Biomedical subjects
Publications and source records attributed to S Bray.
Suppressor of Hairless (Su(H)) is a DNA-binding protein component of the Notch signalling pathway, thought to be required, with a fragment of the Notch receptor, for target gene activation. Recent studies show that this is only one side of the story: target gene enhancers may be regulated by Su(H) in a variety of different ways.
Cell-cell signaling mediated by Notch is critical during many different developmental processes for the specification or restriction of cell fates. Currently, the only known transduction pathway involves a DNA binding protein, Suppressor of Hairless [Su(H)] in Drosophila and CBF1 in mammals, and results in the direct activation of target genes. It has been proposed that in the absence of Notch, Su(H)/CBF1 acts as a repressor and is converted into an activator through interactions with the Notch intracellular domain [1--4]. Recently, we have also suggested that the activation of specific target genes requires synergy between Su(H) and other transcriptional activators [5]. Here we have designed an assay that allows us to directly test these hypotheses in vivo. Our results clearly demonstrate that Su(H) is able to function as the core of a molecular switch, repressing transcription in the absence of Notch and activating in the presence of Notch. In its capacity as an activator, Su(H) can cooperate synergistically with a DNA-bound transcription factor, Grainyhead. These interactions indicate a simple model for Notch target-gene regulation that could explain the precision of gene activation elicited by Notch signaling in different developmental fate decisions.
Suppressor of Hairless [Su(H)] is a DNA-binding protein that is the main intracellular transducer of the Notch signaling pathway in Drosophila. Several different mechanisms have been proposed to account for the activation of Su(H) by Notch. To further investigate how Su(H) activity is regulated we have used misexpression assays with wild-type Su(H) and with modified forms of Su(H) that contained a nuclear localization signal [Su(H)NLS], a transcriptional activation domain [Su(H)VP16], or a deletion of the domain required for interaction with the antagonist Hairless [Su(H)DeltaH]. Only Su(H)VP16 was able to mimic Notch activation effectively in the Drosophila wing, in agreement with the model that Notch activity normally confers coactivator function on Su(H). Neither nuclear localization nor elimination of Hairless binding was sufficient for activation. The phenotypes produced by overexpression of Su(H)wt and Su(H)NLS indicated a mixture of both increased and reduced Notch pathway activity and point to a role for Su(H) in both activation and repression of gene expression, as has been proposed for the mammalian homologue CBF1. Some phenotypes were equivalent to Notch loss-of-function, with wing-nicks and inhibition of a subset of target genes, which is most consistent with the ectopic proteins displacing a Su(H)-coactivator complex. Conversely, other phenotypes were equivalent to Notch gain-of-function, with wing-overgrowths and ectopic target-gene expression. These effects can be explained by the ectopic Su(H)/Su(H)NLS titrating a repressor complex. The wing-overgrowth phenotype is sensitive to the dose of Hairless and the phenotypes produced by coexpressing Su(H) and Hairless suggest that Hairless could form a component of this repressive complex.
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Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.
Wingless is a key morphogen in Drosophila. Although it is evident that Wingless acts at a distance from its site of synthesis, there is considerable debate about how the protein travels across a field of cells. Recent studies have provided important new insights into this process, though the issue is still far from being resolved.
Epithelial structures, such as the wing hairs and ommatidia in Drosophila, are aligned in the plane of the epithelium. This planar polarity requires the transmembrane receptor Frizzled. Recent studies have shed new light on mechanisms that could be involved in generating or transducing the polarity signal.
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Time-related changes in skin thickness have been evaluated in the pig using a noninvasive ultrasound technique after exposure to a range of single doses of 0.97 MeV beta particles from (170)Tm plaques. The reduction in relative skin thickness developed in two phases; the separation into two phases was statistically justified only after 120 Gy (P = 0.04). The first phase was between 12 weeks and 24 weeks after irradiation. No further changes were seen until 48-60 weeks after irradiation, when a second phase of skin thinning was observed. No further changes in relative skin thickness were seen in the follow-up period of 104 weeks. The timing of these phases of relative skin thinning was totally independent of the radiation dose; however, the severity of each phase of radiation-induced skin thinning was related to the dose. The pattern of changes was similar to that reported previously after irradiation with 2.27 MeV beta particles from (90)Sr/(90)Y, but the degree of dermal thinning was less for a similar skin surface dose. From a comparison of the depth-dose distribution of the beta particles from the two radionuclides, it was concluded that the target cell population responsible for both the first and second phase of skin thinning in pig skin after irradiation may be located at approximately 800 microm depth. This corresponds to an area in the reticular dermis in pig skin and may be the appropriate site at which to measure the average dose to the dermal tissue.
The proteins Scalloped and Vestigial are known from genetic studies to play a part in Drosophila wing development. Recent results show how they interact with each other, and in combination with other transcription factors, to confer specific patterns of expression within the wing.
E(spl) bHLH genes are targets of the Notch pathway: they are transcriptionally activated in response to the Notch signal. Yet, during imaginal development, additional regulatory factors appear to modulate transcription resulting in different expression patterns. During early embryogenesis all E(spl) bHLH genes are expressed in roughly the same domain, namely the neurogenic ectoderm. Within this region these seven genes show a highly dynamic, yet distinct transcriptional activity. Our analysis further detected tissue specific expression of some E(spl) genes at later embryonic stages. Prominent differences were observed in the dorsolateral and procephalic neuroectodermal regions as well as in the mesoderm. These observations indicate that other factors in addition to the Notch signal participate in the regulation of the individual E(spl) genes not only in imaginal tissues but also during neuroblast specification and other cell fate determination events in the embryo.
Cell-cell interactions mediated by Notch are critical at multiple stages of development. Our current understanding of the Notch signalling pathway suggests a comparatively simple transduction mechanism. However, this core pathway can be deployed in three different types of developmental process: lateral inhibition, lineage decisions and boundary formation. These illustrate how the activity of the pathway can be modulated both at the cell surface, through availability and effectiveness of ligand interactions, and inside the cell, through effects on the transduction pathway and the responsiveness of target genes.
The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the early Drosophila embryo is identical in thoracic and abdominal segments. However, during late embryogenesis differences in the numbers of neuroblasts and in the extent of neuroblast proliferation arise between these regions. We show that the homeotic genes Ultrabithorax and abdominal-A regulate these late differences, and that misexpression of either gene in thoracic neuroblasts after segregation is sufficient to induce abdominal behaviour. However, in wild type embryos we only detect abdominal-A and Ultrabithorax proteins in early neuroblasts. Furthermore, transplantation experiments reveal that segment-specific behaviour is determined prior to neuroblast segregation. Thus, the segment-specific differences in neuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts, and executed in later programmes controlling neuroblast numbers and proliferation.
The Notch signaling pathway is known to regulate cell fate decisions in a variety of organisms from worms to humans. Although several components of the pathway have been characterized, the actual mechanism and molecular results of signaling remain elusive. We have examined the role of the Notch signaling pathway in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes, whose gene products have been shown to be downstream players in the pathway. Using a reporter assay system in Drosophila tissue culture cells, we have observed a significant induction of E(spl) m gamma and m delta expression after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. We show that Notch-inducible expression of E(spl) m gamma and m delta both in cultured cells and in vivo is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter.
The veins in the Drosophila wing have a characteristic width, which is regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta is restricted to the developing veins, and coincides with places where Notch transcription is lower. We find that this asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins. In these cells, the expression of the Enhancer of split gene m beta is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. We propose that the establishment of vein thickness utilises a combination of mechanisms that include: (1) independent regulation of Notch and Delta expression in intervein and vein territories, (2) Notch activation by Delta in cells where Notch and Delta expression overlaps, (3) positive feedback on Notch transcription in cells where Notch has been activated and (4) repression of veinlet transcription by E(spl)m beta and maintenance of Delta expression by veinlet/torpedo activity.