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The Friend of GATA Transcriptional Co-Regulator, U-Shaped, Is a Downstream Antagonist of Dorsal-Driven Prohemocyte Differentiation in Drosophila.

Gao H, Baldeosingh R, Wu X, Fossett N - PLoS ONE (2016)

Bottom Line: Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs.Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence.Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular and Inflammatory Diseases and the Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, we identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. We showed that Dorsal is required to limit the size of the progenitor pool. Additionally, we showed that activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. We also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. We showed that Dorsal repressed Ush expression levels to promote differentiation, whereas Cactus maintained Ush levels to block differentiation. Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

No MeSH data available.


Related in: MedlinePlus

Lwr maintains Ush to block lamellocyte differentiation.(A-C) Lwr maintains the level of Ush expression. Tep-Gal4 females were crossed to control (+) males or males that carry UAS-lwrRNAi trangenes. (B) Knockdown of Lwr (lwrRNAi) in prohemocytes significantly reduced Ush levels compared to (A) controls (+) in the lymph glands of early-third instar larvae. (C) Histogram showing that the level of Ush expression is significantly reduced in lymph glands in which Lwr was knocked down compared to controls. Student’s t-test; control and lwrRNAi (n = 16); error bars show standard deviation; P value is as shown. (D-G) Ush and Lwr act together to block lamellocyte differentiation in late-third instar larvae. (F) Lamellocyte differentiation in lwr/ush trans-heterozygous lymph glands was significantly increased compared to either (D) lwr/+ or (E) ush/+ lymph glands. Lamellocytes are detected using the specific marker, MSNF9mo-DsRed [69]. Arrows mark lamellocytes (lm). (G) Histogram showing that the number of primary lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in lwr/ush transheterozygous lymph glands compared to either lwr/+ or ush/+ lymph glands. Chi-square test; lwr/ush, lwr/+, and ush/+ (n = 20); P value is as shown. White dotted lines delineate the entire lymph gland. Scale bars: (A,B) 25 μm; (D-F) 50 μm.
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pone.0155372.g007: Lwr maintains Ush to block lamellocyte differentiation.(A-C) Lwr maintains the level of Ush expression. Tep-Gal4 females were crossed to control (+) males or males that carry UAS-lwrRNAi trangenes. (B) Knockdown of Lwr (lwrRNAi) in prohemocytes significantly reduced Ush levels compared to (A) controls (+) in the lymph glands of early-third instar larvae. (C) Histogram showing that the level of Ush expression is significantly reduced in lymph glands in which Lwr was knocked down compared to controls. Student’s t-test; control and lwrRNAi (n = 16); error bars show standard deviation; P value is as shown. (D-G) Ush and Lwr act together to block lamellocyte differentiation in late-third instar larvae. (F) Lamellocyte differentiation in lwr/ush trans-heterozygous lymph glands was significantly increased compared to either (D) lwr/+ or (E) ush/+ lymph glands. Lamellocytes are detected using the specific marker, MSNF9mo-DsRed [69]. Arrows mark lamellocytes (lm). (G) Histogram showing that the number of primary lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in lwr/ush transheterozygous lymph glands compared to either lwr/+ or ush/+ lymph glands. Chi-square test; lwr/ush, lwr/+, and ush/+ (n = 20); P value is as shown. White dotted lines delineate the entire lymph gland. Scale bars: (A,B) 25 μm; (D-F) 50 μm.

Mentions: Our studies show that Dorsal represses Ush expression levels. These findings predict that Toll antagonists, such as Lwr, should maintain Ush expression. Lwr is a small ubiquitin-like modifier (SUMO) conjugase, which functions in a variety of biological processes. Notably, Lwr restricts Toll signaling by limiting the transcriptional activity of Dorsal and maintaining the level of Cact [35,45,53]. Furthermore, like Ush, loss of Lwr function leads to loss of prohemocytes and increased lamellocyte differentiation [68]. To test if Lwr is required to maintain Ush, we used the UAS/Gal4 system coupled with RNAi technology to knockdown Lwr in prohemocytes and assessed Ush levels. Under these conditions, we observed a statistically significant decrease in Ush levels (Fig 7A–7C). Thus, Lwr is required to maintain Ush expression. We then asked if Ush and Lwr interact to control lamellocyte differentiation. To test this hypothesis, we assessed lamellocyte differentiation in lwr/ush trans-heterozygotes. We observed a significant increase in lamellocyte differentiation in the lymph glands of trans-heterozygotes compared to those that were singularly heterozygous for either lwr or ush (Fig 7D–7G). Thus, Lwr interacts with Ush to control lamellcyte differentiation.


The Friend of GATA Transcriptional Co-Regulator, U-Shaped, Is a Downstream Antagonist of Dorsal-Driven Prohemocyte Differentiation in Drosophila.

Gao H, Baldeosingh R, Wu X, Fossett N - PLoS ONE (2016)

Lwr maintains Ush to block lamellocyte differentiation.(A-C) Lwr maintains the level of Ush expression. Tep-Gal4 females were crossed to control (+) males or males that carry UAS-lwrRNAi trangenes. (B) Knockdown of Lwr (lwrRNAi) in prohemocytes significantly reduced Ush levels compared to (A) controls (+) in the lymph glands of early-third instar larvae. (C) Histogram showing that the level of Ush expression is significantly reduced in lymph glands in which Lwr was knocked down compared to controls. Student’s t-test; control and lwrRNAi (n = 16); error bars show standard deviation; P value is as shown. (D-G) Ush and Lwr act together to block lamellocyte differentiation in late-third instar larvae. (F) Lamellocyte differentiation in lwr/ush trans-heterozygous lymph glands was significantly increased compared to either (D) lwr/+ or (E) ush/+ lymph glands. Lamellocytes are detected using the specific marker, MSNF9mo-DsRed [69]. Arrows mark lamellocytes (lm). (G) Histogram showing that the number of primary lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in lwr/ush transheterozygous lymph glands compared to either lwr/+ or ush/+ lymph glands. Chi-square test; lwr/ush, lwr/+, and ush/+ (n = 20); P value is as shown. White dotted lines delineate the entire lymph gland. Scale bars: (A,B) 25 μm; (D-F) 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862636&req=5

pone.0155372.g007: Lwr maintains Ush to block lamellocyte differentiation.(A-C) Lwr maintains the level of Ush expression. Tep-Gal4 females were crossed to control (+) males or males that carry UAS-lwrRNAi trangenes. (B) Knockdown of Lwr (lwrRNAi) in prohemocytes significantly reduced Ush levels compared to (A) controls (+) in the lymph glands of early-third instar larvae. (C) Histogram showing that the level of Ush expression is significantly reduced in lymph glands in which Lwr was knocked down compared to controls. Student’s t-test; control and lwrRNAi (n = 16); error bars show standard deviation; P value is as shown. (D-G) Ush and Lwr act together to block lamellocyte differentiation in late-third instar larvae. (F) Lamellocyte differentiation in lwr/ush trans-heterozygous lymph glands was significantly increased compared to either (D) lwr/+ or (E) ush/+ lymph glands. Lamellocytes are detected using the specific marker, MSNF9mo-DsRed [69]. Arrows mark lamellocytes (lm). (G) Histogram showing that the number of primary lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in lwr/ush transheterozygous lymph glands compared to either lwr/+ or ush/+ lymph glands. Chi-square test; lwr/ush, lwr/+, and ush/+ (n = 20); P value is as shown. White dotted lines delineate the entire lymph gland. Scale bars: (A,B) 25 μm; (D-F) 50 μm.
Mentions: Our studies show that Dorsal represses Ush expression levels. These findings predict that Toll antagonists, such as Lwr, should maintain Ush expression. Lwr is a small ubiquitin-like modifier (SUMO) conjugase, which functions in a variety of biological processes. Notably, Lwr restricts Toll signaling by limiting the transcriptional activity of Dorsal and maintaining the level of Cact [35,45,53]. Furthermore, like Ush, loss of Lwr function leads to loss of prohemocytes and increased lamellocyte differentiation [68]. To test if Lwr is required to maintain Ush, we used the UAS/Gal4 system coupled with RNAi technology to knockdown Lwr in prohemocytes and assessed Ush levels. Under these conditions, we observed a statistically significant decrease in Ush levels (Fig 7A–7C). Thus, Lwr is required to maintain Ush expression. We then asked if Ush and Lwr interact to control lamellocyte differentiation. To test this hypothesis, we assessed lamellocyte differentiation in lwr/ush trans-heterozygotes. We observed a significant increase in lamellocyte differentiation in the lymph glands of trans-heterozygotes compared to those that were singularly heterozygous for either lwr or ush (Fig 7D–7G). Thus, Lwr interacts with Ush to control lamellcyte differentiation.

Bottom Line: Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs.Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence.Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular and Inflammatory Diseases and the Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, we identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. We showed that Dorsal is required to limit the size of the progenitor pool. Additionally, we showed that activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. We also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. We showed that Dorsal repressed Ush expression levels to promote differentiation, whereas Cactus maintained Ush levels to block differentiation. Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

No MeSH data available.


Related in: MedlinePlus