ARF1-GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis

Abstract

Drosophila melanogaster larval hematopoiesis is a well-established model to study mechanisms that regulate hematopoietic niche maintenance and control of blood cell precursor (prohemocyte) differentiation. Molecules that perturb niche function affect the balance between prohemocytes and differentiated hemocytes. The conserved hemocyte-specific endosomal protein Asrij is essential for niche function and prohemocyte maintenance. Elucidating how subcellular trafficking molecules can regulate signaling presents an important challenge. Here we show that Asrij function is mediated by the Ras family GTPase Arf79F, the Drosophila homolog of ADP ribosylation factor 1 (ARF1), essential for clathrin coat assembly, Golgi architecture, and vesicular trafficking. ARF1 is expressed in the larval lymph gland and in circulating hemocytes and interacts with Asrij. ARF1-depleted lymph glands show loss of niche cells and prohemocyte maintenance with increased differentiation. Inhibiting ARF1 activation by knocking down its guanine nucleotide exchange factor (Gartenzwerg) or overexpressing its GTPAse-activating protein showed that ARF1-GTP is essential for regulating niche size and maintaining stemness. Activated ARF1 regulates Asrij levels in blood cells thereby mediating Asrij function. Asrij controls crystal cell differentiation by affecting Notch trafficking. ARF1 perturbation also leads to aberrant Notch trafficking and the Notch intracellular domain is stalled in sorting endosomes. Thus, ARF1 can regulate Drosophila blood cell homeostasis by regulating Asrij endocytic function. ARF1 also regulates signals arising from the niche and differentiated cells by integrating the insulin-mediated and PDGF-VEGF receptor signaling pathways. We propose that the conserved ARF1-Asrij endocytic axis modulates signals that govern hematopoietic development. Thus, Asrij affords tissue-specific control of global mechanisms involved in molecular traffic.

Fig. 1.

ARF1 interacts with the pan hemocyte marker Asrij. (A–D) ARF1 (green) and Asrij (red) colocalization in the lymph gland (A, and magnified boxed region in B), hemocytes in circulation (C), and S2R+ cells (D). Colocalization plots are as indicated. (E) Coimmunoprecipitation (co-IP) of Asrij and ARF1 from S2R+ protein extracts with anti-Asrij antibodies. Immunoblot was probed with anti-ARF1 antibody. (Lane 1) Input control (10% of the total protein). (Lane 2) IP with preimmune serum. (Lane 3) IP with anti-Asrij antibodies. (F) In situ proximity ligation assay on wild-type lymph glands using antibodies against ARF1 and Asrij full length (ArjFL) or ArjN or ArjC. Graph shows PLA signal (red dots/cell). n = 10. Nuclei were stained with DAPI (blue). [Scale bar, 20 μm (A) and 5 μm (B–D and F).]

Fig. 2.

ARF1 perturbation affects blood cell homeostasis. (A–K) Lymph gland whole mounts showing primary (1°) or secondary (2°) lobes of control (e33cGal4GFP) or ARF1 knockdown (e33cGal4GFPUASarf1rnai) third instar larvae. Knockdown shows excess of 2° lobes (B), BrdU+ cells (D), cyclin A staining (F), and H3P+ cells (H), also reduced Antp+ niche with increased differentiation (P1, ProPO staining) (J) compared with controls (A, C, E, G, and I, respectively). Graph shows number of BrdU+ cells (C and D), H3P+ cells (G and H), and Antp+ cells (K). GFP expression (green) in I and J is shown (Lower). (L) Total and differential hemocyte counts in circulation as indicated. (M) Antp+ cells coexpressing GFP (green) seen in control (PCol85Gal4GFP) are absent in PCol85Gal4GFPUASarf1rnai in all larval instars. Graphs represent Antp+ cells/larva. n = 10. P values are as indicated. Red arrows in A, B, G, and H indicate start of the 2° lobes. Genotypes are as indicated. [Scale bar, 50 μm (A, B, G, and H) and 20 μm (C–F, I, J, and M).] Also see Figs. S4 and S5.

Fig. 3.

Perturbation of ARF1–GTP activity leads to aberrant hematopoietic phenotypes. (A–H) Whole mounts showing secondary lobe tissue overgrowth marked with DAPI (blue) and increase in mitotically active cells (H3P+, green) in HmldeltaGal4-mediated gartenzwerg (garz) knockdown and Gap69c (arf1GAP) overexpression lymph glands (C, D, G, and H) compared with controls (A, B, E, and F). Graphs show H3P+ cells/lymph gland (O and W). Antp+ (green) niche cells are reduced in garz knockdown and arf1GAP overexpression (N and V) compared with control (M and U) also represented graphically (P and X). Premature differentiation into plasmatocytes (P1, green) (J and R) and crystal cells (ProPO, green) (L and T) in garz knockdown and arf1GAP overexpression compared with controls (I, K, Q, and S), respectively. Red arrows indicate start of the secondary lymph gland lobes. Genotypes are as labeled. C, control; KD, knockdown. [Scale bar, 50 μm (A–H) and 20 μm (I–N and Q–V).]

Fig. 4.

Perturbation of ARF1 and its activity affects Notch trafficking. NICD expression (green) and Hrs expression (red) in larval circulating hemocytes from control (A, C, and E) and arf1 knockdown (B), garz knockdown (D), and arf1GAP overexpressing (F) larvae. Right panels are merged images and respective colocalization plots. Nuclei were stained with DAPI (blue). Genotypes are as indicated. [Scale bar, 5 μm (A–F).]

Fig. 5.

ARF1–GTP regulates Asrij expression in hemocytes. (A–F) Third instar larval hemocytes showing expression of Asrij (green) and ARF1 (red) in controls (A, C, and E) and (B) arf1 knockdown and (D and F) ARF1-GTP perturbed conditions in (D) garz knockdown and (F) arf1GAP overexpression. Genotypes are as labeled. Right panels are merged images. [Scale bar, 5 μm (A–F).] (G) Proposed model for the role of ARF1 in lymph gland hematopoiesis. ARF1 cycles between GDP- and GTP-bound states depending on the activity of its GAP (Gap69c) and GEF (Garz), respectively. ARF–GDP does not support niche maintenance and promotes premature differentiation possibly due to absence of Asrij interaction. ARF–GTP interacts with and stabilizes Asrij thereby supporting maintenance of blood cell homeostasis by Asrij.

Fig. 6.

ARF1 modulates multiple signaling pathways to control hematopoiesis. (A) Effect of knockdown (KD) or overexpression (OV) of ARF1 in the CZ singly or in combination with Pvr: Whole mount lymph gland primary lobe showing expression of Antp, P1, or ProPo in the different genotypes as indicated. Note increase in P1+ and ProPo+ cells seen in Arf1KD,PvrOV is rescued in Arf1OV,PvrKD. (B) Effect on Antp+ cell numbers. See text for detailed genotypes. (Scale bar, 20 μm.) (C) Proposed model for nonautonomous ARF1 function in progenitor maintenance. Pvf1 emanating from the niche activates Pvr signaling in the CZ. This causes Stat92e activation, which is aided by the ARF1–Asrij complex on endosomes. Thus, signals are generated and/or relayed to the MZ for prohemocyte maintenance. Hence loss of ARF1 from the CZ causes differentiation of MZ progenitors. In addition a signal “X” mediated by non–lymph-gland hemocytes also regulates prohemocyte maintenance by acting on the MZ and/or CZ.