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Cis-interactions between Notch and its ligands block ligand-independent Notch activity.

Palmer WH, Jia D, Deng WM - Elife (2014)

Bottom Line: Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling.Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling.We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, United States.

ABSTRACT
The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

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Endogenous DSL-independent Notch activity in crystal cells is reduced by cis-inhibition.Lz-GAL4-driven green fluorescent protein (GFP) expression is an efficient marker of crystal cells which show a low incidence of bursting (A and E). Misexpressing UAS-Serdel3 increased the frequency of witnessing bursting crystal cells (see arrowheads in B) (B and E). Lymph glands were counted for each genotype (n = 14 for lz > GFP, n = 12 for lz > GFP; UAS-NotchRNAi, and n = 14 for lz > GFP; UAS-Serdel3). Welch's t-test was used to assess significance between wild-type (WT) lymph glands and each of the experimental groups (p = 0.043 and p = 0.029, respectively). To determine whether Serdel3-misexpression induced ‘bursting’ was caused by the cis-inhibitory effect of Ser on ligand-independent Notch activation, we used the Notch activity reporter E(spl):mβ-CD2. We focused our analysis on larger crystal cells, which enter the endocycle as part of their differentiation (Terriente-Felix et al., 2013), and therefore are the ones most probably undergoing ligand-independent Notch activation. For illustrations, all GFP-positive cells were outlined and were filled in with differing shades of red corresponding to Notch reporter staining intensity. E(spl)mβ:CD2 is expressed in 55% of crystal cells and 77.4% of mature crystal cells (C and F). Misexpression of UAS-SerWT significantly (p < 0.0001 for mature crystal cells, p = 0.0457 if all crystal cells were taken into account) reduced the fraction of crystal cells which show E(spl)mβ:CD2 expression, with 34.4% of all cells showing expression and 20.7% of mature cells showing CD2 expression (D and F). For this analysis, the total number of lz > GFP cells were counted, taking into account their size, lz > GFP intensity, and E(spl)CD2 intensity. Mature crystal cells were defined as cells that were both large and had intense lz > GFP. We then took the proportion of either all cells, or mature cells which had E(spl)CD2 staining for each lymph gland (n = 12 for lz > GFP, n = 13 for lz > GFP;UAS-Ser). Grubbs' outlier test was used, which removed one data point (p < 0.05) from the control, which had an unusually small number of crystal cells. Then Welch's t-test was used to assess significance between the mean proportions of crystal cells which showed Notch activity. All error bars represent SD. These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSL-independent Notch activity. Scale bars represent 20 μm.DOI:http://dx.doi.org/10.7554/eLife.04415.014
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fig4s2: Endogenous DSL-independent Notch activity in crystal cells is reduced by cis-inhibition.Lz-GAL4-driven green fluorescent protein (GFP) expression is an efficient marker of crystal cells which show a low incidence of bursting (A and E). Misexpressing UAS-Serdel3 increased the frequency of witnessing bursting crystal cells (see arrowheads in B) (B and E). Lymph glands were counted for each genotype (n = 14 for lz > GFP, n = 12 for lz > GFP; UAS-NotchRNAi, and n = 14 for lz > GFP; UAS-Serdel3). Welch's t-test was used to assess significance between wild-type (WT) lymph glands and each of the experimental groups (p = 0.043 and p = 0.029, respectively). To determine whether Serdel3-misexpression induced ‘bursting’ was caused by the cis-inhibitory effect of Ser on ligand-independent Notch activation, we used the Notch activity reporter E(spl):mβ-CD2. We focused our analysis on larger crystal cells, which enter the endocycle as part of their differentiation (Terriente-Felix et al., 2013), and therefore are the ones most probably undergoing ligand-independent Notch activation. For illustrations, all GFP-positive cells were outlined and were filled in with differing shades of red corresponding to Notch reporter staining intensity. E(spl)mβ:CD2 is expressed in 55% of crystal cells and 77.4% of mature crystal cells (C and F). Misexpression of UAS-SerWT significantly (p < 0.0001 for mature crystal cells, p = 0.0457 if all crystal cells were taken into account) reduced the fraction of crystal cells which show E(spl)mβ:CD2 expression, with 34.4% of all cells showing expression and 20.7% of mature cells showing CD2 expression (D and F). For this analysis, the total number of lz > GFP cells were counted, taking into account their size, lz > GFP intensity, and E(spl)CD2 intensity. Mature crystal cells were defined as cells that were both large and had intense lz > GFP. We then took the proportion of either all cells, or mature cells which had E(spl)CD2 staining for each lymph gland (n = 12 for lz > GFP, n = 13 for lz > GFP;UAS-Ser). Grubbs' outlier test was used, which removed one data point (p < 0.05) from the control, which had an unusually small number of crystal cells. Then Welch's t-test was used to assess significance between the mean proportions of crystal cells which showed Notch activity. All error bars represent SD. These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSL-independent Notch activity. Scale bars represent 20 μm.DOI:http://dx.doi.org/10.7554/eLife.04415.014

Mentions: To explore whether cis-acting ligands might block endogenous raised levels of ligand-independent Notch activation, in addition to the raised levels induced by genetic defects, we examined the effect of increased ligand expression in crystal cells in the larval lymph gland, which have recently been shown to have ligand-independent Notch activation (Mukherjee et al., 2011). Notch activity in crystal cells promotes cell survival, and decreased Notch activity leads to a ‘bursting’ phenotype (Mukherjee et al., 2011) (Figure 4—figure supplement 2B,E). Evidence for this bursting phenotype is provided by the disorganization of membrane-associated GFP (Mukherjee et al., 2011). Using Lozenge (Lz)-GAL4, a crystal cell lineage-specific driver (Terriente-Felix et al., 2013) to misexpress UAS-NotchRNAi or UAS-Serdel3 led to a significantly higher proportion of cells showed the ‘bursting’ phenotype than wild-type crystal cells (NotchRNAi p = 0.0434, Serdel3 p = 0.0286) (Figure 4—figure supplement 2A,B,E). Furthermore, overexpression of UAS-SerWT led to a significant decrease of the Notch reporter E(spl):mβ-CD2expression in mature crystal cells (Figure 4—figure supplement 2C,D,F). Reduced Notch reporter activity was not caused by indirect effects on early ligand-dependent Notch signaling in prohaemocytes, as Hnt, a Notch target in differentiating crystal cells, (Terriente-Felix et al., 2013) was unaffected by ligand misexpression (Figure 4—figure supplement 3A,B). These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSL-independent Notch activity.


Cis-interactions between Notch and its ligands block ligand-independent Notch activity.

Palmer WH, Jia D, Deng WM - Elife (2014)

Endogenous DSL-independent Notch activity in crystal cells is reduced by cis-inhibition.Lz-GAL4-driven green fluorescent protein (GFP) expression is an efficient marker of crystal cells which show a low incidence of bursting (A and E). Misexpressing UAS-Serdel3 increased the frequency of witnessing bursting crystal cells (see arrowheads in B) (B and E). Lymph glands were counted for each genotype (n = 14 for lz > GFP, n = 12 for lz > GFP; UAS-NotchRNAi, and n = 14 for lz > GFP; UAS-Serdel3). Welch's t-test was used to assess significance between wild-type (WT) lymph glands and each of the experimental groups (p = 0.043 and p = 0.029, respectively). To determine whether Serdel3-misexpression induced ‘bursting’ was caused by the cis-inhibitory effect of Ser on ligand-independent Notch activation, we used the Notch activity reporter E(spl):mβ-CD2. We focused our analysis on larger crystal cells, which enter the endocycle as part of their differentiation (Terriente-Felix et al., 2013), and therefore are the ones most probably undergoing ligand-independent Notch activation. For illustrations, all GFP-positive cells were outlined and were filled in with differing shades of red corresponding to Notch reporter staining intensity. E(spl)mβ:CD2 is expressed in 55% of crystal cells and 77.4% of mature crystal cells (C and F). Misexpression of UAS-SerWT significantly (p < 0.0001 for mature crystal cells, p = 0.0457 if all crystal cells were taken into account) reduced the fraction of crystal cells which show E(spl)mβ:CD2 expression, with 34.4% of all cells showing expression and 20.7% of mature cells showing CD2 expression (D and F). For this analysis, the total number of lz > GFP cells were counted, taking into account their size, lz > GFP intensity, and E(spl)CD2 intensity. Mature crystal cells were defined as cells that were both large and had intense lz > GFP. We then took the proportion of either all cells, or mature cells which had E(spl)CD2 staining for each lymph gland (n = 12 for lz > GFP, n = 13 for lz > GFP;UAS-Ser). Grubbs' outlier test was used, which removed one data point (p < 0.05) from the control, which had an unusually small number of crystal cells. Then Welch's t-test was used to assess significance between the mean proportions of crystal cells which showed Notch activity. All error bars represent SD. These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSL-independent Notch activity. Scale bars represent 20 μm.DOI:http://dx.doi.org/10.7554/eLife.04415.014
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Related In: Results  -  Collection

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fig4s2: Endogenous DSL-independent Notch activity in crystal cells is reduced by cis-inhibition.Lz-GAL4-driven green fluorescent protein (GFP) expression is an efficient marker of crystal cells which show a low incidence of bursting (A and E). Misexpressing UAS-Serdel3 increased the frequency of witnessing bursting crystal cells (see arrowheads in B) (B and E). Lymph glands were counted for each genotype (n = 14 for lz > GFP, n = 12 for lz > GFP; UAS-NotchRNAi, and n = 14 for lz > GFP; UAS-Serdel3). Welch's t-test was used to assess significance between wild-type (WT) lymph glands and each of the experimental groups (p = 0.043 and p = 0.029, respectively). To determine whether Serdel3-misexpression induced ‘bursting’ was caused by the cis-inhibitory effect of Ser on ligand-independent Notch activation, we used the Notch activity reporter E(spl):mβ-CD2. We focused our analysis on larger crystal cells, which enter the endocycle as part of their differentiation (Terriente-Felix et al., 2013), and therefore are the ones most probably undergoing ligand-independent Notch activation. For illustrations, all GFP-positive cells were outlined and were filled in with differing shades of red corresponding to Notch reporter staining intensity. E(spl)mβ:CD2 is expressed in 55% of crystal cells and 77.4% of mature crystal cells (C and F). Misexpression of UAS-SerWT significantly (p < 0.0001 for mature crystal cells, p = 0.0457 if all crystal cells were taken into account) reduced the fraction of crystal cells which show E(spl)mβ:CD2 expression, with 34.4% of all cells showing expression and 20.7% of mature cells showing CD2 expression (D and F). For this analysis, the total number of lz > GFP cells were counted, taking into account their size, lz > GFP intensity, and E(spl)CD2 intensity. Mature crystal cells were defined as cells that were both large and had intense lz > GFP. We then took the proportion of either all cells, or mature cells which had E(spl)CD2 staining for each lymph gland (n = 12 for lz > GFP, n = 13 for lz > GFP;UAS-Ser). Grubbs' outlier test was used, which removed one data point (p < 0.05) from the control, which had an unusually small number of crystal cells. Then Welch's t-test was used to assess significance between the mean proportions of crystal cells which showed Notch activity. All error bars represent SD. These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSL-independent Notch activity. Scale bars represent 20 μm.DOI:http://dx.doi.org/10.7554/eLife.04415.014
Mentions: To explore whether cis-acting ligands might block endogenous raised levels of ligand-independent Notch activation, in addition to the raised levels induced by genetic defects, we examined the effect of increased ligand expression in crystal cells in the larval lymph gland, which have recently been shown to have ligand-independent Notch activation (Mukherjee et al., 2011). Notch activity in crystal cells promotes cell survival, and decreased Notch activity leads to a ‘bursting’ phenotype (Mukherjee et al., 2011) (Figure 4—figure supplement 2B,E). Evidence for this bursting phenotype is provided by the disorganization of membrane-associated GFP (Mukherjee et al., 2011). Using Lozenge (Lz)-GAL4, a crystal cell lineage-specific driver (Terriente-Felix et al., 2013) to misexpress UAS-NotchRNAi or UAS-Serdel3 led to a significantly higher proportion of cells showed the ‘bursting’ phenotype than wild-type crystal cells (NotchRNAi p = 0.0434, Serdel3 p = 0.0286) (Figure 4—figure supplement 2A,B,E). Furthermore, overexpression of UAS-SerWT led to a significant decrease of the Notch reporter E(spl):mβ-CD2expression in mature crystal cells (Figure 4—figure supplement 2C,D,F). Reduced Notch reporter activity was not caused by indirect effects on early ligand-dependent Notch signaling in prohaemocytes, as Hnt, a Notch target in differentiating crystal cells, (Terriente-Felix et al., 2013) was unaffected by ligand misexpression (Figure 4—figure supplement 3A,B). These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSL-independent Notch activity.

Bottom Line: Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling.Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling.We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, United States.

ABSTRACT
The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

Show MeSH
Related in: MedlinePlus