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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

Rabex-5 is required in blood cells to restrict proliferation, differentiation and the size of the lymph gland. (A) Rabex-5 RNAi (srp>GFP, Rabex-5IR) caused melanotic masses in 6.7% of larvae compared to 0% in control larvae (srp>GFP) 6 days AEL. (B) Rabex-5 RNAi (srp>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to those in controls (srp>GFP) 4 days AEL. DAPI staining is shown in blue. Scale bars: 50 μm. (C) Rabex-5 RNAi (srp>GFP, Rabex-5IR) did not change circulating hemocyte concentrations compared to those in controls (srp>GFP) 120 h AEL. (D) Rabex-5 RNAi in hemocytes (srp>GFP, Rabex-5IR) increased the percentage of circulating GFP-positive hemocytes compared to that of controls (srp>GFP) 120 h AEL. (E) Rabex-5 RNAi in hemocytes in a Bc1 heterozygous background (Bc1/+; srp>GFP, Rabex-5IR) increased the percentage of melanized crystal cells in the hemolymph compared to that of controls (Bc1/+; srp>GFP) 120 h AEL. (F) Rabex-5 knockdown by using srp-gal4 (srp>GFP, Rabex-5IR) did not change the percentage of circulating plasmatocytes compared to that of controls (srp>GFP) 120 h AEL. (G) Rabex-5 RNAi in embryonic hemocytes (crq>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to that in controls (crq>GFP) 5 days AEL. (H) Rabex-5 RNAi in embryonic hemocytes in a Bc1 heterozygous background (Bc1/+; crq>GFP, Rabex-5IR) did not alter the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; crq>GFP) 120 h AEL. Heat-induced melanization of crystal cells in vivo also showed no difference. (I) Rabex-5 RNAi in the medullary zone of the larval lymph gland (dome>GFP, Rabex-5IR) increased the area of the primary lobes 4 days AEL and increased crystal cell numbers visualized by heating larvae (J) compared to those of controls (dome>GFP). *P≤0.01.
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JCS174433F3: Rabex-5 is required in blood cells to restrict proliferation, differentiation and the size of the lymph gland. (A) Rabex-5 RNAi (srp>GFP, Rabex-5IR) caused melanotic masses in 6.7% of larvae compared to 0% in control larvae (srp>GFP) 6 days AEL. (B) Rabex-5 RNAi (srp>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to those in controls (srp>GFP) 4 days AEL. DAPI staining is shown in blue. Scale bars: 50 μm. (C) Rabex-5 RNAi (srp>GFP, Rabex-5IR) did not change circulating hemocyte concentrations compared to those in controls (srp>GFP) 120 h AEL. (D) Rabex-5 RNAi in hemocytes (srp>GFP, Rabex-5IR) increased the percentage of circulating GFP-positive hemocytes compared to that of controls (srp>GFP) 120 h AEL. (E) Rabex-5 RNAi in hemocytes in a Bc1 heterozygous background (Bc1/+; srp>GFP, Rabex-5IR) increased the percentage of melanized crystal cells in the hemolymph compared to that of controls (Bc1/+; srp>GFP) 120 h AEL. (F) Rabex-5 knockdown by using srp-gal4 (srp>GFP, Rabex-5IR) did not change the percentage of circulating plasmatocytes compared to that of controls (srp>GFP) 120 h AEL. (G) Rabex-5 RNAi in embryonic hemocytes (crq>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to that in controls (crq>GFP) 5 days AEL. (H) Rabex-5 RNAi in embryonic hemocytes in a Bc1 heterozygous background (Bc1/+; crq>GFP, Rabex-5IR) did not alter the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; crq>GFP) 120 h AEL. Heat-induced melanization of crystal cells in vivo also showed no difference. (I) Rabex-5 RNAi in the medullary zone of the larval lymph gland (dome>GFP, Rabex-5IR) increased the area of the primary lobes 4 days AEL and increased crystal cell numbers visualized by heating larvae (J) compared to those of controls (dome>GFP). *P≤0.01.

Mentions: Both srp-gal4-directed expression of Rabex-5 and of dap in Rabex-5- larvae suppressed melanotic mass formation (Fig. 1D,E), indicating a requirement for Rabex-5 specifically in the hematopoietic system and suggesting a role for Rabex-5 to restrict hemocyte proliferation. To investigate a specific requirement for Rabex-5 within the hematopoietic system, we performed RNA interference (RNAi) of Rabex-5 by using srp-gal4 and an inducible inverted repeat allele, Rabex-5IR, we characterized previously (Yan et al., 2010). Surprisingly, reducing Rabex-5 levels by using srp-gal4 was sufficient to cause melanotic masses in 6.7% of larvae (Fig. 3A). Rabex-5 knockdown increased the area and the GFP intensity of the primary lymph gland lobes (Fig. 3B). Although Rabex-5 knockdown was insufficient to increase hemocyte concentration (Fig. 3C), it was sufficient to alter circulating hemocyte proportions. Compared to that of controls, RNAi of Rabex-5 in hemocytes increased the percentage of GFP-positive hemocytes in circulation (Fig. 3D) to an extent similar to that observed in Rabex-5- larvae (Fig. S2C). RNAi of Rabex-5 increased the percentage of circulating crystal cells (melanized cells, Fig. 3E) to an extent similar to that seen in Rabex-5 heterozygous larvae (Fig. 2H). The basement membrane of lymph glands, marked by Trol expression, remained intact upon loss of Rabex-5 (Fig. S3); the increased percentage of circulating hemocytes did not result from rupture or emptying of the lymph gland. In contrast, RNAi of Rabex-5 did not significantly increase the percentage of plasmatocytes in circulation compared to that of controls (P1a/P1b-positive cells, Fig. 3F). These data indicate an intrinsic requirement for Rabex-5 in the hematopoietic system in order to prevent melanotic masses, restrict proliferation in the primary lymph gland and maintain appropriate proportions of hemocytes in the hemolymph.Fig. 3.


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

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

Rabex-5 is required in blood cells to restrict proliferation, differentiation and the size of the lymph gland. (A) Rabex-5 RNAi (srp>GFP, Rabex-5IR) caused melanotic masses in 6.7% of larvae compared to 0% in control larvae (srp>GFP) 6 days AEL. (B) Rabex-5 RNAi (srp>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to those in controls (srp>GFP) 4 days AEL. DAPI staining is shown in blue. Scale bars: 50 μm. (C) Rabex-5 RNAi (srp>GFP, Rabex-5IR) did not change circulating hemocyte concentrations compared to those in controls (srp>GFP) 120 h AEL. (D) Rabex-5 RNAi in hemocytes (srp>GFP, Rabex-5IR) increased the percentage of circulating GFP-positive hemocytes compared to that of controls (srp>GFP) 120 h AEL. (E) Rabex-5 RNAi in hemocytes in a Bc1 heterozygous background (Bc1/+; srp>GFP, Rabex-5IR) increased the percentage of melanized crystal cells in the hemolymph compared to that of controls (Bc1/+; srp>GFP) 120 h AEL. (F) Rabex-5 knockdown by using srp-gal4 (srp>GFP, Rabex-5IR) did not change the percentage of circulating plasmatocytes compared to that of controls (srp>GFP) 120 h AEL. (G) Rabex-5 RNAi in embryonic hemocytes (crq>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to that in controls (crq>GFP) 5 days AEL. (H) Rabex-5 RNAi in embryonic hemocytes in a Bc1 heterozygous background (Bc1/+; crq>GFP, Rabex-5IR) did not alter the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; crq>GFP) 120 h AEL. Heat-induced melanization of crystal cells in vivo also showed no difference. (I) Rabex-5 RNAi in the medullary zone of the larval lymph gland (dome>GFP, Rabex-5IR) increased the area of the primary lobes 4 days AEL and increased crystal cell numbers visualized by heating larvae (J) compared to those of controls (dome>GFP). *P≤0.01.
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JCS174433F3: Rabex-5 is required in blood cells to restrict proliferation, differentiation and the size of the lymph gland. (A) Rabex-5 RNAi (srp>GFP, Rabex-5IR) caused melanotic masses in 6.7% of larvae compared to 0% in control larvae (srp>GFP) 6 days AEL. (B) Rabex-5 RNAi (srp>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to those in controls (srp>GFP) 4 days AEL. DAPI staining is shown in blue. Scale bars: 50 μm. (C) Rabex-5 RNAi (srp>GFP, Rabex-5IR) did not change circulating hemocyte concentrations compared to those in controls (srp>GFP) 120 h AEL. (D) Rabex-5 RNAi in hemocytes (srp>GFP, Rabex-5IR) increased the percentage of circulating GFP-positive hemocytes compared to that of controls (srp>GFP) 120 h AEL. (E) Rabex-5 RNAi in hemocytes in a Bc1 heterozygous background (Bc1/+; srp>GFP, Rabex-5IR) increased the percentage of melanized crystal cells in the hemolymph compared to that of controls (Bc1/+; srp>GFP) 120 h AEL. (F) Rabex-5 knockdown by using srp-gal4 (srp>GFP, Rabex-5IR) did not change the percentage of circulating plasmatocytes compared to that of controls (srp>GFP) 120 h AEL. (G) Rabex-5 RNAi in embryonic hemocytes (crq>GFP, Rabex-5IR) increased the area of the primary lymph gland lobes compared to that in controls (crq>GFP) 5 days AEL. (H) Rabex-5 RNAi in embryonic hemocytes in a Bc1 heterozygous background (Bc1/+; crq>GFP, Rabex-5IR) did not alter the percentage of melanized crystal cells in the hemolymph compared to that in controls (Bc1/+; crq>GFP) 120 h AEL. Heat-induced melanization of crystal cells in vivo also showed no difference. (I) Rabex-5 RNAi in the medullary zone of the larval lymph gland (dome>GFP, Rabex-5IR) increased the area of the primary lobes 4 days AEL and increased crystal cell numbers visualized by heating larvae (J) compared to those of controls (dome>GFP). *P≤0.01.
Mentions: Both srp-gal4-directed expression of Rabex-5 and of dap in Rabex-5- larvae suppressed melanotic mass formation (Fig. 1D,E), indicating a requirement for Rabex-5 specifically in the hematopoietic system and suggesting a role for Rabex-5 to restrict hemocyte proliferation. To investigate a specific requirement for Rabex-5 within the hematopoietic system, we performed RNA interference (RNAi) of Rabex-5 by using srp-gal4 and an inducible inverted repeat allele, Rabex-5IR, we characterized previously (Yan et al., 2010). Surprisingly, reducing Rabex-5 levels by using srp-gal4 was sufficient to cause melanotic masses in 6.7% of larvae (Fig. 3A). Rabex-5 knockdown increased the area and the GFP intensity of the primary lymph gland lobes (Fig. 3B). Although Rabex-5 knockdown was insufficient to increase hemocyte concentration (Fig. 3C), it was sufficient to alter circulating hemocyte proportions. Compared to that of controls, RNAi of Rabex-5 in hemocytes increased the percentage of GFP-positive hemocytes in circulation (Fig. 3D) to an extent similar to that observed in Rabex-5- larvae (Fig. S2C). RNAi of Rabex-5 increased the percentage of circulating crystal cells (melanized cells, Fig. 3E) to an extent similar to that seen in Rabex-5 heterozygous larvae (Fig. 2H). The basement membrane of lymph glands, marked by Trol expression, remained intact upon loss of Rabex-5 (Fig. S3); the increased percentage of circulating hemocytes did not result from rupture or emptying of the lymph gland. In contrast, RNAi of Rabex-5 did not significantly increase the percentage of plasmatocytes in circulation compared to that of controls (P1a/P1b-positive cells, Fig. 3F). These data indicate an intrinsic requirement for Rabex-5 in the hematopoietic system in order to prevent melanotic masses, restrict proliferation in the primary lymph gland and maintain appropriate proportions of hemocytes in the hemolymph.Fig. 3.

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