The data indicate that all midline lineages that normally express sim are absent in N55e11 mutants, while midline lineages that do not normally express sim are present and expanded in zygotic mutants of N55e11. Therefore, similar to the initiation of sim expression early, the maintenance of sim expression at this later time also appears to require zygotic Notch activity. In contrast, the results suggest sim expression persists in zygotic mamΔC mutants (Zhang, 2011).

In the canonical Notch pathway, Mam normally functions as a co-factor and collaborates with both the NICD and Su(H) to activate target genes. Consistent with this role, overexpression of mam alone does not affect the number of AMG generated at mid embryogenesis, whereas the overexpression of the NICD in wild type embryos increases AMG cell number. Overexpression of the NICD in a mamΔC mutant background still increased the number of AMG during this stage, further supporting the idea that zygotic mam is not needed at this time. During late embryogenesis, mamΔC mutants contained extra AMG. Mutations in mam are known to promote neural tissue at the expense of ectoderm and this may result in the production of additional Spi, which inhibits apoptosis and allows extra midline glia to survive (Zhang, 2011).

Altogether, the data suggest a high level of complexity in the regulation of CNS target genes of Notch. Notch likely interacts with additional cell-lineage specific co-activators other than, or in addition to, Mam in certain cells. In this way, combinatorial interactions between components of Notch signaling and other signaling pathways can lead to different outputs in various cell types, increasing cell diversity and function. These result indicate mamΔC mutants contain AMG and PMG, whereas N55e11 mutants do not. While this report describes major disruptions in mam, less severe mutations, such as small deletions, insertions or polymorphisms could also affect the midline and modify its cellular composition. Because mam mutations have more subtle effects on the midline compared to mutations in Notch or Delta, they may be tolerated more than mutations in major components of the pathway and actually contribute to CNS cellular variation in natural populations. Future experiments are needed to fully explore these functional differences between mam and Notch in the midline, as well as other tissues. Such differences can then be exploited to develop progressively specific research and clinical tools to regulate Notch signaling and the cellular composition of tissues from the different sensitivities (Zhang, 2011).

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).

Development of the entero-endocrine lineage and its specification by the Notch signaling pathway

This paper investigated the developmental-genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors (AMPd). As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells (Takashima, 2011).

The function of the intestinal tract of all animals is regulated by two closely interrelated systems, the autonomic nervous system and the endocrine system. The endocrine system is formed by specialized endocrine glands, like the islets of Langerhans in vertebrates' pancreas, or the corpora cardiaca in insects, as well as scattered entero-endocrine cells that form part of the intestinal epithelium. These cells, which typically outnumber cells of all other endocrine organs combined, represent the diffuse endocrine system (DES). Entero-endocrine cells produce different peptide hormones with specific regional distributions and functions. Well known examples from vertebrates are secretin or CKK (produced in the duodenum; stimulates pancreatic bicarbonate secretion), or gastrin (produced in the stomach; increases acid secretion from parietal cells). The peptide hormones produced by entero-endocrine cells also occur as neuro-transmitters in neurons and are therefore frequently referred to as 'brain-gut peptides'. For example, the peptides of the insect tachykinin family are found both in midgut entero-endocrine cells, as well as in neurons of the central nervous system (Winther, 2003). Tachykinin released from neurons locally affects gut muscle contractility; systemic release into the hemolymph acts on many effector organs, including the excretory Malpighian tubules, the heart, and the somatic musculature (Takashima, 2011).

The Drosophila midgut consists of three major cell types, enterocytes, entero-endocrine cells, and progenitors/stem cells. These cells are generated three times during development, when generating a larval midgut, transient pupal midgut, and adult midgut. The mechanisms that specify the cell types, and the morphogenetic process by which these cells actually separate from each other, appear to be highly conserved during each of these phases. Thus, in a first step, a pool of undifferentiated progenitor cells increases its population by cell division. During this phase (early embryonic endoderm; early larval AMPs, late larval AMPs) all cells express esg. In a second step, enterocytes (the outer endoderm layer in embryo; peripheral cells in larva; and prospective adult enterocytes in early pupa) become postmitotic, lose esg expression, and physically split from another group of cells which maintain esg expression and continue to divide (inner layer of embryonic endoderm; inner AMPs in larva; pISCs in pupa). In a third step, the esg-positive cells give off endocrine cells which become Pros-positive and eventually lose esg, as well as progenitor/stem cells that continuously remain esg-positive (Takashima, 2011).

Notch/Delta signaling mediates the decision of midgut cell fates produced by the ISCs in the adult. After each ISC division, Delta is maintained in one of the two daughter cells as a renewed ISC and activates Notch pathway in the other, neighboring daughter cell. This second daughter, called enteroblast, is inhibited by Notch activity from further division. In addition, the level of Notch activity determines whether the enteroblast differentiates as enterocyte (high Notch activity) or entero-endocrine cell (low Notch activity). Thus, a key element of the mechanism underlying midgut fate determination is the level and localization of Delta. In the adult midgut, upstream acting signaling events emanating from the visceral musculature that surrounds the gut are responsible in maintaining (various levels of) Delta expression in the ISCs. It is proposed that the same mechanism could start acting during the pupal stage. During early metamorphosis, the larval visceral musculature de-differentiates; myofibrils and extracellular matrix is lost, even though the muscle cells themselves survive. Around 40 hours APF these cells re-differentiate into adult visceral muscle. This is about the time when pISC change their proliferation pattern from symmetric self renewal to a mode that produces endocrine cells. It is possible that the re-occurrence of visceral muscle structure may differentially activate Delta protein in pISC to change the fate of their daughter cells (Takashima, 2011).

It is likely that the level and spatial distribution of the Delta signal is also instrumental in determining the right number and patterning of enterocytes vs endocrine cells vs progenitors in the larva and embryo. In the larva, complex signaling events between the emerging peripheral cells and the central cells of the AMP islands could focus expression of Delta towards the latter. Again, it is possible that the larval visceral musculature also plays a role in adjusting Delta expression. Delta in turn activates the Notch cascade in the periphery of the AMP islands, resulting in a certain number of transient pupal enterocytes and endocrine cells to develop within each island (Takashima, 2011).

In the embryo, Delta is required to trigger high Notch activity in endoderm cells that separate as outer layer and differentiate into enterocytes. Delta is expressed widely in the endoderm during the stage when inner and outer layers split, and it is not clear how differences in Delta level correlate with different locations of cells within the endoderm. It should be noted that the situation is complicated by the fact that several different types of enterocytes develop which will populate the different segments of the midgut along the antero-posterior axis; for example, one these cell types, the so called interstitial cells which come to lie within the middle of the gut tube, split from the remainder of the enterocytes even earlier than the endocrine cells and the AMPs. More work, based on additional fate-specific markers, is required to reconstruct in detail the mechanics of the Notch-Delta signaling step that specifies fate in the embryonic endoderm (Takashima, 2011).

In the vertebrate gut, entero-endocrine cells are formed within the same endodermal primordium that gives rise to the enterocytes. As in Drosophila, all endodermal cells of the early vertebrate embryo proliferate. Subsequently, as the endodermal epithelium gets folded into villi and crypts, proliferation gets restricted to the crypts. In the mature gut, crypts contain small numbers of slowly cycling stem cells (ISCs), surrounded by a progeny that divides fast (amplifying progenitors) and at the same time adopts different fates. As cells differentiate, they move upward to the apices of the villi, from which they are eventually sloughed off (Takashima, 2011).

Recent studies in vertebrates have started to elucidate the mechanism by which different intestinal cell fates, including enterocytes, entero-endocrine cells (and other secretory cell types), and proliferatory stem cells are related. Labeled clones have been shown to contain both enterocytes and entero-endocrine cells. This suggests that at the level of the progenitor or stem cells in which the clones were induced, a decision between endocrine and enterocyte fate had not yet been made. In line with this conclusion, other studies showed that cell-cell interactions among intestinal cell precursors involving the Notch signaling pathway specify intestinal cell fate. Endocrine precursors (including still proliferatory as well as postmitotic cells) express bHLH transcription factors ('pro-endocrine factors'), in particular Math-1 and neurogenin 3, as well as the Notch ligand, Delta. Loss of Delta or Notch function increases the number of endocrine cells, often at the expense of enterocytes; loss of 'proendocrine factors' has the opposite phenotype. For example, in mouse, Math-1 is expressed in the zone of transient amplifying progenitors and then becomes restricted to postmitotic exocrine and endocrine cells. Loss of Math-1 results in the absence of both cell populations. Another proneural gene, neurogenin 3, may act downstream of Math-1 in a more restricted progenitor populations that include only endocrine cell types. Thus, loss of neurogenin 3 in mouse results in the absence or strong reduction of several endocrine cell populations, in particular glucagon, somatostatin, and gastrin expressing cells. The Notch ligand Delta (DeltaD in zebrafish; Delta1 in mouse) is expressed in enteroendocrine and secretory cells. Disruption of Delta function (by depleting the gene mib in zebrafish) converts gut enterocytes into secretory (exocrine/endocrine) cells. These and other data indicate that the cell fate determining mechanism is highly conserved in vertebrates and Drosophila (Takashima, 2011 and references therein).

A general conclusion that can be drawn from this study, as well as similar studies in vertebrate intestinal development, is that the selection of endocrine cells from epithelial enterocytes follows a similar mechanism, and is controlled by the same gene cassette, as the selection of neural precursors from the neurectoderm. The 'developmental-genetic scenario' that encountered during early neurogenesis (bHLH gene expression-proneural cluster-Notch signaling lateral inhibition-high Notch activity-epithelial cells low Notch activity-delaminating neurons/neuroblasts) appears very similarly in gut development. Here, the epithelial cells depending on high Notch activity are the enterocytes; the cells requiring bHLH genes, and low Notch activity, are the endocrine precursors, which also (at least partially) delaminate from the epithelium. It has long been realized that neurons and endocrine cells share the expression of numerous molecular and ultrastructural features. However, given the developmental findings, the extent of 'genetic overlap' between endocrine and neural cells may be even greater than previously expected. The similarities between neurons and endocrine cells probably reflect a common phylogenetic origin of these cell types. The communication among cells that is mediated by secreted, diffusible signals is phylogenetically older than neural transmission. Animals without nervous system (e.g., sponges) and even protists produce many secreted molecules which are often molecularly homologous to hormones or neural transmitters found in multicellular animals. One may speculate that, during an early step of evolution that occurred in primitive multicellular animals, specialized epithelial cells within the epidermis and the gut reacted to certain stimuli by secreting metabolites that diffused throughout the body and evoked adaptive responses in other tissues. A direct line of evolution led from these cells to the entero-endocrine cells is still found today in all animals. A second line of evolution transformed subsets of the hypothetical 'endocrine-neural forebears' into neurons, cells which elaborate processes and form specialized synaptic contacts. According to this view it would be expected that a conserved genetic mechanism is employed to separate endocrine cells and neurons from the respective epithelium into which they are embedded (Takashima, 2011).

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).

Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and actin protein accumulation in Drosophila embryo

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions (Wesley, 2011).

Data presented in this report show that the loss of hephaestus (dmPTB) function affects the ventral and the dorso-lateral regions of Drosophila embryos very differently. In the ventral region, development of the CNS is suppressed and there was a discernible depletion in the level of the Notch protein. Suppression of the CNS development is consistent with the known role of hephaestus as a negative regulator of canonical Notch signaling during wing and bristle development in the larval and pupal stages. Excess canonical Notch signaling is well known to suppress neurogenesis in embryos. Depletion of the Notch protein in the ventral region that could be explained using data from mammalian systems showing that Nintra/NICD is turned over by a proteolysis process linked to the activity of the transcription factor Mastermind. Thus, excess canonical Notch signaling could result in Notch protein depletion if the rate of Nintra/NICD production and degradation is higher than Notch synthesis. If that were the case, it would suggest that the mechanism responsible for down regulating Notch activity targets not Nintra/NICD production or degradation but Notch synthesis. Combining the data from heph alleles and the Nnd1-dse allele, it appears that most of Notch mRNA transcribed following Notch activation is targeted for degradation by a mechanism that requires the Notch 3' UTR and the dse, a temperature sensitive allele of Notch that produces constitutive and high levels of endogenous Notch activities at the restrictive temperature. It is possible that the Hephaestus protein is part of the RNP complex that regulates this mechanism. In its absence, Notch protein synthesis continues instead of being suppressed. There is growing evidence that ligand-independent canonical Notch signaling in involved in development. It would be interesting to know if this signaling is also affected by the Hephaestus-based down-regulation mechanism. Thus, understanding how exactly hephaestus negatively regulates canonical Notch activity might provide insights into an important aspect of Notch pathway regulation that was hitherto obscure: down-regulation after activation of Notch by a ligand. As many human diseases are linked to gain of canonical Notch signaling, a better understanding of hephaestus and Notch 3' UTR and dse functions might lead to novel mechanistic insights into these diseases (Wesley, 2011).

The surprising finding in this study is the different response of the dorso-lateral regions of the embryo to the loss of hephaestus function or the loss of negative regulation of Notch mRNA 3' processing (due to the Nnd1-dse mutation). The simplest explanation is that Notch function is not required in these regions and de-repression of Notch protein synthesis results in the accumulation of Notch protein in these regions (as there is no signaling dependent depletion). This explanation, however, does not account for Pericardin accumulation, actin accumulation, or the block in dorsal closure. Pericardin level during cardiogenesis is well established to depend on Notch activity. These studies confirm that Pericardin is absent when Notch activity is eliminated (i.e., in Notch null embryos). Studies of others show that Notch activity is associated with higher actin level. Thus, it is very likely that Notch function is required in the dorso-lateral region of the embryo and this function is in excess in mutant heph and Nnd1-dse embryos. (Wesley, 2011).

As Nintra/NICD expression does not lead to actin or Pericardin accumulation in the dorso-lateral region, the simplest explanation is that Notch function in the dorso-lateral region is not completely based on Nintra/NICD activity in the nucleus. There is evidence for the existence of Notch activity independent of Nintra/NICD. For example, a Notch function independent of Presenilin (the enzyme that is required for the release of Nintra/NICD. A similar Notch activity might be functioning in the dorso-lateral regions of the Drosophila embryo. The data suggest that this non-canonical Notch activity might be situated at the cell surface or in the cytoplasm and is involved in regulating actin levels and cell fusion. This inference is consistent with the finding of others that Notch activity other than the one based on Nintra/NICD is associated with actin accumulation in wing discs (Wesley, 2011).

This non-canonical Notch activity could be the predominant Notch activity in the dorso-lateral regions of the embryos but it cannot be the only Notch activity. It is well known that Nintra/NICD and canonical Notch signaling is required for the peripheral nervous system (PNS) development from the dorso-lateral regions. These studies show that the PNS development is also suppressed in heph03429 and Nnd1-dse embryos, which raises the question of why no depletion is seen of Notch and actin proteins in the dorso-lateral regions as a consequence of constitutive Nintra/NICD and canonical Notch signaling. There are two possible explanations. One, a minority of cells are involved in the PNS development. Two, the canonical Notch signaling activity might precede the non-canonical Notch signaling activity and the latter determines the ultimate phenotype. Regardless of which explanation is correct, it is remarkable that cells in the ventral and the dorso-lateral regions respond so differently to the loss of hephaestus function or to the loss of negative regulation of Notch activity due to the Nnd1-dse mutation. At an earlier stage (stage 8 or 9), these cells were all the same, as they all adopt the default neuronal fate in Notch or Delta null embryos. At later stages, the ventral epidermal cells appear to diverge by blocking non-canonical Notch signaling altogether. It appears that this block is not an intrinsic lack of competence because actin and Notch enriched cells are occasionally observed in the ventral region. The block is specific to hephaestus or actin-related Notch activity as the ventral epidermal cells participate in other the actin-dependent processes, for example those involved in producing the denticle belts (Wesley, 2011).

The Notch pathway is long known to be involved in actin and adhesion processes in Drosophila. Interestingly, many processes that depend on Notch or hephaestus activity undergo cell fusion or block them (e.g., myoblast fusion). In this regard, Notch functions in myogenesis are quite instructive. During myogenesis, Nintra/NICD and canonical Notch signaling is required to restrict the number of myoblasts. Not as well known is the fact that Notch activity is also required subsequently for myoblast fusion and differentiation. A Notch activity at these stages is also reported to affect the differentiation of the neighboring epidermal cells and this activity is not based on Nintra/NICD. These reports have not been examined in depth so far because nothing is known about the non-canonical Notch signaling mechanism. The dorso-lateral regions of heph03429 and Nnd1-dse embryos represent an excellent model system for exploring non-canonical Notch mechanism with an unusual empirical power: all aspects of this mechanism in the dorso-lateral regions of the embryos can be compared with the canonical Notch signaling pathway mechanism in the ventral region of the same embryo (Wesley, 2011).

The process that is defective in the ventral region of heph03429 and Nnd1-dse embryos (neuronal cell fate specification) is known precisely, but nothing is known about the process that is defective in the dorso-lateral regions. These data contain two clues to the latter process. One clue is a contrast-enhanced image of wild type and heph03429 embryos probed with the actin antibody. A close examination of this image reveals that the strong actin signals in the heph03429 embryo are more or less amplified and expanded versions of the above-background actin signals in the wild type embryo. The second clue is in heph03429 and Nnd1-dse embryos probed with phalloidin. It appears that these embryos form enlarged versions of the cable-like actin structures that traverse almost the entire length of the dorso-lateral regions in the body of the wild type embryo. It is quite possible that clusters of cells in the dorso-lateral regions undergo partial or full fusion to form actin scaffolds that maintain epithelia integrity during remodeling and migration. Hypertrophy of these actin scaffolds could be the defect in the dorso-lateral regions of heph03429 and Nnd1-dse embryos. At this juncture, the mechanism by which actin protein level is altered in these mutant embryos is unknown (Wesley, 2011).

heph03429 and Nnd1-dse embryos appear to reveal a new level of developmental organization: broad zones that are competent or refractory to non-canonical Notch signaling activity. It is not known what factors or mechanisms determine these zones. A diverse array of mechanisms is known to regulate Notch activity at the protein level, such as glycosylation, trafficking, and proteolytic processing. It is possible that the ventral and the dorso-lateral regions differ in these mechanisms. Understanding the mechanism underlying the zonation of Notch activity in Drosophila embryos might also have practical implications since the Notch pathway is an important regulator of stem cell differentiation and cancer development. It might help in understanding variations within and among stem cell or cancer populations. It is possible that certain populations are composed of cells with potential for only the canonical Notch signaling while others include cells with potential for both canonical and non-canonical Notch signaling. Such differences in potentials might explain why some stem cells just proliferate while others differentiate or why some cancer cells are begin while others are metastatic (Wesley, 2011).

Notch and neoplastic outgrowth in Drosophila

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).

Table of contents

Notch: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | References

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