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Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis.

Reimels TA, Pfleger CM - J. Cell. Sci. (2015)

Bottom Line: Rabex-5 negatively regulates Ras, and we show that Ras activity is responsible for specific Rabex-5 hematopoietic phenotypes.Surprisingly, Ras-independent Notch protein accumulation and transcriptional activity in the lymph gland underlie multiple distinct hematopoietic phenotypes of Rabex-5 loss.Thus, Rabex-5 plays an important role in Drosophila hematopoiesis and might serve as an axis coordinating Ras and Notch signaling in the lymph gland.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

No MeSH data available.


Related in: MedlinePlus

Melanotic mass formation and lamellocyte differentiation are not sensitive to Ras gene dosage. (A) Reducing Ras gene dosage with loss-of-function allele Rase1b in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) decreased larval lethality compared to that of controls (Rabex-5ex42/ex42). (B) Reducing Ras gene dosage restored lymph gland area in a Rabex-5ex42/ex42 background compared to that in controls (Rabex-5ex42/ex42; Rase1b/+ and Rabex-5ex42/ex42) but had no effect on lymph gland area alone (Rase1b/+) 5 days AEL. (C) In a Rabex-5ex42/ex42 background, expressing GEF-inactive Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5DPYT) suppressed larval lethality, and wild-type Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5WT) showed a trend to suppress larval lethality compared to that in controls (Rabex-5ex42/ex42; He>GFP). Reducing Ras gene dosage in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) did not affect the incidence of melanotic masses (D), the percentage of larvae containing lamellocytes (E) 14 days AEL, or the increased crystal cells (F) 9 days AEL. (G) Expressing constitutively active RasV12 in a Bc1 heterozygous background (Bc1/+; srp>GFP, RasV12) did not increase the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; srp>GFP) 120 h AEL. (H) Crystal cells were visualized by heating larvae 5 days AEL. Dominant-negative Serrate (SerBd-3/+) strongly suppressed the increase in the numbers of crystal cells within a Rabex-5ex42/ex42 background. The loss-of-function Delta allele (Dl7/+) subtly suppressed the increased crystal cell numbers. Duplication of Notch (NDpN/+) further increased crystal cell numbers in a Rabex-5ex42/ex42 background. ^P≤0.05, *P≤0.01.
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JCS174433F4: Melanotic mass formation and lamellocyte differentiation are not sensitive to Ras gene dosage. (A) Reducing Ras gene dosage with loss-of-function allele Rase1b in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) decreased larval lethality compared to that of controls (Rabex-5ex42/ex42). (B) Reducing Ras gene dosage restored lymph gland area in a Rabex-5ex42/ex42 background compared to that in controls (Rabex-5ex42/ex42; Rase1b/+ and Rabex-5ex42/ex42) but had no effect on lymph gland area alone (Rase1b/+) 5 days AEL. (C) In a Rabex-5ex42/ex42 background, expressing GEF-inactive Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5DPYT) suppressed larval lethality, and wild-type Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5WT) showed a trend to suppress larval lethality compared to that in controls (Rabex-5ex42/ex42; He>GFP). Reducing Ras gene dosage in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) did not affect the incidence of melanotic masses (D), the percentage of larvae containing lamellocytes (E) 14 days AEL, or the increased crystal cells (F) 9 days AEL. (G) Expressing constitutively active RasV12 in a Bc1 heterozygous background (Bc1/+; srp>GFP, RasV12) did not increase the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; srp>GFP) 120 h AEL. (H) Crystal cells were visualized by heating larvae 5 days AEL. Dominant-negative Serrate (SerBd-3/+) strongly suppressed the increase in the numbers of crystal cells within a Rabex-5ex42/ex42 background. The loss-of-function Delta allele (Dl7/+) subtly suppressed the increased crystal cell numbers. Duplication of Notch (NDpN/+) further increased crystal cell numbers in a Rabex-5ex42/ex42 background. ^P≤0.05, *P≤0.01.

Mentions: To determine whether Ras inhibition underlies Rabex-5- hematopoietic phenotypes, we reduced Ras gene dosage or restored the Rabex-5 E3 ligase domain in the hematopoietic system. Reducing Ras gene dosage by using the loss-of-function allele Rase1b suppressed larval lethality in Rabex-5- larvae (Rabex-5ex42/ex42; Rase1b/+; Fig. 4A) and restored the size of the lymph gland (Fig. 4B). To restore Rabex-5 E3 ligase function, we used He-gal4 in order to express either Rabex-5WT or full-length Rabex-5 with an intact E3 ligase domain and an inactive Rab5 GEF domain (Rabex-5DPYT) that had been characterized previously (Yan et al., 2010). Expressing Rabex-5DPYT suppressed larval lethality in a Rabex-5- background, and expressing Rabex-5WT showed a trend to suppress larval lethality in a Rabex-5- background (Fig. 4C, Fig. S1A).Fig. 4.


Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis.

Reimels TA, Pfleger CM - J. Cell. Sci. (2015)

Melanotic mass formation and lamellocyte differentiation are not sensitive to Ras gene dosage. (A) Reducing Ras gene dosage with loss-of-function allele Rase1b in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) decreased larval lethality compared to that of controls (Rabex-5ex42/ex42). (B) Reducing Ras gene dosage restored lymph gland area in a Rabex-5ex42/ex42 background compared to that in controls (Rabex-5ex42/ex42; Rase1b/+ and Rabex-5ex42/ex42) but had no effect on lymph gland area alone (Rase1b/+) 5 days AEL. (C) In a Rabex-5ex42/ex42 background, expressing GEF-inactive Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5DPYT) suppressed larval lethality, and wild-type Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5WT) showed a trend to suppress larval lethality compared to that in controls (Rabex-5ex42/ex42; He>GFP). Reducing Ras gene dosage in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) did not affect the incidence of melanotic masses (D), the percentage of larvae containing lamellocytes (E) 14 days AEL, or the increased crystal cells (F) 9 days AEL. (G) Expressing constitutively active RasV12 in a Bc1 heterozygous background (Bc1/+; srp>GFP, RasV12) did not increase the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; srp>GFP) 120 h AEL. (H) Crystal cells were visualized by heating larvae 5 days AEL. Dominant-negative Serrate (SerBd-3/+) strongly suppressed the increase in the numbers of crystal cells within a Rabex-5ex42/ex42 background. The loss-of-function Delta allele (Dl7/+) subtly suppressed the increased crystal cell numbers. Duplication of Notch (NDpN/+) further increased crystal cell numbers in a Rabex-5ex42/ex42 background. ^P≤0.05, *P≤0.01.
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JCS174433F4: Melanotic mass formation and lamellocyte differentiation are not sensitive to Ras gene dosage. (A) Reducing Ras gene dosage with loss-of-function allele Rase1b in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) decreased larval lethality compared to that of controls (Rabex-5ex42/ex42). (B) Reducing Ras gene dosage restored lymph gland area in a Rabex-5ex42/ex42 background compared to that in controls (Rabex-5ex42/ex42; Rase1b/+ and Rabex-5ex42/ex42) but had no effect on lymph gland area alone (Rase1b/+) 5 days AEL. (C) In a Rabex-5ex42/ex42 background, expressing GEF-inactive Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5DPYT) suppressed larval lethality, and wild-type Rabex-5 (Rabex-5ex42/ex42; He>GFP, Rabex-5WT) showed a trend to suppress larval lethality compared to that in controls (Rabex-5ex42/ex42; He>GFP). Reducing Ras gene dosage in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; Rase1b/+) did not affect the incidence of melanotic masses (D), the percentage of larvae containing lamellocytes (E) 14 days AEL, or the increased crystal cells (F) 9 days AEL. (G) Expressing constitutively active RasV12 in a Bc1 heterozygous background (Bc1/+; srp>GFP, RasV12) did not increase the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; srp>GFP) 120 h AEL. (H) Crystal cells were visualized by heating larvae 5 days AEL. Dominant-negative Serrate (SerBd-3/+) strongly suppressed the increase in the numbers of crystal cells within a Rabex-5ex42/ex42 background. The loss-of-function Delta allele (Dl7/+) subtly suppressed the increased crystal cell numbers. Duplication of Notch (NDpN/+) further increased crystal cell numbers in a Rabex-5ex42/ex42 background. ^P≤0.05, *P≤0.01.
Mentions: To determine whether Ras inhibition underlies Rabex-5- hematopoietic phenotypes, we reduced Ras gene dosage or restored the Rabex-5 E3 ligase domain in the hematopoietic system. Reducing Ras gene dosage by using the loss-of-function allele Rase1b suppressed larval lethality in Rabex-5- larvae (Rabex-5ex42/ex42; Rase1b/+; Fig. 4A) and restored the size of the lymph gland (Fig. 4B). To restore Rabex-5 E3 ligase function, we used He-gal4 in order to express either Rabex-5WT or full-length Rabex-5 with an intact E3 ligase domain and an inactive Rab5 GEF domain (Rabex-5DPYT) that had been characterized previously (Yan et al., 2010). Expressing Rabex-5DPYT suppressed larval lethality in a Rabex-5- background, and expressing Rabex-5WT showed a trend to suppress larval lethality in a Rabex-5- background (Fig. 4C, Fig. S1A).Fig. 4.

Bottom Line: Rabex-5 negatively regulates Ras, and we show that Ras activity is responsible for specific Rabex-5 hematopoietic phenotypes.Surprisingly, Ras-independent Notch protein accumulation and transcriptional activity in the lymph gland underlie multiple distinct hematopoietic phenotypes of Rabex-5 loss.Thus, Rabex-5 plays an important role in Drosophila hematopoiesis and might serve as an axis coordinating Ras and Notch signaling in the lymph gland.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

No MeSH data available.


Related in: MedlinePlus