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A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.

Sopko R, Lin YB, Makhijani K, Alexander B, Perrimon N, Brückner K - PLoS Genet. (2015)

Bottom Line: In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR.We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions.Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

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Role of EcR and InR in embryonic hemocytes in vivo.(A) In vivo rescue experiment, combining Pvr1 mutant or hemocyte specific expression of UAS-PvrΔC with various UAS-transgenes. Inhibition of EcR signaling by dominant-negative EcR (EcRA dn or EcRB dn) rescues hemocyte numbers in Pvr1 mutant embryos, while co-expression of dominant-negative InR enhances the Pvr1 phenotype. Embryonic hemocyte numbers in embryos of the indicated genetic combinations. Hemocytes were marked by nuclear β-Gal driven by srpHemoGAL4; total hemocytes of individual stage 16 embryos were counted. For full genotype, see Methods. (B-F) Confocal images of representative embryos. (G) Summary model of Pvr Suppressors and Pvr Enhancers as shown in B-F. In Pvr1 mutants, apoptotic hemocytes are phagocytosed by remaining, viable hemocytes, which increase in size [12]. Additional lack of a Pvr Enhancer aggravates the phenotype, while lack of a Pvr Suppressor rescues hemocyte death.
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pgen.1005056.g004: Role of EcR and InR in embryonic hemocytes in vivo.(A) In vivo rescue experiment, combining Pvr1 mutant or hemocyte specific expression of UAS-PvrΔC with various UAS-transgenes. Inhibition of EcR signaling by dominant-negative EcR (EcRA dn or EcRB dn) rescues hemocyte numbers in Pvr1 mutant embryos, while co-expression of dominant-negative InR enhances the Pvr1 phenotype. Embryonic hemocyte numbers in embryos of the indicated genetic combinations. Hemocytes were marked by nuclear β-Gal driven by srpHemoGAL4; total hemocytes of individual stage 16 embryos were counted. For full genotype, see Methods. (B-F) Confocal images of representative embryos. (G) Summary model of Pvr Suppressors and Pvr Enhancers as shown in B-F. In Pvr1 mutants, apoptotic hemocytes are phagocytosed by remaining, viable hemocytes, which increase in size [12]. Additional lack of a Pvr Enhancer aggravates the phenotype, while lack of a Pvr Suppressor rescues hemocyte death.

Mentions: To test the role of InR and EcR in the suppression and enhancement of apoptosis in vivo, we examined the function of these genes in the survival of hemocytes in the Drosophila embryo. Drosophila embryos typically show a developmentally fixed number of ~600 hemocytes post stage 11 until early stage 17, and loss of Pvr signaling causes a rapid decline in hemocytes due to their apoptotic death and phagocytic clearance by the small number of remaining live hemocytes [12]. Based on our findings in Kc cells, we predicted that inhibition of EcR would rescue, and inhibition of InR would enhance, Pvr loss-of-function in embryonic blood cells [12]. Indeed, hemocyte-specific suppression of EcR signaling by expression of dominant-negative forms of EcR [59,60] partially rescued hemocyte counts in Pvr1 mutant embryos (Fig. 4), resembling rescue by the baculovirus inhibitor of apoptosis, p35 [12] (see also Fig. 4A). Conversely, expression of dominant-negative InR in hemocytes enhanced the Pvr phenotype, further reducing embryonic hemocyte numbers (Fig. 4). Consistently, we previously demonstrated that activated PI3K, a positive mediator of the Akt/TOR pathway downstream of InR, can partially rescue the Pvr mutant in vivo phenotype [12]. To confirm hemocyte autonomous effects of EcR and InR, we induced embryonic hemocyte death by hemocyte-specific expression of dominant-negative PvrΔC [12], and examined the effects of co-expressed dominant-negative versions of EcR or InR. Again, we found that dominant-negative EcR rescued apoptotic loss of hemocytes, while dominant-negative InR exacerbated the cell death phenotype (Fig. 4). Expression of the transgenes in the wild type background had no significant effects (Fig. 4A).


A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.

Sopko R, Lin YB, Makhijani K, Alexander B, Perrimon N, Brückner K - PLoS Genet. (2015)

Role of EcR and InR in embryonic hemocytes in vivo.(A) In vivo rescue experiment, combining Pvr1 mutant or hemocyte specific expression of UAS-PvrΔC with various UAS-transgenes. Inhibition of EcR signaling by dominant-negative EcR (EcRA dn or EcRB dn) rescues hemocyte numbers in Pvr1 mutant embryos, while co-expression of dominant-negative InR enhances the Pvr1 phenotype. Embryonic hemocyte numbers in embryos of the indicated genetic combinations. Hemocytes were marked by nuclear β-Gal driven by srpHemoGAL4; total hemocytes of individual stage 16 embryos were counted. For full genotype, see Methods. (B-F) Confocal images of representative embryos. (G) Summary model of Pvr Suppressors and Pvr Enhancers as shown in B-F. In Pvr1 mutants, apoptotic hemocytes are phagocytosed by remaining, viable hemocytes, which increase in size [12]. Additional lack of a Pvr Enhancer aggravates the phenotype, while lack of a Pvr Suppressor rescues hemocyte death.
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pgen.1005056.g004: Role of EcR and InR in embryonic hemocytes in vivo.(A) In vivo rescue experiment, combining Pvr1 mutant or hemocyte specific expression of UAS-PvrΔC with various UAS-transgenes. Inhibition of EcR signaling by dominant-negative EcR (EcRA dn or EcRB dn) rescues hemocyte numbers in Pvr1 mutant embryos, while co-expression of dominant-negative InR enhances the Pvr1 phenotype. Embryonic hemocyte numbers in embryos of the indicated genetic combinations. Hemocytes were marked by nuclear β-Gal driven by srpHemoGAL4; total hemocytes of individual stage 16 embryos were counted. For full genotype, see Methods. (B-F) Confocal images of representative embryos. (G) Summary model of Pvr Suppressors and Pvr Enhancers as shown in B-F. In Pvr1 mutants, apoptotic hemocytes are phagocytosed by remaining, viable hemocytes, which increase in size [12]. Additional lack of a Pvr Enhancer aggravates the phenotype, while lack of a Pvr Suppressor rescues hemocyte death.
Mentions: To test the role of InR and EcR in the suppression and enhancement of apoptosis in vivo, we examined the function of these genes in the survival of hemocytes in the Drosophila embryo. Drosophila embryos typically show a developmentally fixed number of ~600 hemocytes post stage 11 until early stage 17, and loss of Pvr signaling causes a rapid decline in hemocytes due to their apoptotic death and phagocytic clearance by the small number of remaining live hemocytes [12]. Based on our findings in Kc cells, we predicted that inhibition of EcR would rescue, and inhibition of InR would enhance, Pvr loss-of-function in embryonic blood cells [12]. Indeed, hemocyte-specific suppression of EcR signaling by expression of dominant-negative forms of EcR [59,60] partially rescued hemocyte counts in Pvr1 mutant embryos (Fig. 4), resembling rescue by the baculovirus inhibitor of apoptosis, p35 [12] (see also Fig. 4A). Conversely, expression of dominant-negative InR in hemocytes enhanced the Pvr phenotype, further reducing embryonic hemocyte numbers (Fig. 4). Consistently, we previously demonstrated that activated PI3K, a positive mediator of the Akt/TOR pathway downstream of InR, can partially rescue the Pvr mutant in vivo phenotype [12]. To confirm hemocyte autonomous effects of EcR and InR, we induced embryonic hemocyte death by hemocyte-specific expression of dominant-negative PvrΔC [12], and examined the effects of co-expressed dominant-negative versions of EcR or InR. Again, we found that dominant-negative EcR rescued apoptotic loss of hemocytes, while dominant-negative InR exacerbated the cell death phenotype (Fig. 4). Expression of the transgenes in the wild type background had no significant effects (Fig. 4A).

Bottom Line: In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR.We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions.Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

Show MeSH
Related in: MedlinePlus