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The roles of cis-inactivation by Notch ligands and of neuralized during eye and bristle patterning in Drosophila.

Li Y, Baker NE - BMC Dev. Biol. (2004)

Bottom Line: Overexpressed ligands could block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but did not activate Notch nonautonomously in neural cells.High ligand expression levels were required for cis-inactivation, and Serrate was more effective than Delta, although Delta is the ligand normally regulating neural specification.Neuralized was found to act cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10261, USA. yali@aecom.yu.edu

ABSTRACT

Background: The receptor protein Notch and its ligand Delta are expressed throughout proneural regions yet non-neural precursor cells are defined by Notch activity and neural precursor cells by Notch inactivity. Not even Delta overexpression activates Notch in neural precursor cells. It is possible that future neural cells are protected by cis-inactivation, in which ligands block activation of Notch within the same cell. The Delta-ubiquitin ligase Neuralized has been proposed to antagonize cis-inactivation, favoring Notch activation. Cis-inactivation and role of Neuralized have not yet been studied in tissues where neural precursor cells are resistant to nearby Delta, however, such as the R8 cells of the eye or the bristle precursor cells of the epidermis.

Results: Overexpressed ligands could block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but did not activate Notch nonautonomously in neural cells. High ligand expression levels were required for cis-inactivation, and Serrate was more effective than Delta, although Delta is the ligand normally regulating neural specification. Differences between Serrate and Delta depended on the extracellular domains of the respective proteins. Neuralized was found to act cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells.

Conclusions: Delta and Neuralized contribute cell nonautonomously to Notch signaling in neurogenesis, and the model that Neuralized antagonizes cis-inactivation to permit Notch activity and specification of non-neural cells is refuted. The molecular mechanism rendering Notch insensitive to paracrine activation in neural precursor cells remains uncertain.

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Mosaic analysis of ligand-induced neurogenesis A-C. Mosaic thoraces from y hsFLP; FRT42 109-68 [y+]/FRT42; UAS-SESI flies. Yellow bristles (arrows) derive from recombinant cells lacking 109-68-dependent Ser expression. A maximum of one yellow bristle is seen in each proneural region. D. Mosaic thorax derived from f36a hsFLP; [abx>f+>Gal4]; UAS-DESI fly. Forked bristles derive from recombinant cells expressing the Dl transgene. Normal (not forked) bristles lack transgenic Dl expression (arrows). A maximum of one normal bristle is seen in each proneural region. These mosaics show that Ser and Dl overexpression are cell autonomously neurogenic and do not recruit neighboring cells to neural fates. They further suggest that each proneural region contains at least one cell not sensitive to high levels of ligand expression nearby.
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Figure 3: Mosaic analysis of ligand-induced neurogenesis A-C. Mosaic thoraces from y hsFLP; FRT42 109-68 [y+]/FRT42; UAS-SESI flies. Yellow bristles (arrows) derive from recombinant cells lacking 109-68-dependent Ser expression. A maximum of one yellow bristle is seen in each proneural region. D. Mosaic thorax derived from f36a hsFLP; [abx>f+>Gal4]; UAS-DESI fly. Forked bristles derive from recombinant cells expressing the Dl transgene. Normal (not forked) bristles lack transgenic Dl expression (arrows). A maximum of one normal bristle is seen in each proneural region. These mosaics show that Ser and Dl overexpression are cell autonomously neurogenic and do not recruit neighboring cells to neural fates. They further suggest that each proneural region contains at least one cell not sensitive to high levels of ligand expression nearby.

Mentions: Genetic mosaics were used to investigate cell autonomy. FLP-mediated site-specific recombination was used to generate proneural regions mosaic for ectopic ligand expression. scaGal4 was not useful because many mosaic flies died in pharate adulthood when incomplete cuticle tanning prevented scoring the y phenotype being used to identify clones. Instead the Gal4 transgene 109-68 was used to target expression to thoracic proneural regions. 109-68>Ser expression led to extra scutellar bristle differentiation consistent with viability(Figure 3A,3B,3C). Mitotic recombination before 72 h gave clones of cells that never experienced ectopic Ser expression, since 109-68 drives transcription later than 96 h after egg laying, in the whole proneural region(not shown). Nonautonomy might be revealed by recruitment of cells not expressing Ser into the bristle patches. In nearly all cases where mosaic patches of bristles formed, only one of the bristles derived from a cell lacking 109-68(Figure 3A,3B,3C). Rather than nonautonomy, we interpret this to indicate that one cell in each region can undergo neural differentiation without the transgenic ligand (as would happen in normal development), but that ectopic Ser was required cell autonomously for supernumerary bristles. The data also suggest that selection of the favored neural cell is unaffected by large differences in Ser expression between cells.


The roles of cis-inactivation by Notch ligands and of neuralized during eye and bristle patterning in Drosophila.

Li Y, Baker NE - BMC Dev. Biol. (2004)

Mosaic analysis of ligand-induced neurogenesis A-C. Mosaic thoraces from y hsFLP; FRT42 109-68 [y+]/FRT42; UAS-SESI flies. Yellow bristles (arrows) derive from recombinant cells lacking 109-68-dependent Ser expression. A maximum of one yellow bristle is seen in each proneural region. D. Mosaic thorax derived from f36a hsFLP; [abx>f+>Gal4]; UAS-DESI fly. Forked bristles derive from recombinant cells expressing the Dl transgene. Normal (not forked) bristles lack transgenic Dl expression (arrows). A maximum of one normal bristle is seen in each proneural region. These mosaics show that Ser and Dl overexpression are cell autonomously neurogenic and do not recruit neighboring cells to neural fates. They further suggest that each proneural region contains at least one cell not sensitive to high levels of ligand expression nearby.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC420236&req=5

Figure 3: Mosaic analysis of ligand-induced neurogenesis A-C. Mosaic thoraces from y hsFLP; FRT42 109-68 [y+]/FRT42; UAS-SESI flies. Yellow bristles (arrows) derive from recombinant cells lacking 109-68-dependent Ser expression. A maximum of one yellow bristle is seen in each proneural region. D. Mosaic thorax derived from f36a hsFLP; [abx>f+>Gal4]; UAS-DESI fly. Forked bristles derive from recombinant cells expressing the Dl transgene. Normal (not forked) bristles lack transgenic Dl expression (arrows). A maximum of one normal bristle is seen in each proneural region. These mosaics show that Ser and Dl overexpression are cell autonomously neurogenic and do not recruit neighboring cells to neural fates. They further suggest that each proneural region contains at least one cell not sensitive to high levels of ligand expression nearby.
Mentions: Genetic mosaics were used to investigate cell autonomy. FLP-mediated site-specific recombination was used to generate proneural regions mosaic for ectopic ligand expression. scaGal4 was not useful because many mosaic flies died in pharate adulthood when incomplete cuticle tanning prevented scoring the y phenotype being used to identify clones. Instead the Gal4 transgene 109-68 was used to target expression to thoracic proneural regions. 109-68>Ser expression led to extra scutellar bristle differentiation consistent with viability(Figure 3A,3B,3C). Mitotic recombination before 72 h gave clones of cells that never experienced ectopic Ser expression, since 109-68 drives transcription later than 96 h after egg laying, in the whole proneural region(not shown). Nonautonomy might be revealed by recruitment of cells not expressing Ser into the bristle patches. In nearly all cases where mosaic patches of bristles formed, only one of the bristles derived from a cell lacking 109-68(Figure 3A,3B,3C). Rather than nonautonomy, we interpret this to indicate that one cell in each region can undergo neural differentiation without the transgenic ligand (as would happen in normal development), but that ectopic Ser was required cell autonomously for supernumerary bristles. The data also suggest that selection of the favored neural cell is unaffected by large differences in Ser expression between cells.

Bottom Line: Overexpressed ligands could block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but did not activate Notch nonautonomously in neural cells.High ligand expression levels were required for cis-inactivation, and Serrate was more effective than Delta, although Delta is the ligand normally regulating neural specification.Neuralized was found to act cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10261, USA. yali@aecom.yu.edu

ABSTRACT

Background: The receptor protein Notch and its ligand Delta are expressed throughout proneural regions yet non-neural precursor cells are defined by Notch activity and neural precursor cells by Notch inactivity. Not even Delta overexpression activates Notch in neural precursor cells. It is possible that future neural cells are protected by cis-inactivation, in which ligands block activation of Notch within the same cell. The Delta-ubiquitin ligase Neuralized has been proposed to antagonize cis-inactivation, favoring Notch activation. Cis-inactivation and role of Neuralized have not yet been studied in tissues where neural precursor cells are resistant to nearby Delta, however, such as the R8 cells of the eye or the bristle precursor cells of the epidermis.

Results: Overexpressed ligands could block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but did not activate Notch nonautonomously in neural cells. High ligand expression levels were required for cis-inactivation, and Serrate was more effective than Delta, although Delta is the ligand normally regulating neural specification. Differences between Serrate and Delta depended on the extracellular domains of the respective proteins. Neuralized was found to act cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells.

Conclusions: Delta and Neuralized contribute cell nonautonomously to Notch signaling in neurogenesis, and the model that Neuralized antagonizes cis-inactivation to permit Notch activity and specification of non-neural cells is refuted. The molecular mechanism rendering Notch insensitive to paracrine activation in neural precursor cells remains uncertain.

Show MeSH