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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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A schematic depiction of the early stages of Drosophila oogenesis.Oogenesis begins in the germarium, where germline stem cells divide four times, producing a 16-cell germline cyst which is encapsulated by somatic follicle cells (FCs). When the FCs complete encapsulation and bud from the germarium, this is termed a stage 1 egg chamber. The egg chamber then grows and the FCs undergo mitosis until stage 6, and during these stages Cut is expressed and cells remain diploid. At stage 5, Dl is strongly upregulated in the germline. The transition from stage 6 to stage 7 is defined by activation of Notch, upregulation of Hnt, repression of Cut, and the endocycling of the FCs.DOI:http://dx.doi.org/10.7554/eLife.04415.004
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fig1s1: A schematic depiction of the early stages of Drosophila oogenesis.Oogenesis begins in the germarium, where germline stem cells divide four times, producing a 16-cell germline cyst which is encapsulated by somatic follicle cells (FCs). When the FCs complete encapsulation and bud from the germarium, this is termed a stage 1 egg chamber. The egg chamber then grows and the FCs undergo mitosis until stage 6, and during these stages Cut is expressed and cells remain diploid. At stage 5, Dl is strongly upregulated in the germline. The transition from stage 6 to stage 7 is defined by activation of Notch, upregulation of Hnt, repression of Cut, and the endocycling of the FCs.DOI:http://dx.doi.org/10.7554/eLife.04415.004

Mentions: The developing Drosophila egg chamber is a convenient model for dissecting the effects of Notch ligands in cis and in trans, as Dl is the sole signaling source and the signal sending and receiving cells can be easily distinguished (Deng et al., 2001; López-Schier and St Johnston, 2001). (Figure 1—figure supplement 1 provides a brief schematic depiction of the stages of early oogenesis.) At oogenesis stage 7, Notch signaling is activated in the somatic follicle cells by a robust germline Dl upregulation, which leads to the expression of Hindsight (Hnt), downregulation of Cut, and the polyploidization of the follicle cells (Deng et al., 2001; López-Schier and St Johnston, 2001; Sun and Deng, 2005, 2007) (Figure 1A). When Dl germline mutant clones were generated (i.e., trans-activation was removed), the follicle cells failed to downregulate Cut expression, which persisted past stage 7, indicative of a failure to activate Notch (Figure 1B). In contrast, Dl follicle cell mutant clones show precocious Cut downregulation at stage 6 attributable to the relief of cis-inhibition (Poulton et al., 2011) (Figure 1C). Surprisingly, Dl mutant clones in the follicle cells bordering Dl mutant clones in the germline (i.e., a germline with no signaling source, herein referred to as Dl-/Dl- cells) show correct Hnt and Cut expression from stage 7 (Figure 1D,E, Figure 1—figure supplement 2A,B). These Dl-/Dl- clones also correctly transit into the endocycle, as their nuclear volumes are similar to wild-type follicle cells in the later stages of oogenesis after polyploidization (Figure 1F,G), whereas cells neighboring Dl-/Dl- follicle cell clones (retaining a cis-ligand but without a trans-ligand) are comparable to wild-type cells before entry to endocycle (Figure 1F,G). Removal of both cis- and trans-Dl through knockdown of Dl by RNA interference (RNAi) simultaneously in the germline and soma confirmed this finding (Figure 2—figure supplement 1A,B). Together, these observations provide evidence that follicle cells without both cis- and trans-ligand sources can still enter the endocycle stages of oogenesis. This back-up route to the endocycle is not a co-option of Ser in place of Dl, as DlRevF10SerRx82 double clones recapitulated the Dl-/Dl- phenotype (Figure 1E, Figure 1—figure supplement 2A).10.7554/eLife.04415.003Figure 1.Follicle cells without DSL ligand bordering germline cells without DSL ligand show proper Notch activation and downstream differentiation.


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

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

A schematic depiction of the early stages of Drosophila oogenesis.Oogenesis begins in the germarium, where germline stem cells divide four times, producing a 16-cell germline cyst which is encapsulated by somatic follicle cells (FCs). When the FCs complete encapsulation and bud from the germarium, this is termed a stage 1 egg chamber. The egg chamber then grows and the FCs undergo mitosis until stage 6, and during these stages Cut is expressed and cells remain diploid. At stage 5, Dl is strongly upregulated in the germline. The transition from stage 6 to stage 7 is defined by activation of Notch, upregulation of Hnt, repression of Cut, and the endocycling of the FCs.DOI:http://dx.doi.org/10.7554/eLife.04415.004
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4286723&req=5

fig1s1: A schematic depiction of the early stages of Drosophila oogenesis.Oogenesis begins in the germarium, where germline stem cells divide four times, producing a 16-cell germline cyst which is encapsulated by somatic follicle cells (FCs). When the FCs complete encapsulation and bud from the germarium, this is termed a stage 1 egg chamber. The egg chamber then grows and the FCs undergo mitosis until stage 6, and during these stages Cut is expressed and cells remain diploid. At stage 5, Dl is strongly upregulated in the germline. The transition from stage 6 to stage 7 is defined by activation of Notch, upregulation of Hnt, repression of Cut, and the endocycling of the FCs.DOI:http://dx.doi.org/10.7554/eLife.04415.004
Mentions: The developing Drosophila egg chamber is a convenient model for dissecting the effects of Notch ligands in cis and in trans, as Dl is the sole signaling source and the signal sending and receiving cells can be easily distinguished (Deng et al., 2001; López-Schier and St Johnston, 2001). (Figure 1—figure supplement 1 provides a brief schematic depiction of the stages of early oogenesis.) At oogenesis stage 7, Notch signaling is activated in the somatic follicle cells by a robust germline Dl upregulation, which leads to the expression of Hindsight (Hnt), downregulation of Cut, and the polyploidization of the follicle cells (Deng et al., 2001; López-Schier and St Johnston, 2001; Sun and Deng, 2005, 2007) (Figure 1A). When Dl germline mutant clones were generated (i.e., trans-activation was removed), the follicle cells failed to downregulate Cut expression, which persisted past stage 7, indicative of a failure to activate Notch (Figure 1B). In contrast, Dl follicle cell mutant clones show precocious Cut downregulation at stage 6 attributable to the relief of cis-inhibition (Poulton et al., 2011) (Figure 1C). Surprisingly, Dl mutant clones in the follicle cells bordering Dl mutant clones in the germline (i.e., a germline with no signaling source, herein referred to as Dl-/Dl- cells) show correct Hnt and Cut expression from stage 7 (Figure 1D,E, Figure 1—figure supplement 2A,B). These Dl-/Dl- clones also correctly transit into the endocycle, as their nuclear volumes are similar to wild-type follicle cells in the later stages of oogenesis after polyploidization (Figure 1F,G), whereas cells neighboring Dl-/Dl- follicle cell clones (retaining a cis-ligand but without a trans-ligand) are comparable to wild-type cells before entry to endocycle (Figure 1F,G). Removal of both cis- and trans-Dl through knockdown of Dl by RNA interference (RNAi) simultaneously in the germline and soma confirmed this finding (Figure 2—figure supplement 1A,B). Together, these observations provide evidence that follicle cells without both cis- and trans-ligand sources can still enter the endocycle stages of oogenesis. This back-up route to the endocycle is not a co-option of Ser in place of Dl, as DlRevF10SerRx82 double clones recapitulated the Dl-/Dl- phenotype (Figure 1E, Figure 1—figure supplement 2A).10.7554/eLife.04415.003Figure 1.Follicle cells without DSL ligand bordering germline cells without DSL ligand show proper Notch activation and downstream differentiation.

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