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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 prevent melanotic mass formation. (A) Melanotic masses in larvae homozygous for the deletion allele Rabex-5ex42 (arrows; anterior, top). (B) At least one melanotic mass was seen in 3.8% of Rabex-5ex42/ex42 larvae compared to 0% in control larvae 6 days AEL. Incidence of melanotic masses in Rabex-5ex42/ex42 larvae increased to 15% 9 days AEL and 45% 14 days AEL. (C) Serpent- (srp) and croquemort- (crq) gal4 express in embryonic hemocytes. Srp-gal4 also expresses in all hemocytes of the larval lymph gland and in circulating prohemocytes. Hemese- (He) gal4 expresses in larval hemocytes. (D) Expressing wild-type Rabex-5 using srp-gal4, but not He-gal4, in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; srp>GFP, Rabex-5WT and Rabex-5ex42/ex42; He>GFP, Rabex-5WT) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; srp>GFP and Rabex-5ex42/ex42; He>GFP) 14 days AEL. (E) Expressing dap using either He-gal4 or srp-gal4 in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; He>GFP, dap and Rabex-5ex42/ex42; srp>GFP, dap) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; He>GFP and Rabex-5ex42/ex42; srp>GFP) 14 days AEL. ^P≤0.05, *P≤0.01.
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JCS174433F1: Rabex-5 is required in blood cells to prevent melanotic mass formation. (A) Melanotic masses in larvae homozygous for the deletion allele Rabex-5ex42 (arrows; anterior, top). (B) At least one melanotic mass was seen in 3.8% of Rabex-5ex42/ex42 larvae compared to 0% in control larvae 6 days AEL. Incidence of melanotic masses in Rabex-5ex42/ex42 larvae increased to 15% 9 days AEL and 45% 14 days AEL. (C) Serpent- (srp) and croquemort- (crq) gal4 express in embryonic hemocytes. Srp-gal4 also expresses in all hemocytes of the larval lymph gland and in circulating prohemocytes. Hemese- (He) gal4 expresses in larval hemocytes. (D) Expressing wild-type Rabex-5 using srp-gal4, but not He-gal4, in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; srp>GFP, Rabex-5WT and Rabex-5ex42/ex42; He>GFP, Rabex-5WT) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; srp>GFP and Rabex-5ex42/ex42; He>GFP) 14 days AEL. (E) Expressing dap using either He-gal4 or srp-gal4 in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; He>GFP, dap and Rabex-5ex42/ex42; srp>GFP, dap) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; He>GFP and Rabex-5ex42/ex42; srp>GFP) 14 days AEL. ^P≤0.05, *P≤0.01.

Mentions: We previously reported melanotic mass formation (Fig. 1A), and larval and pupal lethality in Drosophila that lack the neoplastic tumor suppressor Rabex-5 (Yan et al., 2010). At least one melanotic mass was found in 3.8% of larvae homozygous for the deletion allele Rabex-5ex42 (referred to as Rabex-5- larvae) 6 days after egg laying (AEL). The incidence of melanotic masses increased over time to 45% 14 days AEL (Fig. 1B). Melanotic masses were of variable size, number and location within the body cavity. In the absence of parasitization, melanotic masses are often associated with abnormalities in the hematopoietic system, including autoimmune-like responses to self-tissue and dysregulation of proliferation leading to excess hemocyte numbers (Watson et al., 1994; Asha et al., 2003; Zettervall et al., 2004; Minakhina and Steward, 2006). To establish whether there is a requirement for Rabex-5 to prevent melanotic mass formation, we expressed wild-type Rabex-5 (Rabex-5WT) using Hemese-gal4 (He-gal4) or Serpent-gal4 (srp-gal4) (Fig. 1C, Table S1). Hemese is a transmembrane protein expressed in all hemocyte lineages beginning in the second larval instar (Kurucz et al., 2003; Jung et al., 2005). He-gal4 expresses in ∼70% of circulating hemocytes, in sessile hemocytes and at low levels in the larval lymph gland (Zettervall et al., 2004), but does not express in the embryo. Serpent is a GATA family member and the earliest known transcription factor required for embryonic and larval hemocyte development (Rehorn et al., 1996; Lebestky et al., 2000). Srp-gal4 expresses in embryonic hemocytes (Narbonne-Reveau et al., 2011) as well as in prohemocytes and all lymph gland cells of the larval stages (Jung et al., 2005). In Rabex-5- larvae, expressing Rabex-5WT by using He-gal4 (Rabex-5ex42/ex42; He>Rabex-5WT) did not reduce the incidence of melanotic masses observed at 14 days AEL; however, expressing Rabex-5WT by using srp-gal4 (Rabex-5ex42/ex42; srp>Rabex-5WT) reduced melanotic mass formation more than twofold (Fig. 1D). This indicates a specific requirement for Rabex-5 during hematopoiesis to prevent melanotic masses. To determine whether hemocyte overproliferation contributes to the melanotic mass phenotype, we utilized Drosophila cyclin-dependent kinase inhibitor dacapo (dap). In Rabex-5- larvae 14 days AEL, expressing dap in the hematopoietic system reduced melanotic mass formation (Rabex-5ex42/ex42; He>dap and Rabex-5ex42/ex42; srp>dap, Fig. 1E). Taken together, these findings suggest a role for Rabex-5 to restrict hemocyte proliferation and prevent melanotic mass formation. Rabex-5- lethality is likely to be pleiotropic; however, He-gal4 directed Rabex-5 expression and He-gal4 or srp-gal4 directed dap expression decreased larval lethality (Fig. S1A,B). This suggests hemocyte overproliferation also contributes to Rabex-5- larval lethality.Fig. 1.


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 prevent melanotic mass formation. (A) Melanotic masses in larvae homozygous for the deletion allele Rabex-5ex42 (arrows; anterior, top). (B) At least one melanotic mass was seen in 3.8% of Rabex-5ex42/ex42 larvae compared to 0% in control larvae 6 days AEL. Incidence of melanotic masses in Rabex-5ex42/ex42 larvae increased to 15% 9 days AEL and 45% 14 days AEL. (C) Serpent- (srp) and croquemort- (crq) gal4 express in embryonic hemocytes. Srp-gal4 also expresses in all hemocytes of the larval lymph gland and in circulating prohemocytes. Hemese- (He) gal4 expresses in larval hemocytes. (D) Expressing wild-type Rabex-5 using srp-gal4, but not He-gal4, in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; srp>GFP, Rabex-5WT and Rabex-5ex42/ex42; He>GFP, Rabex-5WT) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; srp>GFP and Rabex-5ex42/ex42; He>GFP) 14 days AEL. (E) Expressing dap using either He-gal4 or srp-gal4 in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; He>GFP, dap and Rabex-5ex42/ex42; srp>GFP, dap) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; He>GFP and Rabex-5ex42/ex42; srp>GFP) 14 days AEL. ^P≤0.05, *P≤0.01.
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JCS174433F1: Rabex-5 is required in blood cells to prevent melanotic mass formation. (A) Melanotic masses in larvae homozygous for the deletion allele Rabex-5ex42 (arrows; anterior, top). (B) At least one melanotic mass was seen in 3.8% of Rabex-5ex42/ex42 larvae compared to 0% in control larvae 6 days AEL. Incidence of melanotic masses in Rabex-5ex42/ex42 larvae increased to 15% 9 days AEL and 45% 14 days AEL. (C) Serpent- (srp) and croquemort- (crq) gal4 express in embryonic hemocytes. Srp-gal4 also expresses in all hemocytes of the larval lymph gland and in circulating prohemocytes. Hemese- (He) gal4 expresses in larval hemocytes. (D) Expressing wild-type Rabex-5 using srp-gal4, but not He-gal4, in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; srp>GFP, Rabex-5WT and Rabex-5ex42/ex42; He>GFP, Rabex-5WT) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; srp>GFP and Rabex-5ex42/ex42; He>GFP) 14 days AEL. (E) Expressing dap using either He-gal4 or srp-gal4 in a Rabex-5ex42/ex42 background (Rabex-5ex42/ex42; He>GFP, dap and Rabex-5ex42/ex42; srp>GFP, dap) decreased the incidence of melanotic masses compared to those in controls (Rabex-5ex42/ex42; He>GFP and Rabex-5ex42/ex42; srp>GFP) 14 days AEL. ^P≤0.05, *P≤0.01.
Mentions: We previously reported melanotic mass formation (Fig. 1A), and larval and pupal lethality in Drosophila that lack the neoplastic tumor suppressor Rabex-5 (Yan et al., 2010). At least one melanotic mass was found in 3.8% of larvae homozygous for the deletion allele Rabex-5ex42 (referred to as Rabex-5- larvae) 6 days after egg laying (AEL). The incidence of melanotic masses increased over time to 45% 14 days AEL (Fig. 1B). Melanotic masses were of variable size, number and location within the body cavity. In the absence of parasitization, melanotic masses are often associated with abnormalities in the hematopoietic system, including autoimmune-like responses to self-tissue and dysregulation of proliferation leading to excess hemocyte numbers (Watson et al., 1994; Asha et al., 2003; Zettervall et al., 2004; Minakhina and Steward, 2006). To establish whether there is a requirement for Rabex-5 to prevent melanotic mass formation, we expressed wild-type Rabex-5 (Rabex-5WT) using Hemese-gal4 (He-gal4) or Serpent-gal4 (srp-gal4) (Fig. 1C, Table S1). Hemese is a transmembrane protein expressed in all hemocyte lineages beginning in the second larval instar (Kurucz et al., 2003; Jung et al., 2005). He-gal4 expresses in ∼70% of circulating hemocytes, in sessile hemocytes and at low levels in the larval lymph gland (Zettervall et al., 2004), but does not express in the embryo. Serpent is a GATA family member and the earliest known transcription factor required for embryonic and larval hemocyte development (Rehorn et al., 1996; Lebestky et al., 2000). Srp-gal4 expresses in embryonic hemocytes (Narbonne-Reveau et al., 2011) as well as in prohemocytes and all lymph gland cells of the larval stages (Jung et al., 2005). In Rabex-5- larvae, expressing Rabex-5WT by using He-gal4 (Rabex-5ex42/ex42; He>Rabex-5WT) did not reduce the incidence of melanotic masses observed at 14 days AEL; however, expressing Rabex-5WT by using srp-gal4 (Rabex-5ex42/ex42; srp>Rabex-5WT) reduced melanotic mass formation more than twofold (Fig. 1D). This indicates a specific requirement for Rabex-5 during hematopoiesis to prevent melanotic masses. To determine whether hemocyte overproliferation contributes to the melanotic mass phenotype, we utilized Drosophila cyclin-dependent kinase inhibitor dacapo (dap). In Rabex-5- larvae 14 days AEL, expressing dap in the hematopoietic system reduced melanotic mass formation (Rabex-5ex42/ex42; He>dap and Rabex-5ex42/ex42; srp>dap, Fig. 1E). Taken together, these findings suggest a role for Rabex-5 to restrict hemocyte proliferation and prevent melanotic mass formation. Rabex-5- lethality is likely to be pleiotropic; however, He-gal4 directed Rabex-5 expression and He-gal4 or srp-gal4 directed dap expression decreased larval lethality (Fig. S1A,B). This suggests hemocyte overproliferation also contributes to Rabex-5- larval lethality.Fig. 1.

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