Transcriptional Regulation

To date, very little information is available about the regulation of Notch transcription. Suppressor of Hairless exhibits allele specific interaction with Notch, indicating that the Notch pathway may regulate Notch transcription by means of Su(H) (Fortini, 1994).

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch (Hassan, 2000).

Since Ato is expressed in adult brains, ato-Gal4/UAS-lacZ adult brains were stained with anti-beta-galactosidase (anti-beta-gal) and the data were analyzed with confocal microscopy. In adult flies, ato-Gal4 is expressed in a DC and in two ventral clusters (VCs: VLC and VBC) of neurons. Some axons of the DC project ipsilaterally over the lobula. However, most axons of the DC form a bundle that is a component of the dorsal commissure and project contralaterally toward the lobula complex and the medulla. These neurites fan out over the lobula complex and the inner chiasm. Ten to twelve tracts cross the outer chiasm, toward the medulla, in a ladder-like pattern. Over the medulla, the fibers branch and appear to contact one another to form a 'grid-like' lattice. No fibers cross the lamina (Hassan, 2000).

What might the function(s) of ato be in the central brain? To address this question, the consequences of the loss and gain of ato function in the DC in L3 and adult brains was examined using the ato-Gal4 line as a marker and a driver. Surprisingly, all three clusters (DC, VBC, VLC) are present in brains homozygous for the ato1 allele as well as in brains transheterozygous for ato1 and a deficiency that uncovers the ato region. However, several defects are obvious in ato mutant brains. While heterozygous control L3 brains show a normal DC, mutant L3 brains show severe defects in DC position and organization. In addition, probably as a result of the loose morphology of the cluster, the descending axon bundles are defasiculated. These defects were observed with about 15% penetrance and suggest a weak or partially redundant differentiation requirement for ato in the precursors of the DC lineage. Note, however, that the DC axons form a commissural tract, strongly suggesting that their basic identity as commissural neurons is not affected by the ato mutation (Hassan, 2000).

To investigate if ato is required in the postmitotic DC neurons, as the reinitiation of its expression suggests, the morphology of the axonal projections of the DC neurons of adult ato mutant brains was examined. The DC forms a stereotypical axonal pattern, making it simple to detect aberrations that may be caused by the ato mutation. In adult brains, in addition to the aberrant positioning of the cluster seen in L3, the arborization pattern of the DC over the lobula is severely impaired in ato deficiency flies. Most axons enter the lobula either ventrally or dorsally and show very limited branching, failing to form a proper 'fan-shaped' pattern. Since ato mutant brains have a severely reduced medulla, the medullar part of the pattern could not be examined. While these data suggest a role for ato in axon arborization, alternative interpretations are possible: (1) the observed axonal defects may be caused by the significant loss of optic lobe structures associated with loss of ato function (no lamina or medulla, reduced lobula); (2) it is possible that the defects are a reflection of the improper differentiation of the DC cells rather than a reflection of a specific role for ato in arborization (Hassan, 2000).

To understand the mechanism by which ato functions in axonal development, the role of Notch was examined in the development of the DC axonal pattern. Two observations make Notch a logical candidate. (1) ato and Notch interact in an antagonistic fashion during CHO development. For example, in the leg femoral CHO, gain of Notch function reduces the number of precursors selected from the ato-expressing proneural cluster. (2) Notch has been shown to be required for axon guidance, perhaps mediating axon–substrate interactions, in both the PNS and CNS. If ato and Notch act antagonistically in arborization, it is expected that Notch activity levels would be relatively low within the DC, where its function is repressed by ato, and relatively high in the substrate cells, where ato does not antagonize it. Thus, after ato expression is reinitiated in the DC neurons, a differential in Notch activity levels may occur between the arborizing axon and the substrate cells. Perturbations of this imbalance, either by raising Notch activity levels in the DC or by reducing them in the surrounding cells, may result in defective arborization patterns. This model allows for three specific predictions: (1) the generalized loss of Notch function may result in excessive arborization of the DC neurons, whereas the DC-specific loss of function would cause no significant defects; (2) the gain of Notch in the DC neurons is expected to inhibit arborization; (3) if the activation of ato in the DC neurons serves to antagonize Notch activity, then it is expected that the gain of Notch function will be epistatic to the gain of ato function (Hassan, 2000).

To determine the requirements for Notch in DC axon development, two Notch alleles were examined: a temperature-sensitive allele (Nts) and a viable hypomorphic allele (facet notchoid [Nnd3]). In the first set of experiments, Nts;ato-Gal4,UAS-lacZ larvae were raised in a cycling incubator delivering a 30 min, 34°C heat shock every 8 hr from late L1 through wandering L3. L3 brains were examined for DC defects. Reducing Notch activity during larval development has no effects on the number, morphology, or position of the DC neurons or on the formation of the commissure. In contrast, defects in axon branching out of the commissure into the optic lobe were observed. Specifically, excessive branching and defasiculation of the axon bundles entering the optic lobe were observed. Importantly, these defects were not rescued by a wild-type copy of Notch driven by the ato enhancer in the DC (Nts; UAS-N+;ato-Gal4,UAS-lacZ), suggesting that the requirement for Notch in DC axon arborization is nonautonomous in contrast to the requirement for ato. Larvae reared under the cycling heat shock paradigm or at a consistent 28°C temperature between either L1 and adult or L3 and adult do not produce homozygous Nts flies. Therefore, to examine the adult DC innervation pattern in a background of reduced Notch activity, Nnd3;ato-Gal4,UAS-lacZ male flies were used. These flies show excessive branching in the medulla, resulting in an aberrant innervation pattern similar to that caused by ato overexpression in the DC neurons. The disruption is milder due to the fact that Nnd3 is a weak allele of Notch (Hassan, 2000).

Do the defects observed in Notch mutants reflect an independent function for Notch in arborization? It is possible that the conditions used in this study result in mild neurogenic phenotypes generating extra DC target cells. This, in turn, would cause the DC neurons to arborize excessively to innervate new targets. To rule out this possibility, area density (number of cells per unit area) and optic lobe cortex volume (volume occupied by optic lobe cell bodies) analyses were performed on Nts L3 brains and Nnd3 adult brains. By both criteria, there are no significant differences between wild-type and mutant brains. Therefore, these data support a model in which ato generates the branching pattern by antagonizing Notch activity in the DC (Hassan, 2000).

To show that Notch function is not required within the DC neurons themselves, a dominant-negative form of Notch (UAS-NEC) was expressed using ato-Gal4. NEC has no effect on the formation of the larval or adult axonal patterns. In contrast, NEC expression in imaginal discs results in strong loss of Notch function phenotypes and pupal lethality, demonstrating that the construct is active. This shows that while Notch function is required specifically for arborization of the DC neurons, its requirement is nonautonomous. The prediction that ato represses Notch activity in the DC cells implies that gain of Notch function within the DC would result in inhibition of axonal branching, a phenotype similar to that of loss of ato function. The membrane-bound, wild-type form of Notch (N+) is known to be required for the rescue of axonal defects associated with the loss of Notch function. However, in all cases in which ato and Notch appear to interact in the PNS, it is the nuclear form of Notch that is thought to be involved. To evaluate the effects of the gain of Notch function, both forms of Notch were overexpressed in the DC neurons: the membrane-bound N+ and the nuclear form, Nintra. Overexpression of N+ has no effect on the axonal pattern, whereas overexpression of Nintra results, in comparison with controls, in a severe inhibition of axonal branching over the lobula and a complete failure of innervation of the medulla. These data suggest that the nuclear form of Notch, but not the membrane-bound form, affects the arborization pattern of the DC axons. Finally, if ato suppresses Notch signaling within the DC, gain of Notch function should be epistatic to the gain of ato function, placing Notch genetically downstream of ato. Therefore, the combined overexpression of ato and Nintra should result in the same phenotype as the overexpression of Nintra alone. Brains in which both ato and Nintra are overexpressed using ato-Gal4 have a phenotype identical to that of Nintra overexpression alone. Taken together, the data presented above support the hypothesis that ato acts to suppress Notch signaling within the DC and that this suppression is essential for the generation of the proper arborization pattern of the DC axons (Hassan, 2000).

Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).

The expressions of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) protein lacking a basic motif, and Notch (N), were examined because both genes have been shown to be involved in the control of cell proliferation in the wing. In third instar larvae, emc is expressed at a low level throughout the wing disc and at a higher level in two stripes of cells corresponding to the prospective A margin and the AP organizer. Unmodified at the A margin, emc expression is completely lost from the AP organizer cells in either col1 or col1/kn1 mutant discs, showing that Col is required for emc transcription in the L3-L4 intervein primordium. Levels of N protein are high in intervein regions and low in presumptive vein territories in late third instar. In col1 mutants, N is downregulated in the L3m provein domain. col requirement for emc and N upregulation in the AP organizer cells is consistent with the reduced cell number in the central region of col1 mutant discs (Crozatier, 2002).

Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis
In Drosophila, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). This study further characterized MLF's mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. The results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, dnaj-1 loss causes an increase in Lz+ blood cell number and size similarly as in mlf mutant larvae. Moreover dnaj-1 was found to genetically interact with mlf to control Lz level and Lz+ blood cell development in vivo. In addition, mlf and dnaj-1 loss was shown to alter Lz+ cell differentiation, and the increase in Lz+ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, high levels of Lz were shown to be required to repress Notch transcription and signaling. These data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of Notch expression and signaling to prevent aberrant blood cell development. Thus these findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo (Miller, 2017).

Proper blood cell development requires the finely tuned regulation of transcription factors and signaling pathways activity. Consequently mutations affecting key regulators of hematopoiesis such as members of the RUNX transcription factor family or components of the Notch signaling pathway are associated with several blood cell disorders including leukemia. Also, leukemic cells often present recurrent chromosomal rearrangements that participate in malignant transformation by altering the function of these factors. The functional characterization of these genes is thus of importance not only to uncover the molecular basis of leukemogenesis but also to decipher the regulatory mechanisms controlling normal blood cell development. Myeloid Leukemia Factor 1 (MLF1) was identified as a target of the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) more than 20 years ago. Further findings suggested that MLF1 could act as an oncogene or a tumor suppressor depending on the cell context and it was shown that MLF1 overexpression either impairs cell cycle exit and differentiation, promotes apoptosis, or inhibits proliferation in different cultured cell lines. Yet, its function and mechanism of action remain largely unknown (Miller, 2017).

MLF1 is the founding member of a small evolutionarily conserved family of nucleo-cytoplasmic proteins present in all metazoans but lacking recognizable domains that could help define their biochemical activity . Whereas vertebrates have two closely related MLF paralogs, Drosophila has a single mlf gene encoding a protein that displays around 50% identity with human MLF in the central conserved domain. In the fly, MLF was identified as a partner of the transcription factor DREF (DNA replication-related element-binding factor), for which it acts a co-activator to stimulate the JNK pathway and cell death in the wing disc. MLF has been shown to bind chromatin, as does its mouse homolog, and it can either activate or repress gene expression by a still unknown mechanism. MLF also interacts with Suppressor of Fused, a negative regulator of the Hedgehog signaling pathway, and, like its mammalian counterpart, with Csn3, a component of the COP9 signalosome, but the functional consequences of these interactions remain elusive. Interestingly the overexpression of Drosophila MLF or that of its mammalian counterparts can suppress polyglutamine-induced cytotoxicity in fly and in cellular models of neurodegenerative diseases. Moreover phenotypic defects associated with MLF loss in Drosophila can be rescued by human MLF1. Thus MLF function seems conserved during evolution and Drosophila appears to be a genuine model organism to characterize MLF proteins (Miller, 2017).

Along this line, the role of MLF during Drosophila hematopoiesis has been studied. Indeed, a number of proteins regulating blood cell development in human, such as RUNX and Notch, also control Drosophila blood cell development. In Drosophila, the RUNX factor Lozenge (Lz) is specifically expressed in crystal cells and it is absolutely required for the development of this blood cell lineage. Crystal cells account for ±4% of the circulating larval blood cells; they are implicated in melanization, a defense response related to clotting, and they release their enzymatic content in the hemolymph by bursting. The Notch pathway also controls the development of this lineage: it is required for the induction of Lz expression and it contributes to Lz+ cell differentiation as well as to their survival by preventing their rupture. Interestingly, the previous analysis revealed a functional and conserved link between MLF and RUNX factors. In particular, MLF was shown to control Lz activity and prevent its degradation in cell culture, and the regulation of Lz level by MLF is critical to control crystal cell number in vivo. Intriguingly, although Lz is required for crystal cell development, mlf mutation causes a decrease in Lz expression but an increase in crystal cell number. In human, the deregulation of RUNX protein level is associated with several pathologies. For instance haploinsufficient mutations in RUNX1 are linked to MDS/AML in the case of somatic mutations, and to familial platelet disorders associated with myeloid malignancy for germline mutations. In the opposite, RUNX1 overexpression can promote lymphoid leukemia. Understanding how the level of RUNX protein is regulated and how this affects specific developmental processes is thus of particular importance (Miller, 2017).

To better characterize the function and mode of action of MLF in Drosophila blood cells, this study used proteomic, transcriptomic and genetic approaches. In line with recent findings, MLF was found to bind DnaJ-1, a HSP40 co-chaperone, as well as the HSP70 chaperone Hsc70-4, and that both of these proteins are required to stabilize Lz. It was further shown that MLF and DnaJ-1 interact together but also with Lz via conserved domains and that they regulate Lz-induced transactivation in a Hsc70-dependent manner in cell culture. In addition, using a null allele of dnaj-1, it was shown to control Lz+ blood cell number and differentiation as well as Lz activity in vivo in conjunction with mlf. Notably, w mlf or dnaj-1 loss leads to an increase in Lz+ cell number and size due to the over-activation of the Notch signaling pathway. Interestingly, these results indicate that high levels of Lz are required to repress Notch expression and signaling. A model is proposed whereby MLF and DnaJ-1 control Lz+ blood cell growth and number by promoting Lz accumulation, which ultimately turndowns Notch signaling. These findings thus establish a functional link between the MLF/Dna-J1 chaperone complex and the regulation of a RUNX-Notch axis required for blood cell homeostasis in vivo (Miller, 2017).

Members of the RUNX and MLF families have been implicated in the control of blood cell development in mammals and Drosophila and deregulation of their expression is associated with human hemopathies including leukemia. The current results establish the first link between the MLF/DnaJ-1 complex and the regulation of a RUNX transcription factor in vivo. In addition, these data show that the stabilization of Lz by the MLF/DnaJ-1 complex is critical to control Notch expression and signaling and thereby blood cell growth and survival. These findings pinpoint the specific function of the Hsp40 chaperone DnaJ-1 in hematopoiesis, reveal a potentially conserved mechanism of regulation of RUNX activity and highlight a new layer of control of Notch signaling at the transcriptional level (Miller, 2017).

MLF binds DnaJ-1 and Hsc70-4, and these two proteins, like MLF, are required for Lz stable expression in Kc167 cells. In addition, these data show that MLF and DnaJ-1 bind to each other via evolutionarily conserved domains and also interact with Lz, suggesting that Lz is a direct target of a chaperone complex formed by MLF, DnaJ-1 and Hsc70-4. Of note, a systematic characterization of Hsp70 chaperone complexes in human cells identified MLF1 and MLF2 as potential partners of DnaJ-1 homologs, DNAJB1, B4 and B6, a finding corroborated by Dyer (2017). Therefore, the MLF/DnaJ-1/Hsc70 complex could play a conserved role in mammals, notably in the regulation of the stability of RUNX transcription factors. How MLF acts within this chaperone complex remains to be determined. In vivo, this study demonstrated that dnaj-1 mutations lead to defects in crystal cell development strikingly similar to those observed in mlf mutant larvae, and these two genes were shown to act together to control Lz+ cells development by impinging on Lz activity. The data suggest that in the absence of DnaJ-1, high levels of MLF lead to the accumulation of defective Lz protein whereas lower levels of MLF allow its degradation. Thus it is proposed that MLF stabilizes Lz and, together with DnaJ-1, promotes its proper folding/conformation. In humans, DnaJB4 stabilizes wild-type E-cadherin but induces the degradation of mutant E-cadherin variants associated with hereditary diffuse gastric cancer. Thus the fate of DnaJ client proteins is controlled at different levels and MLF might be an important regulator in this process (Miller, 2017).

This work presents the first null mutant for a gene of the DnaJB family in metazoans and the results demonstrate that a DnaJ protein is required in vivo to control hematopoiesis. There are 16 DnaJB and in total 49 DnaJ encoding genes in mammals and the expansion of this family has likely played an important role in the diversification of their functions. DnaJB9 overexpression was found to increase hematopoietic stem cell repopulation capacity and Hsp70 inhibitors have anti-leukemic activity, but the participation of other DnaJ proteins in hematopoiesis or leukemia has not been explored. Actually DnaJ's molecular mechanism of action has been fairly well studied but there are only limited insights as to their role in vivo. Interestingly though, both DnaJ-1 and MLF suppress polyglutamine protein aggregation and cytotoxicity in Drosophila models of neurodegenerative diseases, and this function is conserved in mammals. It is tempting to speculate that MLF and DnaJB proteins act together in this process as well as in leukemogenesis. Thus a better characterization of their mechanism of action may help develop new therapeutic approaches for these diseases (Miller, 2017).

As shown in this study, mlf or dnaj-1 mutant larvae harbor more crystal cells than wild-type larvae. This rise in Lz+ cell number is not due to an increased induction of crystal cell fate as we could rescue this defect by re-expressing DnaJ-1 or Lz with the lz-GAL4 driver, which turns on after crystal cell induction, and it was also observed in lz hypomorph mutants, which again suggests a post-lz / cell fate choice process. Moreover mlf or dnaj-1 mutant larvae display a higher fraction of the largest lz>GFP+ cell population, which could correspond to the more mature crystal cells. It is thus tempting to speculate that mlf or dnaj-1 loss promotes the survival of fully differentiated crystal cells. RNAseq data demonstrate that mlf is critical for expression of crystal cell associated genes, but both up-regulation and down-regulation of crystal cell differentiation markers were observed in mlf or dnaj-1 mutant Lz+ cells. Also these changes did not appear to correlate with crystal cell maturation status since alterations were found in gene expression in the mutants both in small and large Lz+ cells. In addition the transcriptome did not reveal a particular bias toward decreased expression for 'plasmatocyte' markers in Lz+ cells from mlf- mutant larvae. Thus, it appears that MLF and DnaJ-1 loss leads to the accumulation of mis-differentiated crystal cells (Miller, 2017).

The data support a model whereby MLF and DnaJ-1 act together to promote Lz accumulation, which in turn represses Notch transcription and signaling pathway to control crystal cell size and number. Indeed, an abnormal maintenance of Notch expression was observed in the larger Lz+ cells as well as an over-activation of the Notch pathway in the crystal cell lineage of mlf and dnaj-1 mutants or when Lz activity was interfered with. Moreover the data as well as previously published experiments show that Notch activation promotes crystal cell growth and survival. Importantly too the increase in Lz+ cell number and size observed in mlf or dnaJ-1 mutant is suppressed when Notch dosage is decreased. Yet, some of the mis-differentiation phenotypes in the mlf or dnaj-1 mutants might be independent of Notch since changes in crystal cell markers expression seem to appear before alterations in Notch are apparent. At the molecular level, the results suggest that Lz directly represses Notch transcription as this study identified a Lz-responsive Notch cis-regulatory element that contains conserved RUNX binding sites. The activation of the Notch pathway in circulating Lz+ cells is ligand-independent and mediated through stabilization of the Notch receptor in endocytic vesicles. Hence a tight control of Notch expression is of particular importance to keep in check the Notch pathway and prevent the abnormal development of the Lz+ blood cell lineage. Notably, Notch transcription was shown to be directly activated by Notch signaling. Such an auto-activation loop might rapidly go awry in a context in which Notch pathway activation is independent of ligand binding. By promoting the accumulation of Lz during crystal cell maturation, MLF and DnaJ-1 thus provide an effective cell-autonomous mechanism to inhibit Notch signaling. Further experiments will now be required to establish how Lz represses Notch transcription. RUNX factors can act as transcriptional repressors by recruiting co-repressor such as members of the Groucho family. Whether MLF and DnaJ-1 directly contribute to Lz-induced-repression in addition to regulating its stability is an open question. MLF and DnaJ-1 were recently found to bind and regulate a common set of genes in cell culture. They may thus provide a favorable chromatin environment for Lz binding or be recruited with Lz and/or favor a conformation change in Lz that allows its interaction with co-repressors. The scarcity of lz>GFP+ cells precludes a biochemical characterization of Lz, MLF and DnaJ-1 mode of action notably at the chromatin level but further genetic studies should help decipher their mode of action. While the post-translational control of Notch has been extensively studied, its transcriptional regulation seems largely overlooked. The current findings indicate that this is nonetheless an alternative entry point to control the activity of this pathway. Given the importance of RUNX transcription factor and Notch signaling in hematopoiesis and blood cell malignancies, it will be of particular interest to further study whether RUNX factors can regulate Notch expression and signaling during these processes in mammals (Miller, 2017).

Targets of Activity (part 1/3)

Notch signaling, provided by the ligands Delta and Serrate, is carried to the nucleus by Suppressor of Hairless protein.

The Notch pathway is required for Enhancer of split expression during neurogenesis (Jennings, 1994). Whether or not Suppressor of Hairless, and indirectly, Notch, have other targets has not yet been established. Possible Notch targets are suggested by the results of Notch overexpression in the wing disc. Notch is locally activated at the wing margin, as demonstrated by the restricted expression of the Enhancer of split proteins in dorsal and ventral cells abutting the D/V boundary throughout the third larval instar. Notch gain-of-function alleles in which Notch activity is not restricted to the dorsoventral boundary cause mis-expression of cut and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation for wing development (de Celis, 1996a).

Notch targets tramtrack. Experimental evidence suggests that there is an alteration of ttk expression due to reduction or overexpression of Notch. ttk is normally expressed in the sheath cell, one of the products of the sensory organ precursor lineage, but not in the neural cell, the sister of the sheath cell. In Notch mutants, extra neurons have detected resulting from a transformation of sheath cells into neurons. ttk is expressed in most cells in the epidermis of the mutant embryo, but not in neurons, including the supernumerary neurons derived from transformation of sheath cells. Thus Notch function is required not only to specify the sheath cell but also to express ttk in this daughter cell of an asymmetric division. In a reciprocal experiment, overexpression of Notch turns on ttk expression in cells that normally do not express ttk. It is concluded that Notch targets ttk, presumably downstream of Numb (Guo, 1996).

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm (Cowden, 2002).

Neural precursors (or neuroblasts) divide in a stem cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only once to generate two neurons. A screen was carried out for genes expressed in the MP2 neuroblast and its progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. A P-element insertion line was identified that expresses the reporter gene, tau-beta-galactosidase, in the MP2 precursor and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene, mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the MP2 lineage transforms dMP2 to vMP2. Notch signaling requires Mastermind, suggesting that Mastermind acts downstream of Notch to determine the vMP2 cell fate. Mastermind plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies the pCC and RP2sib fates, respectively. This suggests that Notch signaling through Mastermind plays a wider role in specifying neuronal identity in the Drosophila central nervous system. Notch is expressed in both MP2 progeny. Notch signaling is blocked by Numb, which segregates exclusively to dMP2 when the MP2 precursor divides. Numb interacts directly with the intracellular domain of Notch. By antagonizing Notch, Numb promotes the dMP2 cell fate. Thus it is likely that Numb antagonism of Notch signaling in dMP2 confines Mastermind function, acting downstream of Notch, to the vMP2 neuron (Schuldt, 1998).

Cell proliferation in the excretory organs of Drosophila, the Malpighian tubules (MT), is under the control of a neural tip cell. This unique cell is singled out from equivalent MT primordial cells in response to Notch signalling. The gene Kruppel (Kr), best known for its segmentation function in the early embryo, is under the control of the Notch-dependent signalling process. Lack-of-function and gain-of-function experiments demonstrate that Kr activity determines the neural fate of tip cells by acting as a direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. A unique cis-acting element has been identified that mediates all spatial and temporal aspects of Kr gene expression during MT development. This element contains functional binding sites for the restricted proneural bHLH factors and Fork head protein which is expressed in all MT cells. These results suggest a mechanism in which these transcription factors cooperate to set up a unique cell fate within an equivalence group of cells by restricting the activity of the developmental switch gene Kr in response to Notch signalling (Hoch, 1998).

Notch signaling may regulate the restricted expression of buttonless. buttonless is expressed in dorsal median cells, mesodermal cells that are arranged as a single pair within each segment along the dorsal midline, just above the central nervous system. Dorsal midline cells in Notch mutant cells differentiate with approximately twice the wild-type number. The phenocritical period for hypertrophy of the DM cells in the temperature sensitive Notch mutant occurs between 4 and 6 hours after fertilization (Chiang, 1994 and Hartenstein, 1992).

lame duck expression was examined in wg and N mutant embryos to determine the relative position of lmd within the genetic hierarchy that controls somatic muscle specification. In wgcx4 mutant embryos, lmd RNA expression is not detectable in dorsolateral and lateral somatic mesodermal cells although there is residual expression in cells located in the ventral region. Thus, activation of lmd expression is mediated via wg-dependent and wg-independent pathways. Significantly, lmd expression in the somatic mesoderm is completely abolished in N5419 mutant embryos, indicating that activation of lmd expression in presumed fusion-competent myoblasts requires active Notch signaling. By contrast, founder cell formation is promoted in the absence of Notch function (Duan, 2001).

What is the function of Sparkling in the differentiation of bristles? All bristles or mechanosensory organs of the adult fly arise in a simple, stereotyped manner by three consecutive asymmetric divisions during which a single SOP gives rise to a neuron and three support cells. The first division generates different siblings, one of which by subsequent division produces the cells that form the socket (tormogen) and shaft (trichogen); the other sibling produces the neuron and glial cell (thecogen). Sparkling appears to be required at various stages during bristle development. Initially, it is observed in SOPs and all four bristle precursor cells (Fu, 1997). However, by mid-pupal stages it is no longer expressed in the tormogen and neuron, but continues to be expressed in the thecogen and is strongly elevated in the trichogen. If thecogen and trichogen, in response to opposite Notch (N) signaling, arise from their precursors by analogous asymmetric divisions, Sparkling expression must also react to N signaling in these cells in opposite ways. For example, while N signaling may activate sparkling through Su(H) in glial cells, it may repress sparkling in socket cells. Comparison of Spa protein levels in the developing eye bristles of svn (svn is an allele of sparkling) mutants with those of wild type suggests that its expression needs to be maintained at least to mid-pupal stages for proper differentiation of trichogens and the formation of a shaft of normal size. However, trichogen cells are able to differentiate to some extent in hypomorphic svn mutants that express Spa during early pupal stages. If the levels of Spa are further reduced, the shafts are also shortened or completely missing. In that case, the shaft is replaced by a second socket, which indicates that the trichogen is no longer able to differentiate properly but becomes a tormogen. It appears therefore that Sparkling protein is not only required during differentiation of a shaft cell, but also serves at an earlier time to specify the fate of shaft versus socket cell. This would be consistent with Sparkling being a target of the N signaling pathway. The roles that Sparkling plays in developing cone and primary pigment cells or in bristle cells, which manifest themselves in the various enhancer mutant alleles of spa and sv, are probably not the only functions of Sparkling as evident from several additional locations where Spa protein can be detected during embryonic and larval development. The expression of Sparkling in the embryonic PNS may indicate that Sparkling plays a function in the development of larval sense organs analogous to that of sv in the development of the mechanosensory bristles of the adult (Fu, 1998).

The adult peripheral nervous system of Drosophila includes a complex array of mechanosensory organs (bristles) that cover much of the body surface of the fly. The four cells (shaft, socket, sheath, and neuron) that compose each of these organs adopt distinct fates as a result of cell-cell signaling via the Notch (N) pathway. However, the specific mechanisms by which these cells execute their conferred fates are not well understood. shaven, called in this paper D-Pax2, the Drosophila homolog of the vertebrate Pax2 gene, has an essential role in the differentiation of the shaft cell. In flies bearing strong loss-of-function mutations in shaven, shaft structures specifically fail to develop. Consistent with this, Shaven protein is expressed in all cells of the bristle lineage during the mitotic (cell fate specification) phase of bristle development, but becomes sharply restricted to the shaft and sheath (glial) cells in the post-mitotic (differentiative) phase. Three cell types, early on the pIIA secondary precursor and later the tormogen, and the thecogen, are responsive to the N-mediated signals sent by their sister cells, but pIIB, the trichogen, and the neuron are made resistant to the reciprocal signal by N pathway antagonists such as Numb and Hairless. Interestingly, while the tormogen/socket and thecogen/glial/sheath cells are recipients of Notch signals, it is the trichogen/shaft and thecogen that express Shaven (Kavaler, 1999).

An anti-Shaven antiserum was used to examine the temporal and spatial pattern of Shaven accumulation in the pupal notum during microchaete development. Shaven expression is first apparent in the SOP nucleus before division (14 hours after puparium formation (APF). After SOP division (16 hours APF), Shaven is present at similar levels in the nuclei of the two daughter cells, the secondary precursors pIIA and pIIB. Following the completion of the pIIA and pIIB divisions (18 hours APF), all four cells of the microchaete lineage express Shaven at fairly comparable levels, though one cell (the presumptive trichogen/shaft) is regularly distinguishable at this stage by its slightly elevated accumulation of the protein. Subsequently, the pattern of Shaven expression is refined so that by 32 hours APF, Shaven protein is present in only two cells, one containing a large polyploid nucleus (either the trichogen or the tormogen/socket) and one containing a small nucleus (either the neuron or thecogen/glia). By double-labeling nota with anti-Shaven antibody and the neuron/shaft marker mAb 22C10, the two cells that express Shaven at 32 hours APF could be identified. Microchaete neurons (clearly identifiable by their 22C10-positive cell bodies and axons) are Shaven-negative, but are positioned adjacent to small, Shaven-positive nuclei in 22C10-negative cells, which by inference are thecogens. This interpretation is confirmed by a second double-labeling experiment using anti-Elav (a specific nuclear marker for post-mitotic neurons) and anti-D-Shaven antibodies at 32 hours APF. In both macrochaetes and microchaetes, the small nucleus that labels with anti-Shaven is clearly distinct from the small nucleus that labels with anti-Elav. Thus, the two bristle cells that exhibit specific nuclear expression of Shaven at 32 hours APF are the thecogen and trichogen. These results distinguish two phases of Shaven expression in the bristle lineage: an early stage, in which the protein appears at similar levels in the SOP and all of its progeny as cell division proceeds, and a late stage, in which Shaven expression is restricted to the postmitotic trichogen (shaft) and thecogen (sheath) cells (Kavaler, 1999).

Two lines of evidence described here indicate that shaven expression and function is at least in part downstream of cell fate specification mechanisms such as N signaling. (1) The lack of late shaven expression in the socket cell (the sister of the shaft cell) is controlled by N pathway activity; (2) loss of shaven function is epistatic to the socket-to-shaft cell fate transformation caused by reduced N signaling. Overexpression of H after the completion of the microchaete cell divisions results in the expression of the shaft cell fate by both the normal trichogen and the transformed tormogen; that is, reduction of N pathway function promotes the expression of the shaft differentiation program. Loss of shaven function has the opposite effect: the stronger shaven mutant genotypes cause a broad failure of shaft development while permitting the normal or nearly normal expression of the other bristle cell fates. The epistatic relationship between excess H activity and loss of shaven function was examined by combining these two conditions and observing the effect on shaft differentiation. Homozygosity for even the comparatively weak shaven allele effectively suppresses the double shaft phenotype of heat-treated Hs-H transgenic flies. Thus, in the combined genotype large territories devoid of external bristle structures are observed. This effect may be explained as follows: excess H activity alone causes both the normal trichogen and the transformed tormogen to adopt the shaft fate, but reduction of shaven function prevents the expression of this fate by either cell, so no external cuticular structures are produced by the double mutant bristles. In other words, loss of shaven activity is epistatic to the effects of reduced N pathway function on the expression of the shaft differentiation program. This result strongly suggests that shaven acts at least in part downstream of the N-dependent trichogen/tormogen cell fate decision in the bristle lineage, and plays a major role in the differentiation phase of shaft cell development. Additional experiments show that misexpression of shaven is sufficient to induce the production of ectopic shaft structures. From these results, it is proposed that Shaven is a high-level transcriptional regulator of the shaft cell differentiation program, and acts downstream of the N signaling pathway as a specific link between cell fate determination and cell differentiation in the bristle lineage (Kavaler, 1999).

atonal is a proneural gene for the development of Drosophila chordotonal organs and photoreceptor cells. atonal expression is controlled by modular enhancer elements located 5' or 3' to the atonal-coding sequences. During chordotonal organ development, the 3' enhancer directs expression in proneural clusters; whereas successive modular enhancers located in the 5' region drive tissue-specific expression in chordotonal organ precursors in the embryo and larval leg, wing and antennal imaginal discs. Similarly, in the eye disc, the 3' enhancer directs initial expression in a stripe anterior to the morphogenetic furrow. These atonal-expressing cells are then patterned through a Notch-dependent process into initial clusters, representing the earliest patterning event yet identified during eye morphogenesis. A distinct 5' enhancer drives expression in intermediate groups and R8 cells within and posterior to the morphogenetic furrow. Both enhancers are required for normal atonal function in the eye. The 5' enhancer, but not the 3' enhancer, depends on endogenous atonal function, suggesting a switch from regulation directed by other upstream genes to atonal autoregulation during the process of lateral inhibition. The regulatory regions identified in this study can thus account for atonal expression in every tissue and essentially in every stage of its expression during chordotonal organ and photoreceptor development (Sun, 1998).

Restriction of proneural gene expression from proneural clusters to SOPs is usually Notch (N) dependent. During eye development, N is known to function within and posterior to the MF in restricting ato expression to R8 cells within intermediate groups. Anterior to the morphogenetic furrow, N has been shown to promote ato expression. To test the function of N in the formation of the ato prepattern anterior to the MF and in regulating the 3' enhancer, lacZ expression was examined from the 3' enhancer-lacZ reporter gene in a temperature-sensitive N mutant background. When larvae carrying the temperature sensitive N allele and the 3' enhancer-lacZ fusion gene are shifted to the restrictive temperature for 2 hours, the 3' enhancer-directed lacZ expression anterior to the MF becomes continuous and appears broader and stronger than that in wild type, and the initial clusters normally seen within the initial stripe fail to form. The endogenous ato gene responds similarly to N inactivation in the initial stripe. It is concluded that N is involved in refining ato expression anterior to the MF from a continuous band to patterned initial clusters, which prefigure the future ommatidia (Sun, 1998).

The adult external sense organ precursor (SOP) lineage is a model system for studying asymmetric cell division. Adult SOPs divide asymmetrically to produce IIa and IIb daughter cells; IIa generates the external socket (tormogen) and hair (trichogen) cells, while IIb generates the internal neuron and sheath (thecogen) cells. The expression and function of prospero has been examined in the adult SOP lineage. Although Prospero is asymmetrically localized in embryonic SOP lineage, this is not observed in the adult SOP lineage: Prospero is first detected in the IIb nucleus; during IIb division, it is cytoplasmic and inherited by both neuron and sheath cells. Subsequently, Prospero is downregulated in the neuron but maintained in the sheath cell. Loss of prospero function leads to double bristle sense organs (reflecting a IIb-to-IIa transformation) or single bristle sense organs with abnormal neuronal differentiation (reflecting defective IIb development). Conversely, ectopic prospero expression results in duplicate neurons and sheath cells and a complete absence of hair/socket cells (reflecting a IIa-to-IIb transformation). It is concluded that (1) despite the absence of asymmetric protein localization, prospero expression is restricted to the IIb cell but not its IIa sibling; (2) prospero promotes IIb cell fate and inhibits IIa cell fate, and (3) prospero is required for proper axon and dendrite morphology of the neuron derived from the IIb cell. Thus, prospero plays a fundamental role in establishing binary IIa/IIb sibling cell fates without being asymmetrically localized during SOP division. Finally, in contrast to previous studies, the IIb cell is found to divide prior to the IIa cell in the SOP lineage (Manning, 1999).

What mechanisms lead to prospero expression in the IIb cell but not in the IIa cell? Specification of IIa/IIb cell fates is determined by the relative activity of Notch signaling. Productive Notch signaling results in IIa cell fate; asymmetric localization of Numb protein into the IIb cell blocks Notch signaling and results in the IIb cell fate. It is proposed that productive Notch signaling prevents prospero expression in the IIa cell, whereas lack of Notch signaling allows prospero expression in the IIb cell. Consistent with this model, SOP lineages with unregulated Notch signaling produce a pair of IIa cells that both fail to express prospero, while SOP lineages lacking Notch function produce two IIb cells that both express prospero (Reddy, 1999). One effector of Notch signaling in the IIa cell is the zinc-finger transcriptional repressor Tramtrack, which may directly or indirectly repress prospero expression. Interestingly, prospero is expressed in the R7 neuron during eye development and tramtrack mutants have supernumerary R7 neurons, while tramtrack misexpression reduces R7 differentiation. Thus, a similar Notch-, tramtrack-dependent pathway may repress prospero expression in both the R7 photoreceptor neuron and the IIa cell. It should be noted that a somewhat different mechanism must be involved in repressing prospero in the neuron but not the sheath cell; in this case, Notch signaling is required for sheath cell fate, the cell that maintains prospero expression. The lack of Notch-mediated repression of prospero expression in the sheath cell may reflect the fact that Notch signaling is SuH-dependent in the IIa cell, but SuH-independent in the sheath cell. prospero is essential for distinguishing IIa and IIb cell fates (Manning, 1999 and references).

A role for prospero in establishing different IIa/IIb cell fates has been demonstrated based on both loss-of-function and misexpression experiments. A significant fraction of the SOP lineages lacking prospero function show a duplication of the external bristle (a progeny of the IIa cell) and a loss of the neuron (a progeny of the IIb cell) (Reddy, 1999). Socket cell fate could not be adequately scored, because multiple socket cells can generate a single, fused socket structure. The simplest interpretation of the double bristle prospero minus sense organs is that the IIb cell has become partially or fully transformed into a IIa cell, resulting in duplicate hair/sockets and loss of neuron/sheath cell. It is unlikely, but it cannot be rule out, that the neuron is transformed into a duplicate hair cell and the sheath cell is unaffected. In both notum and eye, however, there are still many single bristle sense organs that have an associated neuron and, in these sense organs, the IIb cell must have been specified relatively normally. Thus prospero is not strictly necessary for IIb cell specification, but its function is important for the high-fidelity specification of IIb cell fate. While the presence of prospero in the IIb cell is important for reliable IIb cell specification, the absence of prospero from the IIa cell is absolutely essential for IIa cell specification. Misexpression of prospero in the IIa cell and its progeny results in a fully penetrant loss of a socket cell marker (SuH) as well as the morphological external socket and hair structures; there is a corresponding increase in the internal Elav+ neurons and BarH1+ sheath cells. The misexpression experiments show that absence of Prospero in the IIa cell is required for normal IIa development, and that the presence of Prospero in the IIa transforms it partially or fully to the IIb cell fate. Thus, differential expression of prospero between IIa and IIb siblings is essential for normal SOP development. Similar results were observed using transient heat-shock-induced misexpression of prospero, although in these experiments a very low frequency of double and triple bristle sense organs at the borders of the bald areas was observer. The cell lineage of these rare sense organs is unknown (Manning, 1999).

It is interesting to consider the different mechanisms by which prospero acts to distinguish sibling cell fate. During embryonic neuroblast cell division, localization of Prospero into the daughter GMC is necessary for GMC development, but exclusion of Prospero from the neuroblast is relatively unimportant for neuroblast development (this is because neuroblast development is fairly normal in miranda mutants where Prospero remains in the neuroblast; Chris Doe, unpublished results cited in Manning, 1999). In contrast, during the adult SOP lineage, it appears equally important to remove Prospero from the IIa cell as well as provide it to the IIb cell. Another key difference between the adult SOP lineage and the embryonic SOP and neuroblast lineages is the timing of cell divisions. There are several hours between each cell division in the adult SOP lineage, considerably longer than the 40-60 minutes cell cycle of embryonic neuroblasts and SOPs. The shorter cell cycles of the embryonic lineages may require asymmetric localization of Prospero for efficient specification of sibling cell fate, whereas the longer adult SOP cell cycles may provide time for the action of other regulatory mechanisms (e.g. Notch-mediated repression of prospero expression) (Manning, 1999).

In single bristle prospero minus sense organs, a single neuron was observed with profound defects in neurite outgrowth. The defects in axon and dendrite outgrowth and connectivity could be due to lack of prospero function in the IIb cell, a non-autonomous effect due to lack of prospero function in the sheath cell, or the absence of prospero function in the neuron itself. The first possibility is unlikely because axon outgrowth defects can be observed in R7 neurons, which do not arise from a Prospero+ precursor cell. The second possibility is unlikely because lack of sheath cells (in glial cells missing embryos) does not generate similar axon outgrowth defects. A third model is favored, in which prospero has a direct function in the neuron, because many neurons with different origins (CNS, PNS, eye) transiently express prospero and all show a similar prospero mutant phenotype: stunted and misrouted axons (Manning, 1999).

hibris is regulated by Notch and Ras in a Toll10b mutant background. This regulation was confirmed in vivo in wild-type embryos. hbs expression was examined in Notch and Ras loss-of-function embryos and embryos overexpressing activated forms of Notch and Ras in the mesoderm. A dominant negative Ras construct activates hbs expression in the somatic mesoderm. Zygotic null Notch embryos show lower hbs transcription. Conversely, an activated form of Notch upregulates hbs in the mesoderm, while an activated form of Ras almost completely inhibits hbs expression. These results argue that, upon stimulation, Notch activates hbs, while Ras acts as a negative signal, and predicts that hbs expression in the somatic mesoderm would be restricted to fusion-competent cells (Notch dependent) and excluded from founder cells (Ras dependent). It is not known whether this regulation is direct, that is, Notch or Ras effectors act directly on the hbs promoter, or indirect, that is, Notch/Ras converts cell fate, which in turn would lead to hbs upregulation/downregulation by some other effector (Artero, 2001).

Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. The single Drosophila Hey orthologue is primarily expressed in a subset of newly born neurons that receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed the study of Notch signalling during the genesis of secondary neurons in the larval central nervous system (Monastirioti, 2010).

Among the superfamily of basic-helix-loop-helix (bHLH) transcription factors, several structurally distinct classes are discerned. One of these, the bHLH-Orange (bHLH-O) class, is characterized by the 'Orange' domain, a protein interaction domain perhaps serving as an extended dimerization surface. bHLH-O proteins are important developmental and physiological regulators in processes ranging from neurogenesis to circadian rhythm control (Monastirioti, 2010).

In a number of invertebrate and vertebrate species, bHLH-O repressors are known to inhibit neural differentiation. In Drosophila, the seven E(spl) bHLH-O proteins are expressed in the neuroectoderm, where they inhibit cells from differentiating as neuroblasts (NBs). In vertebrates, a number of Hes bHLH-O proteins, notably Hes1, Hes3 and Hes5 in the mouse, are also expressed in the neuroectoderm; in this case it is the neural stem cells that express the Hes genes, which are subsequently downregulated in the differentiating neuronal progeny. Triple Hes1, Hes3, Hes5 knock-out causes premature neural differentiation, disruption of the neuroepithelium and a hypoplastic nervous system owing to stem cell depletion. In Drosophila, loss of the entire E(spl) locus results in supernumerary NB specification from the neuroectoderm and a hyperplastic nervous system. Despite these differences, owing to the different mode of neural precursor specification between vertebrates and insects, the generalization can be made that E(spl)/Hes proteins antagonize neuronal differentiation. At most developmental settings across metazoan phylogeny, neural expression of E(spl)/Hes genes is a direct response to Notch signalling (Monastirioti, 2010).

Expression of another subfamily of bHLH-O genes has been detected in the progenitor cell zones of the developing vertebrate central nervous system (CNS). These genes encode the three Hey proteins, so named after a characteristic tyrosine residue in their C-terminal domain (Hairy/enhancer-of-split like with a Y); they are also known as Hrt, Herp, Hesr, Chf or Gridlock. Although neural defects are minor in Hey knock-out mice, overexpression studies have suggested that Hey and Hes proteins might synergize with each other in suppressing neural differentiation and maintaining the neural stem cell fate. Hey1 has even been linked to the pathogenesis and aggressiveness of gliomas. Hey knock-out mice have highlighted their roles in developmental processes outside the nervous system, in particular, heart and vasculature development. In these contexts, all three mammalian Hey genes appear to respond to Notch signalling, similar to E(spl)/Hes genes in neurogenesis. Biochemical data support Hes-Hey heterodimer formation, raising the possibility that these two subclasses of bHLH-O proteins might synergize in some developmental contexts as Notch effectors (Monastirioti, 2010).

The Drosophila genome contains a single Hey orthologue (Kokubo, 1999), which had not been studied to date. This study characterized it in the hope of better understanding the process of neural precursor specification, based on the assumption that, by analogy to vertebrates, Hey might display protein-protein interactions with E(spl). Surprisingly, Hey was not co-expressed with the E(spl) proteins in the neuroectoderm, rather was restricted to differentiating neurons, suggesting a radically different role in neurogenesis than was assumed. Once NBs are specified in Drosophila, they undergo cycles of asymmetric cell divisions that give rise to a secondary precursor, called a ganglion mother cell (GMC), in addition to self-renewing. GMCs divide once to give rise to two neurons or, less often, glia. The majority of GMC divisions are asymmetric, with the fates of the two daughters dictated by unequal levels of Notch signalling. The 'A' sibling neuron requires high Notch signalling, whereas the 'B' sibling neuron downregulates Notch reception, which is usually achieved by asymmetric segregation of a Notch inhibitor, Numb, into the nascent 'B' neuron. This study describes a complex pattern of Hey expression in relation to these divisions during both neurogenic phases of the animal, early embryogenesis and larval life, where thousands of new neurons are added to generate the adult CNS. In all sibling pairs that were identified, Hey was expressed in the 'A' neuron. Genetic analysis confirmed that Hey is a Notch target gene in most instances. These results extend the Hey-Notch relationship to Drosophila in support of an ancient connection between bHLH-O genes and Notch activity and, for the first time, implicate a bHLH-O protein in the process of GMC asymmetric division (Monastirioti, 2010).

A full-length Hey cDNA was amplified from a Drosophila cDNA library, which was used as a probe for in situ hybridization, and for cloning in prokaryotic expression vectors. Bacterially expressed full-length Hey protein was used to raise anti-Hey antibodies. There were no obvious differences between the RNA and protein patterns. Hey protein showed nuclear accumulation, as expected for a transcription factor, and was primarily detected in a segmentally repeated pattern within the CNS starting at stage 10. Later, more Hey-positive cells gradually appear in the CNS. The neuroectodermal epithelium, where the related E(spl) bHLH-O proteins are expressed already starting at stage 8, is devoid of Hey expression, which instead is detected at deeper levels overlapping with the GMC/immature neuron marker Pros. From double-staining with the neuronal antigen Elav it was clear that the vast majority of Hey-positive cells represent neurons rather than GMCs, confirmed as lack of colocalization with the NB/GMC marker Asense. Besides neurons, Hey expression was detected in a subset of Repo-positive glia of the CNS and peripheral nervous system (PNS). Of note, Eve staining, which was used to visualize particular neurons, also marks the dorsally located pericardial cells. No Hey immunoreactivity was detected within or near these heart precursors, contrary to the strong expression of mammalian Hey genes during cardiogenesis. Finally, a few Hey-positive cells per segment were detected in the embryonic PNS. Most of these were also neurons, by virtue of being Elav-positive, but were not characterized further (Monastirioti, 2010).

Lineage-specific markers were used to characterize Hey expression in more detail. One was Even skipped, which marks a subset of neurons: the aCC/pCC sibling pair, the RP2 motoneuron, the cluster of U motoneurons and the cluster of EL interneurons. Another was the AJ96-lacZ enhancer trap, which marks the MP2 precursor and its progeny, the dMP2/vMP2 neurons. With AJ96-lacZ, strong Hey accumulation was detected in vMP2 but not in dMP2. Weak Hey expression was detected shortly before mitosis of the MP2 progenitor during late stage 10. Among the Eve-positive neurons, pCC and the U neurons expressed Hey. aCC, RP2 and the EL neurons were Hey-negative. At stage 11, the sibling of RP2, RP2sib, a smaller cell, which only transiently expresses Eve, was Hey-positive. Hey expression in all these neurons appeared transient. For example, whereas immunoreactivity in vMP2 was strong at stage 12, it was downregulated and barely detectable by stage 14. Similarly, by stage 14 no Hey could be detected in pCC cells, although it was still expressed strongly in some of the later-born U motorneurons. Transient Hey expression was also observed in the two identical progeny of MP1, a midline precursor, which are marked by Odd (Monastirioti, 2010).

Most of the neurons described above belong to well-characterized lineages, in which sibling fates arise through differential Notch signalling. In each of the RP2/RP2sib, aCC/pCC and dMP2/vMP2 pairs, the second cell requires Notch signalling in order to acquire the 'A' fate, distinct from that of its sibling cell ('B' fate). Also in the U lineages, which arise from sequential GMCs from neuroblast NB7-1, the U neurons require Notch, whereas their Eve-negative Usib neurons do not. All Notch-requiring cells, namely RP2sib, pCC, vMP2 and the U cells, robustly express Hey, whereas none of their 'B'-fate siblings do so. This raises the possibility that Hey is expressed in response to Notch (Monastirioti, 2010).

Thus Hey was detected almost exclusively in the CNS in young postmitotic neurons and glia, specifically those that receive a Notch signal at birth. It has long been appreciated that Notch signalling plays an important role in the acquisition of neuronal/glial cell fate after GMC division, with most GMCs producing two different progeny, an 'A' cell with high Notch activity and a 'B' cell with no Notch activity. Still, no Notch target genes had been identified in this process. This study shows that Hey is such a target gene in many, and perhaps all, GMC asymmetric divisions. These conclusions are based on the expression pattern of Hey, its response to Notch pathway perturbation and on the ability of ectopic Hey to block development of RP2 and dMP2, two 'B'-type neurons (Monastirioti, 2010).

Although these is good evidence that Hey expression can recapitulate the effect of Notch signalling, Hey loss-of-function has only a mild phenotype. The trivial possibility that the transposon insertion allele used has residual activity is unlikely as (1) no Hey protein is detectable in homozygous mutants and (2) the Heyf06656 allele results in recessive lethality. Nevertheless, the issue will be permanently decided with the generation and analysis of more Hey alleles. The alternative hypothesis, which seems more probable, is that one or more additional factors besides Hey can also act as nuclear effectors downstream of Notch in the 'A' GMC progeny. No Hey paralogues exist in the D. melanogaster genome, but structurally divergent proteins, even outside the bHLH-O family, could share similar functional characteristics. At the moment, there are no good candidate for such a factor; however, a number of bHLH-O factors have been excluded that do not seem to be co-expressed with Hey in neurons, namely E(spl)mγ and m8, Hairy and Dpn (Monastirioti, 2010).

Besides GMCs, a number of other neural progenitors, namely NBs, sensory organ precursors (SOPs) and SOP progeny cells, all undergo asymmetric cell divisions with Notch involvement. No Hey expression was detected in either the NB/GMC pair or in the SOP progeny cells of external sensory organs, suggesting that Hey expression is turned on exclusively in GMC asymmetric divisions. Hey-positive glia could also be the progeny of asymmetrically dividing GMCs. It is yet unclear which cells might be the immediate progenitors of the few Hey-positive PNS neurons (Monastirioti, 2010).

Until the present work and the recent paper by Krejci (2009), the only Drosophila bHLH-O genes known to be targets of Notch were the seven of the E(spl) complex. Hey and two other bHLH-O genes, dpn and Her, had been predicted as candidate Notch targets based on nearby clustering of putative Su(H) binding sites, the DNA elements via which activated Notch is tethered to its target genes. Although HES-related (Her) does not seem to be a true Notch target have shown that dpn is a Notch target in the muscle-progenitor-like Drosophila DmD8 cell line; an in vivo context for such a response has yet to be determined. Together with Hey, this makes a total of 9 out of 13 bHLH-O genes in the Drosophila genome which are regulated by Notch. It should be stressed that Notch has a number of additional (non-bHLH-O) targets, depending on the species and cellular context, but few, if any, show such widespread association as the bHLH-O genes. The latter are activated by Notch in a multitude of unrelated contexts, such as neuroectoderm, mesoderm, wing epithelium, leg segmentation and now GMC asymmetric cell divisions in Drosophila, and in neural progenitors, presomitic mesoderm, cardiogenesis and vasculogenesis in vertebrates (Monastirioti, 2010 and references therein).

In addition to its widespread Notch-dependent expression, this study detected a clear instance of Notch-independent expression of Hey within the GMCs and neurons of the MB precursors. Other examples where Hey expression does not correlate with known events of Notch signalling are the MP2 NB and the two MP1 midline neurons. It is also clear that in embryos with severe Notch signalling defects, a small number of Hey-positive cells is still seen in the CNS, suggesting that there are additional neural lineages, where Hey is likely to be expressed independently of Notch. Analysis of the cis regulatory regions of Hey should shed light on Notch-dependent and Notch-independent enhancer elements (Monastirioti, 2010).

The bHLH-O family has undergone considerable diversification during evolution. Although sequence analysis can unambiguously assign genes to this family, it cannot identify orthologues in distantly related species. A classic example is the Drosophila to mammals comparison, where no clear orthologue relationships exist between Hairy, Dpn and the seven E(spl) in Drosophila and Hes1, 2, 3, 5, 6 and 7 in mammals, suggesting that the diversification of these proteins occurred separately after divergence of protostomes and deuterostomes. Hey proteins are the singular exception, being particularly well conserved. The bHLH domain of Drosophila Hey shows 97-98% similarity to that of its mammalian counterparts. This might lead one to expect substantial conservation of Hey function, which, strangely enough, was not observed (Monastirioti, 2010).

First, mammalian Hey genes have a very broad expression pattern, including presomitic mesoderm, embryonic heart, vascular precursors, developing brain and spinal cord, neural crest etc (Kokubo, 1999; Leimeister, 1999). Fly Hey, by contrast, seems confined within the CNS and PNS. Although there is complexity in its expression, as documented in this study with its contextual Notch dependence/independence, the great majority of its expression pattern seems to be in the newly born Notch-dependent 'A'-type neurons. The absence of Hey expression from the developing Drosophila heart is most striking, given the foremost importance of Hey genes in vertebrate cardiogenesis. A second indicator of functional non-conservation comes from comparing the role of Hey within the nervous systems of mammals versus Drosophila. In the former, Hey has been proposed to act in the maintenance of progenitor fate and to antagonize neuronal differentiation, similar to Hes proteins. In fact, it has been proposed that Hey-Hes heterodimers mediate these effects. In the fly, Hey expression was not detected within progenitor cells, with the few rare GMC exceptions, noted above. Hey-E(spl) or Hey-Dpn co-expression could not be detected, although all seven E(spl) genes were not tested for lack of specific reporter lines. To overcome any doubt, functional tests were made by ectopically expressing Hey. Instead of suppressing sensory organ formation, it mildly increased the number of bristles, showing an opposite phenotype from that of E(spl) or hairy ectopic expression. It can therefore be confidently said that Hey does not antagonize neural differentiation in the fly (Monastirioti, 2010).

This leaves a puzzle of why Hey is so strongly conserved. Perhaps some yet uncharacterized molecular aspect of its role in chromatin recognition/transcriptional regulation is conserved, despite considerable diversification in cellular and developmental contexts. These contexts have diverged greatly between insects and vertebrates, the only unifying theme being their regulation by Notch signalling. A homologous function might be that of promoting gliogenesis, as Hey2 was shown to promote Müller glia formation in the murine retina. Further comparative studies encompassing more species will no doubt shed light on the function of this highly conserved bHLH-O protein (Monastirioti, 2010).

The bHLH factor deadpan is a direct target of Notch signaling and regulates neuroblast self-renewal in Drosophila

A defining feature of stem cells is their capacity to renew themselves at each division while producing differentiated progeny. How these cells balance self-renewal versus differentiation is a fundamental issue in developmental and cancer biology. The Notch signaling pathway has long been known to influence cell fate decisions during development. Indeed, there is a great deal of evidence correlating its function with the regulation of neuroblast (NB) self-renewal during larval brain development in Drosophila. However, little is known about the transcription factors regulated by this pathway during this process. This study shows that deadpan (dpn), a gene encoding a bHLH transcription factor, is a direct target of the Notch signaling pathway during type II NB development. Type II NBs undergo repeated asymmetric divisions to self-renew and to produce immature intermediate neural progenitors. These cells mature into intermediate neural progenitors (INPs) that have the capacity to undergo multiple rounds of asymmetric division to self-renew and to generate GMCs and neurons. The results indicate that the expression of dpn at least in INPs cells depends on Notch signaling. The ectopic expression of dpn in immature INP cells can transform these cells into NBs-like cells that divide uncontrollably causing tumor over-growth. In addition to dpn, Notch signaling must be regulating other genes during this process that act redundantly with dpn (San-Juán, 2011).

During the division of type II NBs the asymmetric sequestration of Numb into one daughter cell ensures that the activity of Notch signaling is restricted to the NBs, whereas it is blocked in the other daughter cell, the immature INP cell. The down-regulation of Notch signaling in this latter cell prevents it being transformed into NBs. When Notch signaling is ectopically expressed in the immature INP, it cannot mature into an INP and it then over-proliferates as NB-like cells. The results indicate that this function of Notch signaling is through Su(H). Evidence is presented that suggests that the bHLH factor dpn is one direct target of Notch signaling during this process. Alterations in the activity of this gene reproduce the effects found when Notch signaling is ectopically activated. In addition, a regulatory region upstream of the transcriptional initiation site was identified that drives the expression of dpn in NBs and INPs, that is directly regulated by Su(H). Altogether, these results lead to the following model: After the asymmetric division of NBs, the down-regulation of Notch signaling in the immature INP prevents the activation of dpn in this cell. This process, which likely occurs in conjunction with other mechanisms that promote Dpn degradation, rapidly eliminates Dpn in immature INP. The loss of Dpn permits the maturation of the immature INP into an INP cell. When Dpn is continuously expressed during asymmetric NB division, either by the activation of Notch signaling or by ectopic expression of Dpn, both recently born cells will express high levels of Dpn. This event can cause the prevention of maturation of immature INP into INP that would cause this cell to adopt its parental NB fate, entering mitosis and initiating over-growth of NB-like cells. Although, these over-growths are mostly constituted by NB-like cells, occasionally INPs (Ase+) and also few Elav positive cells were found. It is thought that the diversity of cell types within clones of dpn-expressing cells is likely due to differentiation of some of the immature INPs contained in these clones. These few escaper cells can give rise to all cell types found in a type II NB lineage (San-Juán, 2011).

A Notch-responsive enhancer has been identified that is contained in a regulatory region upstream of the transcriptional initiation site of dpn. This enhancer drives the expression of dpn in all NBs as well as in INPs, reproducing the pattern of expression of the endogenous Dpn. These data suggest that Notch regulates the expression of dpn in all these cells. However, it was found that in clones of a null allele of Su(H), dpn expression is not altered in NB and is only eliminated in the INPs. This clonal phenotype suggests that Notch signaling might function redundantly with other signals in NB. Thus, it is possible that in NBs multiples enhancers act redundantly to regulate the expression of dpn, and therefore its regulation depends on different signals. For instance, numb and brain tumor seem to function cooperatively to ensure the maturation of immature INP cells. Brat function appears independent of Notch signaling, suggesting that additional signals are required to promote the progression of recently born cells to INPs. Thus, it is possible that several signals function redundantly to ensure that NBs would be generated in appropriate numbers even in the absence of one or more genes (San-Juán, 2011).

According to this model, if dpn were the only target of Notch signaling during NBs proliferation, it would be expected that the loss of dpn would be sufficient to suppress the effects caused by the ectopic activation of the pathway. However, although it was found that in dpn mutant brains the total number of NBs is reduced and type II NBs are not found, clones of dpn mutant cells always contained a single neuroblast. In addition, the loss of dpn is not sufficient to suppress the effects caused by the ectopic activation of Notch. Although the reasons for these relatively mild clonal phenotypes are not fully understood, one possibility is that the system ensures its robustness by the existence of genetic redundancy. This redundancy may occur with other bHLH genes. Some members of the E(spl) complex were tested, and therefore a possible requirement of other members of this complex cannot be ruled out. This redundancy could ensure that neurons would be generated in appropriate numbers (San-Juán, 2011).

Notch and gut dorsovental axis determination

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

To investigate the role of the genes expressed in the large intestine, lack- and gain-of-function studies were performed. In amorphic Notch and Delta mutant embryos, kni/knrl, rho, and high levels of Crb expression on the apical plate are absent in the large intestine, and the boundary cell fate is not established. In contrast, ventral cell morphologies are normal in Notch or Delta mutant embryos, and En expression and dorsal cell fates are unchanged. This indicates that Notch signaling is required to establish the boundary cells but not for dorsal or ventral cell fates. To further test this, gain-of-function experiments were performed using the UAS/Gal4 system. As driver lines, the G445.2 or the 14-3-fkhGal4 strains were used -- they mediate ubiquitous gene expression in the developing hindgut from the extended germ band stage onward until late stage 16. In order to ectopically activate the Notch signaling pathway, flies carrying the Notch intracellular domain fragment, Nicd, under the control of UAS sequences were used. Expressing Nicd ubiquitously in the hindgut results in an ectopic induction of kni and of rho. In addition, the cellular localization of the Crb protein is affected in these embryos. In dorsal and ventral cells of the large intestine of wild-type embryos, Crb is localized to the apical cell margins, whereas it is localized to the entire apical plates of the boundary cells. In the embryos, in which Nicd is ectopically expressed, Crb protein is found on the apical plates of all the hindgut cells; in addition, it is found in high concentrations in vesicles, especially on the baso/lateral sides of the cells. A similar but less intensive ectopic expression of Crb can also be induced if both Kni and Rho are coexpressed in all the hindgut cells, suggesting that crb may be a downstream effector gene of Kni/Knrl and Rho activities. This is consistent with the analysis of rho7M; Df(3L) riXT1 mutants [Df(3L) riXT1 is a deficieny encompassing the kni and knrl transcription units] in which the expression of crb in the boundary cells is strongly reduced. In summary, these results suggest that rho, kni/knrl, and Crb are target genes which are activated in response to Notch signaling in the boundary cells (Fusse, 2002).

In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).

These results suggest that the activation of the Notch receptor in the boundary cells of the hindgut is triggered by the binding of Delta, which is expressed at high levels in adjacent ventral cells. If Delta levels are uniform and this boundary condition is lost, as in enE mutants, Notch signaling fails to occur. To further obtain insight into how the spatial control of Notch receptor activation is mediated, the localization of the receptor was determined using antibody stainings to Notch. In ventral and dorsal cells, Notch is expressed in the apical cell margins, as can be demonstrated using coimmunostainings with Neurexin IV. However, in the boundary cells, the Notch receptor is positioned to the entire apical plate where it is colocalized with Crb or Discs-lost. To test whether the apical localization of the receptor is necessary for its signaling activity, amorphic crb mutants were studied in which the sorting of proteins to the apical domain of the cells is affected. In these mutants, a strong reduction of the number of boundary cells was found, although hindgut morphogenesis is only slightly affected. In addition, the remaining boundary cells are mislocalized, and two rows of cells are often found instead of a single row as is found in wild-type. Anti-Notch/anti-Kni double immunostainings of crb mutants demonstrate the reduction of apical Notch receptor localization in crb mutants. Furthermore, in cells in which the Notch receptor is not localized along the apical plate of the cells, the activation of Notch target genes fails to occur. These results indicate that apical localization of the receptor may be important for boundary cell fate determination (Fusse, 2002).

These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).

These results provide evidence that Notch signaling in the Drosophila hindgut controls the fate of a single row of boundary cells separating the dorsal and ventral halves of the gut tube. Activation of the Notch receptor in the boundary cells is mediated by its ligand Delta that is expressed in adjacent ventral cells. The induction of Notch target genes activate the expression of the apical polarity organizer Crb, which may be required, in turn, for apical Notch receptor localization. These findings identify a simple mechanism that controls the specification of a single row of DV boundary cells in an animal gut (Fusse, 2002).

Notch function in myogenesis

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined. Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).

This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2 divides asymmetrically to give rise to two founder cells, one specific for a pair of persistently Eve-positive heart-associated or pericardial cells (EPCs) in every hemisegment and a second of previously undetermined identity. This second founder coexpresses Eve along with the gap gene Runt, with Eve levels rapidly fading but Runt persisting as development proceeds. By the time that Eve is evident in the EPCs, Runt labels a single somatic muscle, dorsal oblique muscle 2 (DO2). Runt is also detected in the muscle DO2 precursor during germband retraction (Carmena, 2002).

The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably expressed marker for it is unavailable (Carmena, 2002).

To further substantiate the lineage relationships among these progenitors and founders, observations related to RTK signaling dependence of P2 and P15 specification were used: whereas P15 requires the activities of both Egfr and Htl, only Htl is involved in P2 formation. In this way, targeted mesodermal expression of a dominant negative form of Egfr strongly blocks formation of DA1 but not the EPCs. Also, consistent with DO2 and EPC founders being the progeny of P2, DO2 development, like that of the EPCs, is not affected by dominant negative Egfr. Additional support for the sibling relationship between the DO2 and EPC founders derived from the analysis of targeted expression of a dominant negative form of Htl. Under conditions in which early mesoderm migration is not perturbed, dominant negative Htl generates an incompletely penetrant phenotype in which different hemisegments lose derivatives of P2, P15, or both progenitors. With such partial inhibition of Htl activity, muscle DO2 and the EPCs are consistently either both present or both absent from any given hemisegment; in no cases did one of these cell types develop without the other, as expected for cells derived from a common progenitor. In contrast, muscle DA1 frequently forms in the absence of muscle DO2 and the EPCs, consistent with its derivation from an independent progenitor. Taken together, these data establish that the EPC and DO2 founders are sibling cells of the P2 division, whereas the other Eve-expressing muscle founder arises from a different progenitor (Carmena, 2002).

This model differs from one derived on the basis of clonal analysis in which it was proposed that the two Eve-positive mesodermal cell types originate from the same progenitor. This discrepancy may relate to the fact that muscles form by sequential cell fusions involving both founders and fusion-competent cells of potentially different parental cell origins, thereby confounding the interpretation of clonal analysis in which the cytoplasm of a single myotube is labeled by the lineage tracing marker (Carmena, 2002).

Autoregulation of a signal transduction cascade can cause either enhancement or attenuation of the transduced signal, depending on whether the feedback loop acts positively or negatively. Both types of feedback control occur during the Ras- and N-mediated specification of Eve mesodermal progenitors. Ras activation leads to increased expression of several proximal components of both the Fgfr and Egfr pathways that serve to amplify and/or prolong both fate-inducing RTK/Ras signals in the emerging Eve progenitors. A similar amplification of Egfr signaling occurs via induction of Rho during Drosophila oogenesis and mesothoracic bristle formation, and via upregulation of Egfr expression during C. elegans vulva development. The present analysis also uncovers a positive feedback mechanism for inductive Fgfr signaling, in this case via increased expression of not only the Htl receptor but also its specific signal transducer, Heartbroken (Hbr). Interestingly, the data suggest that the downstream components may respond to different thresholds of Ras activity since Rho exhibits a less robust response than either Htl or Hbr to Ras activation (Carmena, 2002).

A negative feedback loop occurs in the Egfr pathway through autoactivation of the inhibitory ligand, Aos. Aos cooperates with Dl to block the progenitor-inducing Ras signal in both adjacent and more remote cells of the cluster. Aos could also exert a late inhibitory effect on the progenitor by terminating the inductive Egfr signal since Spi levels decrease following the establishment of cellular identity. Consistent with this possibility, MAPK activation fades from the singled out progenitor prior to its asymmetric division, suggesting that prolonged RTK signaling does not occur (Carmena, 2002).

Positive and negative feedback also occur during N function in the mesoderm. N activation both downregulates its ligand Dl and upregulates its own expression, thereby enhancing the potential for inhibitory signaling in cells not destined for the progenitor fate. Together, these opposing changes in Dl and N expression produce a unidirectional inhibitory signal emanating from the prospective progeni\tor and directed toward the adjacent nonprogenitor cells. Similar feedback mechanisms regulate the N pathway in the Drosophila embryonic CNS, adult PNS and wing vein-forming cells, and also apply to the N receptor-ligand combinations controlling gonadal and vulval cell fates in C. elegans (Carmena, 2002 and references therein).

Competitive cross-talk between Ras and N is manifest by the ability of the latter to block the expression of proximal components of the two RTK pathways—namely Htl/Hbr and Rho -- as well as to prevent the associated activation of MAPK. An antagonistic relationship between the RTK and N pathways is also revealed by the strong genetic interaction between Dl and Egfr, in agreement with previously reported genetic studies. Collectively, these results establish that the RTK and N pathways are not simply acting in parallel to exert opposing influences on progenitor specification; rather, N must be interfering with the generation and/or transmission of the inductive RTK signal. This effect could occur at multiple levels. The ability of activated N to at least partially block MAPK activation induced by constitutive Ras argues that N functions downstream of Ras. An additional direct effect of N on expression of Ras-responsive target genes cannot be excluded, particularly since Enhancer of split repressors are involved in the specification of progenitor cell fates. Such targets could include eve itself, or, given positive autoregulation of RTK signaling, one or more RTK pathway components (Carmena, 2002).

During C. elegans vulva development, Lin-12/N inhibits Egfr activity by stimulating the expression of a MAPK phosphatase. This is an attractive explanation for the effect of N observed here. However,while stimulation of a MAPK phosphatase could contribute in part to N inhibition of Ras signaling in the Drosophila embryonic mesoderm, it cannot be the only explanation since a constitutively activated form of Pointed is completely unable to reverse the activity of constitutive N. This is in marked contrast to the substantial reversal of N exhibited by activated Ras and occurs even though Pnt is a major Ets domain activator involved in RTK-dependent eve regulation (Carmena, 2002).

To account for the differential abilities of constitutive Ras and Pnt to compete effectively with constitutive N, the idea is favored that an additional, as yet uncharacterized, Pnt-independent function of Ras may be a target of N inhibition. Hence, there exist at least four potential sites of competitive interaction between these pathways: (1) direct regulation of target gene enhancers by pathway-specific transcriptional activators and repressors; (2) regulation of MAPK phosphorylation; (3) inhibition by N of RTK pathway component expression; and (4) an additional level of Pnt-independent cross-talk between Ras and N. These mechanisms modulate the relative flux through the competing Ras- and N-dependent processes and determine which pathway predominates, thereby achieving a critical threshold for a given cell fate. The importance of relative activity thresholds is underscored by results with different combinations of activated Ras and N insertions in which different numbers of Eve-expressing cells were induced, presumably reflecting slight fluctuations in the relative strengths of each pathway in individual cells. Similar levels of control may underlie the antagonistic effects of Ras and N in other developmental contexts (Carmena, 2002 and references therein).

Although the net effect of Ras and N signaling in the present system is the result of their antagonistic relationship, several forms of cooperative cross-talk also occur. For example, Ras activation induces the expression of Dl. Since the Ras signal is amplified by a positive feedback loop, this has the effect of biasing Dl expression to the emerging progenitor, thereby generating a polarized, nonautonomous inhibitory signal that acts on adjacent cells of the cluster. Aos is also a target of Ras activation, and Aos acts synergistically with the neurogenic pathway to block inductive RTK signaling. Thus, through its effects on the two inhibitory ligands, Dl and Aos, Ras cooperates with N to ensure that only one cell segregates as a progenitor from each equivalence group (Carmena, 2002).

Further cooperation is evident in the N-mediated down-regulation of Dl and Aos in prospective nonprogenitors, a combination of negative feedback and cross-talk that effectively prevents neighboring cells from sending an inhibitory signal to the emerging progenitor. Yan is yet another Ras-dependent component that reinforces the effect of N: when MAPK is suppressed in cells in which N is active, Yan is a functional repressor that blocks progenitor fate. Other examples of cooperation between Ras and N signaling include mammalian cell tumorigenesis and photoreceptor specification in the Drosophila eye (Carmena, 2002).

One seemingly paradoxical signaling interaction is N expression upregulation by Ras. Since Ras output is amplified in the progenitor, N protein might be expected to decrease in this cell, thereby restricting lateral inhibition to the appropriate direction. However, increased N in the Eve progenitor does not actually affect the polarity of lateral inhibition because the activating ligand, Dl, is downregulated by N in the adjacent nonprogenitors. Of further relevance, Dl may inhibit N activity when the two proteins are expressed in the same cell. Moreover, upregulation of N normally occurs very late in progenitor specification, as opposed to Dl which increases in one cell of the cluster at an earlier stage. Lastly, increased N has independent biological significance in progenitors since N is required for an asymmetric division that immediately follows the specification of these cells. In this respect, the response of the N receptor to Ras activation is an efficient, anticipatory 'feed forward' mechanism for insuring that this cell division is appropriately regulated (Carmena, 2002).

A model is presented that summarizes the progressive changes in Ras and N signaling and the cellular events corresponding to each stage. The model emphasizes that, while clusters of equivalent cells begin with the same signaling repertoires, they acquire distinct biochemical states which uniquely determine progenitor and nonprogenitor identities. Most important, this complex circuitry drives the reciprocal alterations in Ras and N activities toward the requisite thresholds that are essential for determining these fates. What biases one cell in an equivalence group toward the imbalance in Ras and N signaling that initiates the entire mechanism remains an open question. One possibility is that localized expression of the RTK ligands may provide the initiating event. Similarities of other developmental systems reinforce the general relevance of these conclusions (Carmena, 2002).

The reinforcing effects of Aos and Dl are essential since neither is fully capable of insuring that only one progenitor segregates from each Eve cluster. Furthermore, simultaneous loss of both inhibitory pathways leads to the formation of additional Eve cells within the competence domain but outside of the normal Eve equivalence groups. This suggests that the combined actions of both inhibitors prevent the spreading of the inductive signal beyond the normal cluster boundaries, as might occur through positive feedback of Rho expression and the associated increase in secreted Spi production. Such a remote inhibitory mechanism is particularly relevant to Aos, which is hypothesized to act at a longer range than Spi. Of note, synergistic inhibition by Aos and the N pathway has not been observed in other systems (Carmena, 2002).

These results also revealed an effect of Aos on Htl- but not Egfr-dependent C2 cluster development. Although this could be interpreted as indicating a role for Aos in the inhibition of the Htl Fgfr, the idea that Aos is actually blocking basal and/or spontaneous levels of Egfr activation in C2 cells is favored. This interpretation is supported by the finding that a dominant negative form of Egfr suppressed the effect of aos loss-of-function not only in Egfr-dependent C15, but also in C2, which does not require Egfr for its specification. In this cluster, the requisite Aos expression is dependent on Htl activity (Carmena, 2002).

Another advantage of combining Aos and Dl relates to their differing properties. Aos is a secreted inhibitor capable of acting over several cell diameters, whereas Dl -- although subject to proteolytic processing -- is generally considered a membrane-bound ligand requiring cell contact for its activity. If a progenitor emerges from the center of a cluster such that it is in close proximity to all of its initially equivalent neighbors, then Dl alone might be sufficient for the segregation of only one progenitor. However, if a progenitor forms on the periphery of a cluster, then the addition of Aos would compensate for the inability of Dl to inhibit its more distant neighbors. Thus, two distinct modes of lateral inhibition have complementary and reinforcing functions (Carmena, 2002).

The involvement of RTK/Ras and N pathways in the specification of Eve muscle and heart progenitors exemplifies the complex regulatory interactions that can occur between two antagonistic signaling pathways acting in concert. These findings demonstrate RTK/Ras and N signals do not function independently, converging only at the most distal step leading to a particular biological response. Rather, their effects are intertwined at multiple levels to form an integrated network of cross-talk nodes and feedback loops. The combination of cell autonomous and nonautonomous components of both pathways affords the high degree of regulatory versatility and specificity required to generate the polarized signaling activities that distinguish progenitors from their nonprogenitor neighbors. These interactions are especially remarkable since, once initiated, they propagate into self-sustaining cascades that differentially drive equipotent cells to their individual fates. Of further significance, the mesodermal cells produced by these mechanisms give rise to the differentiated derivatives that compose the stereotyped structures of the embryonic heart and body wall muscles. Thus, the signaling circuitry uncovered here not only establishes the finely tuned balance between the inductive and inhibitory influences which coordinately generate progenitor cell patterns, but also sets the stage for subsequent morphogenetic events (Carmena, 2002).

Specificity of Notch pathway activation: twist controls the transcriptional output in adult muscle progenitors

Cell-cell signalling mediated by Notch regulates many different developmental and physiological processes and is involved in a variety of human diseases. Activation of Notch impinges directly on gene expression through the Suppressor of Hairless [Su(H)] DNA-binding protein. A major question that remains to be elucidated is how the same Notch signalling pathway can result in different transcriptional responses depending on the cellular context and environment. This study investigated the factors required to confer this specific response in Drosophila adult myogenic progenitor-related cells. This analysis identifies Twist (Twi) as a crucial co-operating factor. Enhancers from several direct Notch targets require a combination of Twi and Notch activities for expression in vivo; neither alone is sufficient. Twi is bound at target enhancers prior to Notch activation and enhances Su(H) binding to these regulatory regions. To determine the breadth of the combinatorial regulation Twi occupancy was mapped on a genome-wide level in DmD8 myogenic progenitor-related cells by chromatin immunoprecipitation. Comparing the sites bound by Su(H) and by Twi in these cells revealed a strong association, identifying a large spectrum of co-regulated genes. It is concluded that Twi is an essential Notch co-regulator in myogenic progenitor cells and has the potential to confer specificity on Notch signalling at over 170 genes, showing that a single factor can have a profound effect on the output of the pathway (Bernard, 2010).

To determine whether the Twi co-regulation could be extrapolated to a broad spectrum of Notch targets in muscle progenitors, whether there was a significant association between Su(H) and Twi binding in the muscle progenitor-related DmD8 cells was assessed. Comparison of the binding regions genome-wide revealed a strong association of Twi and Su(H) among these targets: 71% of Su(H) peaks directly overlapped with Twi peaks. This association was highly significant based on random models that constrained the positions of peaks across the genome to take account of the non-random distribution of transcription factor binding sites and in comparison to several other ChIP data sets. Expression of putative Notch-Twi targets in myogenic precursors was dependent on Twi, as predicted by the association. Together, these data indicate that one transcription factor, Twi, has the potential to co-ordinate the expression of a broad cross-section of Notch targets in muscle progenitors (84% of previously assigned Notch targets are associated with a Twi peak) and thus to confer a specific context on the Notch response (Bernard, 2010).

Further evidence in support of the instructive role of Twi comes from its ability to confer Notch responsiveness on some muscle precursor targets when expressed in a heterologous cell line. Twi itself was found to occupy sites on the target enhancers prior to Notch activation, and in the heterologous cells it was accompanied by increased Su(H) binding after Notch activation. This suggests that Twi binding precedes Su(H) recruitment. However, the co-regulation does not appear to require the Twi partner Da [the E47 (TCF3) homologue], which was previously reported to contact Notch. Furthermore, there does not appear to be any specific organisation or spacing of Twi and Su(H) motifs among the co-regulated targets, in contrast to the conserved motif, consisting of paired Su(H) sites closely linked to an A-class bHLH binding site, that is thought to underlie Notch-proneural bHLH synergy. Nevertheless, in most of the co-regulated targets, the Su(H) and Twi mid-peaks are separated by less than 500 bp, suggesting that the interaction operates over a limited range. The Twi-Su(H) co-regulation appears, therefore, more in keeping with models in which binding sites for transcription factors are flexibly disposed and act independently with targets in the basal transcriptional machinery (the so-called 'billboard' model). However, mutation of a single Twi binding motif in the aos enhancer is sufficient to compromise activity, despite the fact that there are several matches to the Twi consensus site, suggesting that only a subset of the possible binding motifs are crucial. In addition, as the characterised Notch-Twi-dependent enhancers do not have identical patterns of expression, it is likely that their activity is further constrained by others factors. This is most evident for E(spl)m6, which is only expressed in a small patch of the AMPs but nevertheless responds very robustly to Twi and NICD. What are the likely characteristics conferred on cells by the Twi-Notch combination (Bernard, 2010)?

The AMPs have the capacity for self-renewal and are not committed to a particular muscle lineage, characteristics similar to those of mammalian muscle satellite cells. The genes regulated by the combination of Twi and Notch might therefore be important for maintaining these cells as progenitors with myogenic potential. Normally, Twi expression declines as the muscles differentiate. Interfering with this regulation by persistent expression of Twi or Notch inhibits the development of mature fibres. Conversely, ablating Notch results in premature differentiation. The genes regulated by the combination of Twi and Notch include those with proven roles in myogenic regulation that are relevant to the maintenance of muscle progenitors. These include twi itself, Him [an inhibitor of Mef2 (Liotta, 2007)] and zfh1 [a repressor of myogenesis. As Notch signalling and Twi homologues also inhibit vertebrate myogenic differentiation, and overexpression of Twi in terminally differentiated myotubes can induce reversal of cell differentiation, it will be interesting to test whether homologues of the identified co-regulated targets of Notch and Twi are similarly regulated (Bernard, 2010).

The Notch-Twi combination might also be required to confer properties on the adult myogenic precursors, such as differential adhesion, migration and proliferation. Besides the genes with proven roles in myogenic regulation, many of the Notch-Twi co-regulated genes are implicated in morphogenesis. These include the Ig-domain proteins Roughest, Kirre and Dscam, the netrin receptor Unc-5 and leucine repeat protein Capricious. Notch and Twi have both been found to contribute to the regulation of the epithelial-mesenchymal transition (EMT), and Twi is proposed to affect malignant progression by inducing EMT and suppressing the senescence response. Therefore, it is possible that the co-regulated genes might also confer specialised behaviours that are required in the adult precursors and in cells undergoing EMT (Bernard, 2010).

In conclusion, this study found that Notch and Twi potentially co-regulate a broad spectrum of genes required for the maintenance of muscle progenitors. This suggests that a single co-regulatory relationship can account for a significant component (>170 genes) of the Notch output in one cell type (Bernard, 2010).

Continued: Notch Targets of Activity part 2/3 | part 3/3

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

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