strawberry notch

Targets of Activity

Subjecting temperature sensitive strawberry notch to heat shock results in a down regulation of wg at the wing margin. Expression of wg in other regions of the wing disc as well as in other imaginal discs is unaffected by the loss of sno function. Likewise sno is required for the expression of vestigial, cut and E(spl)-m8 at the wing margin (Majumdar, 1997).

In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch target gene activation (Nagel, 2001).

These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by N^act. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by N^act/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of N^act are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).

A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of N^act as co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer (Nagel, 2001).

These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).

These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).

DEVELOPMENTAL BIOLOGY

Embryonic

The Sno protein is nuclear. Sno is not detected in mesodermal and endodermal tissues, therefore, the expression of Sno is not as wide as Notch, a gene that is found in all three germ layers in the embryo. Sno is first detected in nuclei at the syncytial blastoderm stage. This expression is evident before the onset of zygotic transcription. This expression represents the translation of maternally derived SNO mRNA. In the cellular blastoderm, all nuclei of the embryo, except for the pole cells, appear to contain Sno. At gastrulation, Sno is seen transiently in the mesodermal precursors but shuts off as soon as these cells invaginate inside the embryo. At stage 8, when the formation of the midline precursor cells depends on Notch signaling, higher levels of Sno are observed in the midline precursor cells than in the surrounding epidermal cells. Between stages 11 and 14, Sno is seen uniformly throughout the epidermis, and finally, at around stage 16, high level of expression is restricted to the central nervous system (Majumdar, 1997).

Larval

Sno is expressed in all of the larval imaginal discs. In the leg disc, Sno expression is ubiquitous, although the central tarsal region shows higher levels of expression as compared with those seen in surrounding cells. All cells in wing and eye discs also show high levels of Sno. During oogenesis, Sno protein is detected in follicle cell nuclei, but not in nurse cell or oocyte nuclei (Majumdar, 1997).

Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye

In the developing eye of Drosophila, the EGFR and Notch pathways integrate in a sequential, followed by a combinatorial, manner in the specification of cone-cell fate. This study demonstrates that the specification of primary pigment cells requires the reiterative use of the sequential integration between the EGFR and Notch pathways to regulate the spatiotemporal expression of Delta in pupal cone cells. The Notch signal from the cone cells then functions in the direct specification of primary pigment-cell fate. EGFR requirement in this process occurs indirectly through the regulation of Delta expression. Combined with previous work, these data show that unique combinations of only two pathways -- Notch and EGFR -- can specify at least five different cell types within the Drosophila eye (Nagaraj, 2007).

Unlike photoreceptor R cells, cone cells do not express Delta at the third instar stage of development. However, these same cone cells express Delta at the pupal stage. In addition, correlated with this Delta expression, the upregulation of phosphorylated MAPK was observed in these cells. This is very similar to the earlier events seen in R cells during larval development, in which the activation of MAPK causes the expression of Delta. Also, as in the larval R cells, the pupal upregulation of Delta in cone cells is transcriptional. A Delta-lacZ reporter construct, off in the larval cone cell, is detected in the corresponding pupal cone cells. To determine whether EGFR is required for the activation of Delta in the pupal cone cells, the temperature-sensitive allele EGFR^ts1 was used. Marked clones of this allele were generated in the eye disc using ey-flp at permissive conditions and later, in the mid-pupal stages, shifted the larvae to a non-permissive temperature. Cells mutant for EGFR, but not their adjacent wild-type cells, showed a loss of Delta expression. However, both mutant and wild-type tissues showed normal cone-cell development, as judged by Cut (a cone-cell marker) expression. As supporting evidence, ectopic expression of a dominant-negative version of EGFR (EGFR^DN) in cone cells using spa-Gal4 after the cells have already undergone initial fate specification also causes a complete loss of Delta expression without compromising the expression of the cone-cell-fate-specification marker (Nagaraj, 2007).

Gain-of-function studies further support the role of EGFR signaling in the regulation of Delta expression in cone cells. Although weak EGFR activation is required for cone-cell fate, activated MAPK is not detectable in cone-cell precursors of the third instar larval eye disc. When spa-Gal4 (prepared by cloning the 7.1 kb EcoRI genomic fragment of D-Pax2) is used to express an activated version of EGFR in larval cone cells, detectable levels of MAPK activation in these cells were found and the consequent ectopic activation of Delta in the larval cone cells occurred. Taken together, these gain- and loss-of-function studies show that, during pupal stages, EGFR is required for the activation of Delta. However, this Delta expression is not essential for the maintenance of cone-cell fate (Nagaraj, 2007).

In larval R cells, the activation of Delta transcription in response to EGFR signaling is mediated by two novel nuclear proteins, Ebi and Sno. To determine the role of these genes in wild-type pupal-cone-cell Delta expression, sno and ebi function were selectively blocked in the pupal eye disc. A heteroallellic combination of the temperature-sensitive allele sno^E1 and the null allele sno⁹³ⁱ exposed to a non-permissive temperature for 12 hours caused a significant reduction in Delta expression. Similarly, a dominant-negative version of ebi also caused the loss of Delta expression. Importantly, pupal eye discs of neither spa-Gal4, UAS-ebi^DN nor sno^E1/sno⁹³ⁱ showed any perturbation in cone-cell fate, as judged by the expression of Cut. Thus, as in the case of larval R cells, the loss of ebi and sno in the pupal cone cells causes the loss of Delta expression without causing a change in cone-cell fate (Nagaraj, 2007).

To test whether the expression of Delta in pupal cone cells is required for the specification of primary pigment cells, N^ts pupae were incubated at a non-permissive temperature for 10 hours during pupal development and pigment-cell differentiation was monitored using BarH1 (also known as Bar) expression as a marker. Loss of Notch signaling during the mid-pupal stages caused a loss of Bar, further demonstrating the requirement of Notch signaling in the specification of primary pigment-cell fate. Similarly, when the 54CGal4 driver line, which is activated in pigment cells, was used to drive the expression of a dominant-negative version of Notch, pupal eye discs lost primary pigment-cell differentiation, again suggesting an autonomous role for Notch in pigment-cell precursors. In neither the N^ts nor the 54C-Gal4, UAS-N^DN genetic background was perturbation observed in cone-cell fate specification. It is concluded that Delta activation mediated by EGFR-Sno-Ebi in pupal cone cells is essential for neighboring pigment-cell fate specification (Nagaraj, 2007).

Delta-protein expression in pupal cone cells is initiated at 12 hours and is downregulated by 24 hours of pupal development. To determine the functional significance of this downregulation, the genetic combination spa-Gal4/UAS-Delta was used, in which Delta is expressed in the same cells as in wild type, but is not temporally downregulated. Whereas, in wild type, a single hexagonal array of pigment cells surrounded the ommatidium, in the pupal eye disc of spa-Gal4, UAS-Delta flies, multiple rows of pigment cells were observed surrounding each cluster. Furthermore, in wild type, only two primary pigment cells were positive for Bar expression in each cluster, whereas, in spa-Gal4, UAS-Delta pupal eye discs, ectopic expression of Bar was evident in the interommatidial cells. Therefore, the temporal regulation of Notch signaling and its activation, as well as its precise downregulation, are essential for the proper specification of primary pigment-cell fate (Nagaraj, 2007).

By contrast to the autonomous requirement for Notch signaling in primary pigment cells, the function of the EGFR signal appears to be required only indirectly in the establishment of primary pigment-cell fate through the regulation of Delta expression in the pupal cone cells. When a dominant-negative version of EGFR was expressed using hsp70-Gal4 at 10-20 hours after pupation, no perturbation was observed in the specification of primary pigment cells, as monitored by the expression of the homeodomain protein Bar. By contrast, the expression of dominant-negative Notch under the same condition resulted in the loss of Bar-expressing cells. Thus, in contrast to Notch, blocking EGFR function at the time of primary pigment-cell specification does not block the differentiation of these cells. Importantly, blocking EGFR function in earlier pupal stages caused the loss of Delta expression in cone cells and the consequent loss of pigment cells. Based on these observations, it is concluded that, in the specification of primary pigment-cell fate, the Notch signal is required directly in primary pigment cells, whereas EGFR function is required only indirectly (through the regulation of Delta) in cone cells (Nagaraj, 2007).

The Runt-domain protein Lz functions in the fate specification of all cells in the developing eye disc arising from the second wave of morphogenesis. At a permissive temperature (25°C), lz^TS114 pupal eye discs showed normal differentiation of primary pigment cells. lz^TS114 is a sensitized background in which the Lz protein is functional at a threshold level. When combined with a single-copy loss of Delta, a dosage sensitive interaction caused the loss of primary pigment cells. By contrast, under identical conditions, a single-copy loss of EGFR function had no effect on the proper specification of primary pigment-cell fate. This once again supports the notion that the specification of primary pigment cells directly requires Lz and Notch, whereas EGFR is required only indirectly to activate Delta expression in cone cells (Nagaraj, 2007).

This study highlights two temporally distinct aspects of EGFR function in cone cells. First, this pathway is required for the specification of cone-cell fate at the larval stage, and EGFR is then required later in the pupal cone cell for the transcriptional activation of Delta, converting the cone cell into a Notch-signaling cell. Delta that was expressed in the cone cell through the activation of the Notch pathway functioned in combination with Lz in a cell autonomous fashion and promoted the specification of the primary pigment-cell fate (Nagaraj, 2007).

Studies using overexpressed secreted Spitz have shown that ectopic activation of the EGFR signal in all cells of the pupal eye disc results in excess primary pigment cells. This study shows that EGFR activation in the pupal eye disc is required for the transcriptional activation of Delta in cone cells, but that the loss of EGFR function at the time when primary pigment cells are specified does not perturb their differentiation. It is concluded that the ectopic primary pigment cells seen in an activated-EGFR background result from the ectopic activation of Delta, which then signals adjacent cells and promotes their differentiation into primary pigment cells. Indeed, it has been shown that excessive Delta activity results in the over specification of primary pigment cells. The results are also consistent with the previous observation that the EGFR target gene Argos is not expressed in primary pigment cells in pupal eye discs. Additionally, loss of EGFR function in pupal eye discs does not perturb the normal patterning of interommatidial bristle development, which develop even later than the primary pigment cells (Nagaraj, 2007).

The elucidation of the Sevenless pathway for the specification of R7 led to the suggestion that different cell types within the developing eye in Drosophila will require combinations of dedicated signaling pathways for their specification. However, studies from several laboratories have suggested that the Sevenless pathway seems to be an exception, in that cell-fate-specification events usually require reiterative combinations of a very small number of non-specific signals. Cone-cell fate is determined by the sequential integration of the EGFR and Notch pathways in R cells followed by the parallel integration of the EGFR and Notch pathways in cone-cell precursors. This study shows that the most important function of EGFR in the specification of primary pigment cells is to promote the transcriptional activation of Delta in cone cells through the EGFR-Ebi-Sno-dependent pathway. The sequential integration of the EGFR and Notch pathways, first used in the larval stage for Delta activation in R cells, is then reused a second time in cone cells to regulate the spatiotemporal expression of Delta, converting the cone cells at this late developmental stage to Notch-signaling cells. Delta present in the cone cell then signals the adjacent undifferentiated cells for the specification of primary pigment cells. For this process, the Notch pathway functions directly with Lz but indirectly with EGFR. Through extensive studies of this system it now seems conclusive that different spatial and temporal combinations of Notch and EGFR applied at different levels can generate all the signaling combinations needed to specify the neuronal (R1, R6, R7) and nonneuronal (cone, pigment) cells in the second wave of morphogenesis in the developing eye disc (Nagaraj, 2007).

The EGFR and Notch pathways are sequentially integrated, in a manner similar to that described here, in multiple locations during Drosophila development. In the development of wing veins, EGFR that is activated in the pro-vein cells causes the expression of Delta, which then promotes the specification of inter-vein cells. Similarly, these two pathways are sequentially integrated in the patterning of embryonic and larval PNS, and during muscle development. Indeed, there are striking similarities between the manner in which the EGFR and Notch pathways are integrated in the developmental program in the C. elegans vulva and the Drosophila eye. During vulval fate specification in the C. elegans hermaphrodite gonad, anchor cells are a source of EGFR signal (Lin3), which induces the specification of the nearest (P6) cell to the primary cell fate from within a group of six equipotent vulval precursor cells (VPC). This high level of EGFR activation induces the transcriptional activation of Notch ligands in the primary cells in what can be considered sequential integration of the two pathways - the Notch signal from the primary cell both inhibits EGFR activity in the VPCS on either side of P6.p and also promotes the secondary cell fate. Thus, the reiterative integration of these two signals, in series and in parallel, can be used successfully to specify multiple cell fates in different animal species. Given that the RTK and Notch pathways function together in many vertebrate developmental systems, it is likely that similar networks will be used to generate diverse cell fates using only a small repertoire of signaling pathways (Nagaraj, 2007).

Effects of mutation or deletion

Flies bearing a temperature sensitive allele of sno show a modest loss of wing margin tissue when raised at 23 degrees C. When such flies are also made heterozygous for a single copy loss of wingless, extensive loss of wing margin tissue is observed, suggesting a dominant synergistic interaction between wg and sno. In similar experiments, temperature sno combined with a single copy loss of vestigial also results in a dominant enhancement of wing margin defects; mutants exhibit extensive loss of wing margin tissue. Genetic combination of a weak allele of cut with a sno mutation shows extensive loss of wing margin tissue, suggesting a synergistic interaction between sno and cut. These results are reminiscent of the interaction of Notch with wg, vg and ct and further extablish that sno, like Notch, has a crucial role in the establishment of D/V boundary fate by participating in a common genetic pathway that regulates wing margin-specific genes. In addition to the wing margin defects, sno mutants also exhibit thickening of wing veins. This is likely to be a secondary consequence of defective wing pouch development caused by improper D/V boundary specification. This same phenotype can also be seen in some of the other D/V boundary genes, such as vestigial and Serrate (Majumdar, 1997).

A single copy of Hairless, is able to suppress the wing defects of heterozygous sno, suggesting that Hairless and sno exhibit related antagonistic activities downstream of the Notch pathway. In a similar fashion, a single copy of Suppressor of Hairless and a single copy of sno show enhanced defects, indicating that Su(H) and Sno cooperate closely in patterning the wing. As Su(H) and Sno have not been shown to physically interact, this may mean that the two proteins work in parallel or that the interaction is too weak to be detected (Majumdar, 1997).

Temperature sensitive sno mutants raised at nonpermissive temperatures show a severe phenotype, while those raised at an intermediate temperature (23 degrees) show an intermediate phenotype consisting of a notched blade and thick veins which end in large deltas. Legs of mutant flies grown at nonpermissive temperatures show fused second and third tarsal segments as well as a less severe fusion of fourth and fifth tarsal segments. Mutant flies have rough eyes. Eye bristles, normally present at alternative corners of each facet, are often misplaced and sometimes duplicated. At the 5-precluster stage, 20% of the ommatidia in sno mutants contain six R cells. The extra cell is positioned between R3 and R4 where a mystery cell would have been in an earlier column. This suggests that in the mutant, a mystery cell fails to leave the precluster, giving rise to a 6-cell precluster. In mutant females the number of ovarioles is reduced by 25% to an average of 30 per female (Coyle-Thompson, 1993).

sno synergistically affects Notch phenotypes in a tissue-specific manner. For example, split alleles of Notch affect the eye, while notchoid alleles affect the wing. In double mutant combinations with sno, the phenotypes in these respective tissues are enhanced. This suggests a role for sno in many independent Notch-related pathways (Coyle-Thompson, 1993)

Brou, C., et al. (1994). Inhibition of the DNA-binding activity of Drosophila Suppressor of Hairless and of its human homolog, KBF2/RBP-Jk, by direct protein-protein interaction with Drosophila Hairless. Genes Dev. 8: 2491-2503.

Coyle-Thompson, C. A. and Banerjee, U. (1993). The strawberry notch gene functions with Notch in common developmental pathways. development 119: 377-395.

Fitzgerald, K., Wilkinson, H. A. and Greenwald, I. (1993). glp-1 can substitute for lin-12 in specifying cell fate decisions in Caenorhibditis elegans. Development 119: 1019-1027.

Majumdar, A., Nagaraj, R. and Banerjee, U. (1997). strawberry notch encodes a conserved nuclear protein that functions downstream of Notch and regulates gene expression along the developing wing margin in Drosophila. Genes Dev. 11: 1341-1353.

Nagaraj, R. and Banerjee, U. (2007). Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye. Development 134(5): 825-31. Medline abstract: 17251265

Nagel, A. C., Wech, I. and Preiss, A. (2001). scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila. Mech. Dev. 109: 241-251. 11731237

Tsuda, L., et al. (2002). An EGFR/Ebi/Sno pathway promotes Delta expression by inactivating Su(H)/SMRTER repression during inductive Notch signaling. Cell 110: 625-637. 12230979

Watanabe, Y., Miyasaka, K. Y., Kubo, A., Kida, Y. S., Nakagawa, O., Hirate, Y., Sasaki, H. and Ogura, T. (2017). Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm. Sci Rep 7: 46135. PubMed ID: 28401892

date revised: 25 June 2007 

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