Rab5 and Rab11 maintain hematopoietic homeostasis by restricting multiple signaling pathways in Drosophila
- Department of Genetics, College of Life Sciences, Northeast Forestry University, China
Figures
Figure 1 with 2 supplements
Inhibiting Rab5 or Rab11 promoted cell proliferation in circulating hemocytes and lymph glands.
(A) The number of circulating hemocytes increased upon the loss of Rab5 or Rab11 but not Rab7 in hemocytes. (B–P) Immunostaining for PH3 was performed in lymph glands from third instar larvae. The …
Figure 1—figure supplement 1
Analyses of lymph gland size and cell proliferation in circulating hemocytes as well as the sessile hemocyte pattern.
(A–F, P) Lymph glands were labeled with DAPI to measure the anterior lobe area. The anterior lobe areas in different genotypes are shown in (P). (G–L, Q) Cell proliferation was analyzed in hemocytes …
Figure 1—figure supplement 2
Localization of the Rab5 and Rab11 proteins in lymph glands and hemocytes.
(A–D”) The localization of Rab5 and Rab11 was analyzed in Hml>UAS-GFP and col>UAS-GFP lymph glands with anti-GFP (green) and anti-Rab5 (red) or anti-Rab11 (red) antibodies as appropriate. (E–H’) No …
Figure 2 with 6 supplements
Inactivation of Rab5 or Rab11 promoted differentiation in circulating hemocytes and lymph glands.
(A–D) The percentage of the GFP-positive area in anterior lobes was increased in Hml>UAS-GFP>UAS-Rab5DN and Hml>UAS-GFP>UAS-Rab11DN lymph glands; the quantification is shown in (D). (E–G’) …
Figure 2—figure supplement 1
Evaluation of the plasmatocyte count and medullary zone (MZ) area in the lymph gland after Rab5/Rab11 inactivation.
(A–C’) The plasmatocyte count was increased in the anterior (A–C) and posterior (A’–C’) lobes in lymph glands from Hml>UAS-Rab5 RNAi and Hml>UAS-Rab11 RNAi larvae, as determined by anti-P1 …
Figure 2—figure supplement 2
Overexpression of STAT92E or Adgf-A did not rescue the increased P1-positive area in Hml>UAS-Rab5/11DN lymph glands.
(A–H) P1 staining was performed in Hml>WT (A), Hml>UAS-Rab5/11DN>WT (B, E), Hml>UAS-Rab5/11DN>UAS-STAT92E (C, F), and Hml>UAS-Rab5/11DN>UAS-Adgf-A (D, G) lymph glands. The quantifications of the …
Figure 2—figure supplement 3
Analysis of the crystal cell count and rescue assays of lamellocyte differentiation in lymph glands.
(A–D) The crystal cell count (as evaluated by anti-ProPO staining) was unchanged in lymph glands upon the inactivation of Rab5 or Rab11. (E–G) Aberrant lamellocyte formation was observed in Hml>UAS-G…
Figure 2—figure supplement 4
Inactivation of Rab5/Rab11 with different hemocyte-specific Gal4 drivers resulted in massive lamellocyte formation.
(A–O) Aberrant lamellocyte formation was observed in pxn>UAS-GFP>UAS-Rab5/11DN, He>UAS-GFP>UAS-Rab5/11DN, and srp>UAS-Rab5/11DN lymph glands and in pxn>UAS-GFP>UAS-Rab5/11DN and He>UAS-GFP>UAS-Rab5/1…
Figure 2—figure supplement 5
Active Rab11 GTPase activity could restore the aberrant lamellocyte differentiation in lymph glands after inhibition of Rab5.
(A and B) Immunostaining of lamellocytes was performed with anti-L1 antibodies. The increased lamellocyte count was rescued in Hml>UAS-Rab5DN>UAS-Rab11CA lymph glands. Scale bar: 50 μm.
Figure 2—figure supplement 6
Analysis of the medullary zone (MZ) and posterior signaling center (PSC) upon the inactivation of Rab5 or Rab11.
PSC cells and the MZ were analyzed with anti-Antp antibodies or labeled with GFP after the inactivation of Rab5 or Rab11 using the Hml-Gal4 (A–C, Y), dome-Gal4 (J–L, Z), Antp-Gal4 (S–U, AA), and col-…
Figure 3 with 2 supplements
JNK signaling was activated upon Rab5 or Rab11 inactivation in the lymph gland.
(A–I) The JNK pathway activity was elevated in lymph glands upon Rab5 or Rab11 inactivation, as elucidated by the monitoring of JNK signaling with puc-lacZ (A–C) and anti-p-JNK antibodies (D–F). …
Figure 3—figure supplement 1
Knocking down hop or blocking apoptosis did not repress aberrant lamellocyte differentiation.
Knocking down hop (A–F) or overexpressing UAS-p35 (G–J) failed to repress the aberrant lamellocyte differentiation in Hml>UAS-GFP>UAS-Rab5/11 RNAi or Hml>UAS-Rab5/11DN lymph glands. Scale bar: 50 μm.
Figure 3—figure supplement 2
Knocking down bsk repressed aberrant lamellocyte differentiation in circulating hemocytes.
(A–D) Knocking down bsk repressed the aberrant lamellocyte differentiation in Hml>UAS-Rab5/11 RNAi circulating hemocytes. Quantification of the lamellocyte count is shown in (E). Scale bar: 20 μm. …
Figure 4 with 2 supplements
Inhibiting Rab5 or Rab11 induced high p-JNK levels in endosomes.
(A–C’’) An increased degree of Hrs (green) and p-JNK (red) colocalization was observed in Hml>UAS-Rab5DN and Hml>UAS-Rab11DN hemocytes. Merged images (Hrs+p-JNK+DAPI) are displayed in (A–C). (D–K) …
Figure 4—figure supplement 1
Analysis of the p-JNK, p-Jun, and Mmp1 levels in circulating hemocytes.
(A–U) Circulating hemocytes were stained with antibodies against p-JNK (A–G), p-Jun (H–N), and Mmp1 (O–U). Hml>UAS-hepAct, Rab5>UAS-bsk, and Rab11>UAS-bsk hemocytes showed elevated levels of p-JNK, …
Figure 4—figure supplement 2
Overexpression of bsk in hemocytes and fat bodies or in hemocytes and the midgut simultaneously induced lamellocyte formation.
(A–D) Hemocytes from Hml>ppl>UAS-bsk, Hml>NP3084>UAS-bsk, and Hml>elav>UAS-bsk larvae were stained with anti-L1 antibodies. The lamellocyte frequency in circulating hemocytes is shown in (D). Scale …
Figure 5
Inhibiting Rab5 or Rab11 in hemocytes increased the p-Jun and Mmp1 levels.
(A–F) Immunostaining of circulating hemocytes showed that the levels of p-Jun (A–C) and Mmp1 (D–F) were increased after the inactivation of Rab5 or Rab11. (G–M) The lamellocyte frequency, as …
Figure 6 with 1 supplement
Ras/EGFR signaling was enhanced upon Rab5 or Rab11 inactivation.
(A–C) Immunostaining of lymph glands showed high p-Erk (red) signals upon Rab5 or Rab11 inactivation. (D–I) Anterior lobe enlargement (visualized by DAPI staining) in Hml>UAS-Rab5/11DN lymph glands …
Figure 6—figure supplement 1
Knocking down Ras85D restored the aberrant lamellocyte differentiation in circulating hemocytes.
(A–D) Knocking down Ras85D repressed the aberrant lamellocyte differentiation in Hml>UAS-Rab5/11DN circulating hemocytes. (E) The lamellocyte frequency in circulating hemocytes. Scale bar: 20 μm. …
Figure 7 with 1 supplement
The Toll pathway was activated upon Rab5 or Rab11 inactivation.
(A–H) Immunostaining of circulating hemocytes showed that Dif and Dorsal were activated in Hml>UAS-Rab5/11DN (B–C, F–G) and Hml>UAS-hepAct (D, H) hemocytes. (I–X) Detection of lamellocyte formation …
Figure 7—figure supplement 1
The Toll signaling pathway was activated upon the loss of Rab5 or Rab11.
The Dif (A–C) and Dorsal (D–F) levels were analyzed with immunostaining and found to be elevated in Hml>UAS-Rab5 RNAi and Hml>UAS-Rab11 RNAi hemocytes. Scale bar: 10 μm.
Figure 8 with 2 supplements
Loss of Rab5 or Rab11 activated autophagy in hemocytes.
(A–C) Many autophagosomes were observed in Cg;Atg8a-mCherry>UAS-Rab5DN and Cg;Atg8a-mCherry>UAS-Rab11DN hemocytes. (D–I) Circulating hemocytes of Hml-Gal4, Hml>UAS-Rab5DN, and Hml>UAS-Rab11DN larvae …
Figure 8—figure supplement 1
The dFOXO levels and autophagy activity were enhanced after the inactivation of Rab5 or Rab11 in hemocytes.
(A and B) Overexpression of dFOXO in hemocytes using pxn>GFP induced lamellocyte formation. (C–F) The dFOXO (green) levels were increased in circulating hemocytes from Hml>UAS-Rab5DN, Hml>UAS-Rab11DN…
Figure 8—figure supplement 2
Repressing JNK signaling restored the increased lamellocyte count in Hml>UAS-Rab5/11DN larvae.
(A–F) The increased lamellocyte counts were restored in Hml>UAS-Rab5/11DN>UAS-bsk RNAi (B, E) and Hml>UAS-Rab5/11DN>UAS-bskDN (C, F) circulating hemocytes. The quantification of lamellocytes is …
Figure 9
The lamellocyte formation upon the loss of Rab5 or Rab11 was autophagy-dependent.
(A–I, U–X) The lamellocyte count was determined in circulating hemocytes by anti-L1 immunostaining. Overexpression of Atg1 in two sources of UAS-Atg1 flies resulted in massive lamellocyte production …
Figure 10
Schematic diagram of lymph gland morphology and the regulatory network of signaling pathways upon Rab5/Rab11 inactivation.
(A) Schematic diagram of lymph glands from control and Rab5/Rab11-deficient third instar larvae. When Rab5 or Rab11 was downregulated in the cortical zone (CZ), the formation of many premature …
Author response image 1
Author response image 2
