Gcn5 is expressed in all compartments of the Drosophila larval LG

Cartoon of the third instar larval LG, indicating the anterior primary lobes and the posterior lobes (A). The primary lobe of the LG depicting different subpopulations: the Posterior Signaling Centre (PSC: blue) functioning as the niche, Medullary zone (MZ: green) houses the progenitors, Intermediate progenitor zone (IZ: yellow) which contains the cells transitioning from progenitors to specific lineages, and the Cortical zone (CZ: brown) containing the differentiated hemocytes population – plasmatocytes and crystal cells (B). Gcn5 expression in different compartments of lymph gland (C-F’). Gcn5 (red) expression in the - Posterior Signaling Centre (PSC) using Collier- GFP (green) (C-C’, E-E’), Medullary zone prohemocytes using Dome-GFP (green) (D-D’, F-F’). Nuclei are stained with DAPI (Blue). Scale Bar: 50µm (C-D’), 30µm (E-F’).

Whole animal gcn5 mutants show defective LG homeostasis.

Posterior Signaling Center (PSC) cell numbers marked by Antennapedia (red) in gcn5[C137Y/C137Y] (B-B’), gcn5[E333st/E333st] (C-C’) and gcn5[E333st/C137Y] (D-D’’) as compared to wildtype (A-A’). Graphical representation of PSC cell numbers of the gcn5 mutants as compared to the wildtype (E), n=24 for wildtype, n=37 for gcn5[C137Y/C137Y], n=29 for gcn5[E333st/E333st] and n=20 for gcn5[E333st/C137Y] for PSC cell number quantification. Plasmatocyte differentiation was marked by P1 (red) in gcn5[C137Y/C137Y] (G-G’), gcn5[E333st/E333st] (H-H’) and gcn5[E333st/C137Y] (I-I’) as compared to the wildtype (F-F’). Graphical representation of plasmatocyte differentiation index of the gcn5 mutants compared to wildtype (J), n=20 for wildtype, n=29 for gcn5[C137Y/C137Y], n=33 for gcn5[E333st/E333st] and n=26 for gcn5[E333st/C137Y] for quantification. Crystal cell differentiation marked by Hnt (red) in gcn5[C137Y/C137Y] (L-L’), gcn5[E333st/E333st] (M-M’) and gcn5[E333st/C137Y] (N-N’) as compared to wildtype (K-K’). Graphical representation of crystal cell differentiation index for gcn5 mutants compared to wildtype (O), n=34 for wildtype, n=29 for gcn5[C137Y/C137Y], n=38 for gcn5[E333st/E333st] and n=28 for gcn5[E333st/C137Y] for quantification. Cells undergoing DNA damage marked by γH2AX (red) in gcn5[C137Y/C137Y] (Q-Q’), gcn5[E333st/E333st] (R-R’) and gcn5[E333st/C137Y] (S-S’) as compared to wildtype (P-P’). Graphical representation of γH2AX positive cells undergoing DNA damage in the gcn5 mutants compared to the wildtype (T), n=24 for wildtype, n=54 for gcn5[C137YC137Y], n=41 for gcn5[E333st/E333st]and n=31 for gcn5[E333st/C137Y] for quantification. Nuclei are stained with DAPI (Blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-S’). Nuclei are stained with DAPI (blue). Scale Bar: 50µm (A-S’).

Modulation of Gcn5 levels in the prohemocytes alters blood cell homeostasis in the lymph gland

Posterior Signalling Center (PSC) population marked by Antennapedia (red) upon Dome-Gal4 mediated knockdown (B-B’) or over-expression (C-C’) of Gcn5 as compared to wildtype (A-A’). Plasmatocyte differentiation marked by P1 (red) upon Dome-Gal4 mediated knockdown (E-E’) or over-expression (F-F’) of Gcn5 as compared to wildtype (D-D’). Crystal cell differentiation marked by Hnt (red) upon Dome-Gal4 mediated knockdown (H-H’) or over-expression (I-I’) of Gcn5 as compared to wild type (G-G’). Cells undergoing DNA damage marked by γH2AX (red) upon Dome-Gal4 mediated knockdown (K-K’) or over-expression (L-L’) of Gcn5 as compared to wild type (J-J’). Graphical representation of PSC numbers upon modulation of Gcn5 levels in the Dome positive population compared to wildtype (M), n=28 for wildtype, n=28 for Gcn5 knockdown, and n=20 for Gcn5 over-expression. Graphical representation of prohemocyte index upon modulation of Gcn5 levels in dome population compared to wildtype (N), n=30 for wildtype, n=30 for Gcn5 knockdown, and n=30 for Gcn5 over-expression. Graphical representation of plasmatocyte differentiation index upon modulation of Gcn5 levels in dome population compared to wildtype (O), n=35 for wildtype, n=23 for Gcn5 knockdown, and n=25 for Gcn5 over-expression. Graphical representation of Crystal cell differentiation index upon modulation of Gcn5 levels in the dome population compared to wildtype (P), n=36 for wildtype, n=26 for Gcn5 knockdown, and n=34 for Gcn5 over-expression. Graphical representation of γH2AX positive cells upon modulation of Gcn5 levels in the dome population compared to wildtype (Q), n=33 for wildtype, n=27 for Gcn5 knockdown, and n=38 for Gcn5 over-expression. Nuclei are stained with DAPI (blue). Dome>GFP positive population (Green) indicates the expression domain of Dome-Gal4 activity in the LG. n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the Lymph Gland. Values are mean ± SD, and asterisk marks statistically significant differences (**p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-L’).

Prohemocyte-specific expression of Gcn5 domain deletion constructs affects LG homeostasis.

Schematic of the SAGA HAT complex containing different modules – Sgf29, Ada2, Gcn5, and Ada3. Gcn5 is an 813 aa protein in Drosophila containing the Pcaf homology domain, HAT domain, Ada domain, and Bromodomain (A). Posterior Signalling Center (PSC) numbers marked by Antennapedia (red) upon expression of different Gcn5 domain deletion constructs in the tep-GFP (green) population using tep4-Gal4 (C-F), compared to wildtype (B). Progenitor index marked by tep (green) upon expression of different Gcn5 domain deletion constructs (C’-F’), compared to wildtype (B’). Plasmatocyte differentiation marked by P1 (red) upon expression of different Gcn5 domain deletion constructs in the tep-GFP population using tep4-Gal4 (green) (C”-F”), compared to wildtype (B”). Crystal cell differentiation marked by Hnt (red) upon expression of different Gcn5 domain deleted constructs in the tep-GFP population using tep4-Gal4 (green) (C”’-F”’), compared to wildtype (B”’). Graphical representation of PSC numbers upon expression of Gcn5 domain deletion constructs, compared to wildtype (G), n=40 for wildtype, n=29 for Gcn5ΔHAT expression, n=35 for Gcn5ΔPcaf expression, n=33 for Gcn5ΔBromo expression, and n=26 for Gcn5ΔAda expression in the tep population. Graphical representation of Prohemocyte index upon expression of Gcn5 domain deletion constructs, compared to wildtype (H), n=24 for wildtype, n=24 for Gcn5ΔHAT expression, n=34 for Gcn5ΔPcaf expression, n=24 for Gcn5ΔBromo expression, and n=24 for Gcn5ΔAda expression in the tep population. Graphical representation of Plasmatocyte differentiation index upon expression of Gcn5 domain deleted constructs, compared to wildtype (I), n=38 for wildtype, n=30 for Gcn5ΔHAT expression, n=29 for Gcn5ΔPcaf expression, n=27 for Gcn5ΔBromo expression, and n=27 for Gcn5ΔAda expression in the tep population. Graphical representation of Crystal cell numbers upon expression of Gcn5 domain deleted constructs, compared to wildtype (J), n=23 for wildtype, n=35 for Gcn5ΔHAT expression, n=26 for Gcn5ΔPcaf expression, n=26 for Gcn5ΔBromo expression, and n=26 for Gcn5ΔAda expression in the tep population. Graphical representation of Crystal cell Differentiation index upon expression of Gcn5 domain deleted constructs, compared to wildtype (K), n=20 for wildtype, n=34 for Gcn5ΔHAT expression, n=25 for Gcn5ΔPcaf expression, n=15 for Gcn5ΔBromo expression, and n=27 for Gcn5ΔAda expression in the tep population. Nuclei are stained with DAPI (blue). n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (B-F’’’).

Autophagic flux in Drosophila blood cells is negatively regulated by Gcn5

p62 (red) labeling the autophagy adaptor protein upon Hml-Gal4 mediated Gcn5 knockdown (B), or overexpression (C) as compared to wildtype (A). Atg8 (red), the autophagosomal marker upon Hml-Gal4 mediated Gcn5 knockdown (B’) or over-expression (C’) as compared to wildtype (A’). Nuclei were stained with DAPI (blue). Graphical representation of the p62 positive puncta/cell (D). Graphical representation of Atg8 positive puncta/cell (E). Values are mean ± SD, and asterisk marks statistically significant differences (***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 20µm (A-E’).

Genetic and chemical ablation of autophagy leads to aberrant blood cell differentiation.

Plasmatocyte differentiation marked by P1 (red) upon tep4-Gal4 mediated knockdown of TFEB (C-C’), Atg8a (E-E’), Atg5 (G-G’), or Atg18 (I-I’) in the tep population (green) as compared to wildtype (A-A’). Crystal cell differentiation marked by Hnt (red) upon tep4-Gal4 mediated knockdown of TFEB (D-D’), Atg8a (F-F’), Atg5 (H-H’), or Atg18 (J-J’) in the tep population (green) as compared to wildtype (B-B’). Graphical representation of Plasmatocyte differentiation index upon knockdown of TFEB (n=33), Atg8a (n=28), Atg5 (n=24), or Atg18 (n=22) in the tep population, compared to the wildtype (n=22) (K). Graphical representation of Crystal cell differentiation index upon knockdown of TFEB (n=22), Atg8a (n=35), Atg5 (n=24), or Atg18 (n=22) in the tep population, compared to the wildtype (n=19) (L). Plasmatocyte differentiation(red) upon Chloroquine treatment (N) as compared to control (M). Crystal cell differentiation upon Chloroquine treatment (P) as compared to control (O). Graphical representation of Plasmatocyte Differentiation Index upon Chloroquine treatment (n=30), compared to control (n=25) (Q). Graphical representation of Crystal Cell differentiation index upon Chloroquine treatment (n=26), compared to control (n=23) (R). Nuclei are stained with DAPI (blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-P).

Modulation of mTORC1 activity regulates blood cell differentiation in the Lymph gland.

Plasmatocyte differentiation marked by P1 (red) upon 3BDO treatment (B-B’) as compared to control (A-A’). Crystal cell differentiation marked by Hnt (red) upon 3BDO treatment (D-D’) compared to control (C-C’). Graphical representation of Plasmatocyte Differentiation Index upon 3BDO treatment (n=28) compared to control (n=24) (E). Graphical representation of Crystal cell differentiation index upon 3BDO treatment (n=19) compared to control (n=21) (F). Plasmatocyte differentiation marked by P1 (red) upon Rapamycin treatment (H-H’) compared to control (G-G’). Crystal cell differentiation marked by Hnt (red) upon Rapamycin treatment (J-J’) compared to control (I-I’). Graphical representation of Plasmatocyte Differentiation Index upon Rapamycin treatment (n=28) compared to control (n=20) (K). Graphical representation of Crystal cell differentiation index upon Rapamycin treatment (n=23) compared to control (n=22) (L). Immunoblot showing GCN5 (93 kDa) protein levels in different diet conditions – fed, starved, and high-fat diet (M). Immunoblot showing GCN5 (93 kDa) protein levels upon Rapamycin treatment compared to wildtype (N). β-actin (42kDa) was used as the loading control. Nuclei are stained with DAPI (blue). Quantification of GCN5 protein levels in the fed, starved and High Fat diet scenario (O). Quantification of GCN5 protein levels in the wildtype as compared to rapamycin treatment (P). n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-J’).

mTORC1 can override the effect of Gcn5 level modulation over autophagy.

Plasmatocyte differentiation marked by P1 (red) upon 3BDO treatment in tep4-Gal4 mediated (green) Gcn5 knockdown background (B-B’) compared to control treatment in the same genetic background (A-A’). Crystal cell differentiation marked by Hnt (red) upon 3BDO treatment in the tep-mediated (green) Gcn5 knockdown background (D-D’) compared to control treatment in the same genetic background (C-C’). Plasmatocyte differentiation marked by P1 (red) upon Rapamycin treatment in Dome-mediated (green) Gcn5 over-expression background (F-F’) compared to control treatment in the same genetic background (E-E’). Crystal cell differentiation marked by Hnt (red) upon Rapamycin treatment in Dome-Gal4 mediated (green) Gcn5 over-expression background (H-H’) as compared to control treatment in the same genetic background (G-G’). Graphical representation of Plasmatocyte Differentiation Index upon 3BDO treatment (n=21) and control treatment (n=20) in the tep-mediated Gcn5 knockdown background (I). Graphical representation of Crystal cell differentiation index upon 3BDO treatment (n=19) and control treatment (n=21) in the tep-mediated Gcn5 knockdown background (J). Graphical representation of Plasmatocyte Differentiation Index upon Rapamycin treatment (n=27) and control treatment (n=21) in the Dome-mediated Gcn5 over-expression background (K). Graphical representation of Crystal Cell differentiation index upon Rapamycin treatment (n=43) and control treatment (n=21) in the Dome-mediated Gcn5 over-expression background (L). Nuclei are stained with DAPI (blue). n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (**p<0.01; ***p<0.001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-H’).

Gcn5-mTORC1 mediated autophagy regulation controls blood cell homeostasis.

A cartoon summarizing how Gcn5-mTORC1-signaling axis regulates autophagy to control blood cell homeostasis in the Drosophila LG. Created in BioRender.com.