1. Developmental Biology

Fatty acid β-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila

  1. Satish Kumar Tiwari
  2. Ashish Ganeshlalji Toshniwal
  3. Sudip Mandal
  4. Lolitika Mandal  Is a corresponding author
  1. Developmental Genetics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, India
  2. Molecular Cell and Developmental Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, India
https://doi.org/10.7554/eLife.53247
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Cite this article
  1. Satish Kumar Tiwari
  2. Ashish Ganeshlalji Toshniwal
  3. Sudip Mandal
  4. Lolitika Mandal
(2020)
Fatty acid β-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila
eLife 9:e53247.
https://doi.org/10.7554/eLife.53247
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16 figures, 1 video, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
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FAO genes are expressed in hemocyte progenitors of lymph gland.

Age and genotype of the larvae are mentioned in respective panels. (A–A') Model of lymph gland of third early and third late instar stages depicting anterior primary lobes and posterior lobes. (A’). Primary lobe showing different subpopulations: Pvf2+ Dome- pre-progenitor, Dome+ progenitors and Dome+ Pxn+ Hml+ Intermediate progenitors (IPs) in early third and late third instar larval stages. Progenitors are present in the core of the primary lobe called the medullary zone (MZ), and differentiated cells (Plasmatocytes and crystal cells) are present in the outer zone called cortical zone (CZ). (B–B'') Expression of Hnf4-GAL4 > UAS-GFP in Pvf2+ pre-progenitors of the early third instar lymph gland. (C–C'') Expression of Hnf4-GAL4 > UAS-GFP in Dome+ progenitors and Dome+ Hml+ Intermediate progenitors (IPs) shown in dome-MESO-EBFP2/+; Hml-DsRed/+ genotype. (D). Quantitative analysis of B–C''- reveals that the Dome+ progenitors have higher levels of Hnf4 expression. p-Value for Hnf4-GAL4 > UAS-GFP expression in Dome+ progenitors is 9.55 × 10−9 compared to control Pvf2+ pre-progenitors. p-Value for Hnf4-GAL4 > UAS-GFP expression for Dome+ Hml+ IPs is 7.34 × 10−3 compared to control Pvf2+ pre-progenitors. (E–E'') Nile red staining in Pvf2+ pre-progenitors of early third instar stage lymph gland. (F–F'') Expression of Nile red in Dome+ progenitors and Dome+ Hml+ Intermediate progenitors (IPs) shown in dome-MESO-EBFP2/+; Hml-DsRed/+ genotype (Dome+: blue, Hml+: green). (G). Quantitative analysis of E–F'' shows higher levels of neutral lipids in the Dome+ progenitors. Compared to control Pvf2+ pre-progenitors, p-Values for nile red expression in Dome+ progenitors is 1.39 × 10−7 and Dome+ Hml+ IPs pre-progenitors is 9.11 × 10−3. Five optical sections of 1 µm thickness from the middle of the Z-stack were merged into a single section. (H) Schematic representation of FAO and the constituent enzymes. (I) Transcripts of β-oxidation enzymes, whd, Mcad, Mtpα, scully, Mtpβ, and yip2 (Refer to H) can be detected in the third late instar lymph gland. eL3 and lL3 refer to the early and late instar lymph glands. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 1—source data 1

Contains numerical data plotted in Figure 1D and G.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
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Temporal analysis of pre-progenitors in the lymph gland and mitochondrial analysis in Dome+ progenitors.

Age and genotype of the larvae are mentioned in respective panels. (A–B) Expression of Pvf2 in Dome- pre-progenitors in third early instar lymph gland (A) late third instar lymph glands lack Pvf2 expression as well as Dome- pre-progenitors (B). (C–C') Super-resolution image of mitochondria in Dome+ progenitors visualized by dome-GAL4, UAS::mCD8RFP/+; UAS-mito-HA-GFP. Five optical sections of 0.33 µm thickness from the middle of the Z -stack were merged into a single section. (C') is the higher magnification of region of interest (ROI) from C). (D–D') Image of mitochondria in Hml+ differentiated hemocytes and Hml- progenitors of the lymph gland visualized by Streptavidin-Cy3. (D') is the higher magnification of ROI from D. eL3 and lL3 refer to the early and late instar lymph glands. Scale bar: 20 µm.

Figure 1—figure supplement 2
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FAO components are expressed in hemocyte progenitor subpopulations in the lymph gland.

Age and genotype of the larvae are mentioned in respective panels. (A–A'') LipidTOX labeling neutral lipids in Pvf2+ pre-progenitors in the early third instar lymph gland. (B–B''). LipidTOX labeling in Dome+ progenitors and Dome+ Hml+ Intermediate progenitors (IPs) shown in dome-MESO-EBFP2/+; Hml-DsRed/+ genotype. (C). Quantitative analysis of results from A–B'' showing a higher level of neutral lipids in Dome+ progenitors. Compared to control Pvf2+ pre-progenitors, p-Values for lipidTOX labeling in Dome+ progenitors is 6.20 × 10−6 and Dome+ Hml+ IPs is 6.84 × 10−5. Five optical sections of 1 µm thickness from the middle of a the Z-stack were merged into a single section. (D–D'') Expression of acyl-CoA dehydrogenase, CG3902-YFP expression in Pvf2+ pre-progenitors in the early third instar lymph gland. (E–E''). CG3902-YFP expression in Dome+ progenitors and Dome+ Hml+ Intermediate progenitors (IPs) shown in dome-MESO-EBFP2/+; Hml-DsRed/+ genotype. (F). Quantitative analysis of results from D–E'' reveals high levels of CG3902-YFP expression in the Dome+ progenitors. p-value for CG3902-YFP labeling in Dome+ progenitors is 6.02 × 10−5 compared to control Pvf2+ pre-progenitors and for Dome+ Hml+ IPs is 2.70 × 10−3. Five optical sections of 1 µm thickness from the middle of the Z-stack were merged into a single section. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 1—figure supplement 2—source data 1

Contains numerical data plotted in Figure 1—figure supplement 2C and F.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 2 supplements
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Loss of fatty acid β-oxidation affected differentiation of hemocyte progenitors of the lymph gland.

(A) Schematic representation of fatty acid β-oxidation within the mitochondria of a cell. (B–D) Compared to control (B) decrease in differentiation (red, reported by P1 immunostaining) and increment in progenitor number (dome > GFP) is observed in the lymph gland of a homozygous null allele of whd (C). (D) Quantitative analysis of B–C reveals a significant increment in the number of Dome+ progenitors. p-Value for dome-GAL4, UAS-GFP; whd1/whd1=2.67×10−10 compared to control. (E–G) Compared to control (E) decrease in crystal cell number (red, reported by proPO immunostaining) and increment in the progenitor cell population (dome > GFP) is observed in the lymph gland of the homozygous null allele of whd (F). (G). Quantitative analysis of results from E–F shows a significant drop in the number of crystal cells. p-Value for dome-GAL4, UAS-GFP; whd1/whd1=4.38×10−7 compared to control. (H–I'') The hemocyte progenitor subpopulation dynamics (red, reported by Pxn immunostaining and green marking dome > GFP) of Dome+ progenitors and Dome+ Pxn+ (IPs) in the late third instar lymph gland of control (H–H'') and homozygous null allele of whd (I–I''). (J–S) Spatio-temporal analysis of hemocyte progenitor subpopulations of Dome- pre-progenitors, Dome+ progenitors, and Dome+ Pxn+ (IPs) (red, reported by Pxn immunostaining and green marking dome > GFP) observed in the lymph gland of control (J–N) and homozygous null allele of whd (O–S). Insets in K, L, and M show pre-progenitors, progenitors and intermediate progenitors respectively in control and inset in Q shows abundant progenitors in the homozygous null allele of whd. (T–X) Compared to control (T) decrease in differentiation (red, reported by P1 immunostaining) and increase in progenitor number (dome > GFP) is observed in lymph gland upon progenitor specific RNAi based down-regulation of whd (U) CRISPR-Cas9 based knock-out of whd (V) and miRNA based knockdown of Hnf4 (W). (X) Quantitative analysis of the results from T–W, illustrating the significant increase in Dome+ progenitors upon targeted loss of FAO. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-whd RNAi = 2.84×10−15 compared to control. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-dCas9; U-6: sgRNA-whd = 3.84×10−19. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-Hnf4.miRNA =6.04×10−14. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 2—source data 1

Contains numerical data plotted in Figure 2D,G and X.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
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Fatty acid β-oxidation is essential for lymph gland progenitor differentiation.

(A) Quantitative analysis of results from Figure 2H–I''. p-Value for progenitors of dome-GAL4, UAS-GFP; whd1/whd1 = 5.7×10−14 compared to control. p-Value for Intermediate progenitors of dome-GAL4, UAS-GFP; whd1/whd1 = 1.33×10−6 compared to control. (B) Quantitative analysis of results from Figure 2J–N. p-Value for pre-progenitors of eL3 (54 hr AEH) dome-GAL4, UAS-GFP = 5.95×10−2 compared to pre-progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for progenitors of eL3 (54 hr AEH) dome-GAL4, UAS-GFP = 2.34×10−1 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for IPs of eL3 (54 hr AEH) dome-GAL4, UAS-GFP = 1.7×10−2 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for pre-progenitors of eL3 (70 hr AEH) dome-GAL4, UAS-GFP = 3.2×10−2 compared to pre-progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for progenitors of eL3 (70 hr AEH) dome-GAL4, UAS-GFP = 2.74×10−6 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for IPs of eL3 (70 hr AEH) dome-GAL4, UAS-GFP = 4.5×10−3 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for progenitors of mL3 (84 hr AEH) dome-GAL4, UAS-GFP = 3.2×10−9 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for IPs of mL3 (84 hr AEH) dome-GAL4, UAS-GFP = 4.999×10−6 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for progenitors of lL3 (96 hr AEH) dome-GAL4, UAS-GFP = 6.117×10−12 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. p-Value for IPs of lL3 (96 hr AEH) dome-GAL4, UAS-GFP = 2.8×10−11 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP. (C) Quantitative analysis of results from Figure 2O–S. p-Value for pre-progenitors of eL3 (54 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 3.3×10−2 compared to pre-progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for progenitors of eL3 (54 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 6.3×10−3 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for IPs of eL3 (54 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 2.46×10−4 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for pre-progenitors of eL3 (70 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 7.85×10−7 compared to pre-progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for progenitors of eL3 (70 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 1.78×10−1 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for IPs of eL3 (70 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 1.8×10−1 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for progenitors of mL3 (84 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 4.78×10−1 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for IPs of eL3 (84 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 1.75×10−8 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for progenitors of lL3 (96 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 4.47×10−5 compared to progenitors of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. p-Value for IPs of lL3 (96 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1 = 5.25×10−8 compared to IPs of mL2 (36 hr AEH) dome-GAL4, UAS-GFP; whd1/whd1. (D) Schematic representation of the timeline of experiments involving dome-GAL4, UAS-GFP; tubGAL80ts20 mediated targeted knockdown of specific gene expression for this study. (E–G) Compared to control (E), the status of differentiation {red, CZ indicated by Pxn and progenitors of MZ marked by shg/DE-cadherin (green)} of hemocyte progenitor are compromised upon down-regulation of whd function. Independent progenitor specific driver TepIV-GAL4, along with a UAS-whd RNAiKK obtained from VDRC endorses our previous findings (F). (G). Quantitative analysis of the results from E–G. p-Value for TepIV-GAL4 >UAS whd RNAi = 1.43×10−10 compared to control. (H–M) Compared to control (H), the status of differentiation {red, CZ indicated by Pxn and progenitors of MZ marked by shg/DE-cadherin (green)} of hemocyte progenitor are compromised in heteroallelic null loss of function of Hnf4 (I), homozygous null loss of function alleles of Mtpα (J), Mtpβ (K), and whd (L). (M) Quantitative analysis of results from H–L depicting less differentiation in FAO null. p-Value for Hnf4Δ33/Hnf4Δ17 = 9.55×10−10 and p-Value for MtpαKO/MtpαKO =1.01×10−09 and p-value for MtpβKO/MtpβKO =6.37×10−10 and p-Value for whd1/whd1 = 9.33×10−10 compared to control. (N–Q) Blocking Fatty Acid β-oxidation by feeding Etomoxir (O) and Mildronate (P) decreases progenitor differentiation compared to control dome > GFP (N). (Q). Quantitative analysis of the results from N–P) shows a drop in differentiation upon pharmaceutical inhibition of FAO. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 treated with Etomoxir = 6.803×10−14 compared to control and dome-GAL4, UAS-GFP; tubGAL80ts20 treated with Mildronate = 1.17×10−13. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 2—figure supplement 1—source data 1

Contains numerical data plotted in Figure 2—figure supplement 1A,B,C,G,M and Q.

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Figure 2—figure supplement 2
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Model depicting the posible role of Hnf4 and FAO in hemocyte progenitor differentiation.

Hnf4 is a transcription factor implicated in mobilization of fatty acids and its oxidation in Drosophila. Hnf4 as well as members of FAO expresses in the hemocyte progenitors, loss of either one of them affects differentiation.

Figure 3 with 1 supplement
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Loss of fatty acid β-oxidation causes an increase in proliferation of hemocyte progenitors of the lymph gland primary lobe.

Genotype of the larvae are mentioned in respective panels. (A–C) The difference in proliferation status (reported by EdU incorporation) in the lymph gland of third late instar control larvae (A–A') compared to whd null mutant (B–B'). (C). Quantitative analysis of the results from A–B' illustrates a significant increase in proliferation in whd1 Dome+ progenitors. p-Value for dome-GAL4, UAS-GFP; whd1/whd1=1.71×10−6 compared to control. (D–F) Difference in cell cycle status (reported by Fly-FUCCI) in the lymph gland of third late instar control larvae (D) compared to the progenitor-specific RNAi-based down-regulation of whd (E). (D'–E'): Pie chart depicting the fraction of G1 (green), S(red), and G2/M (yellow) progenitors in J–K. (F) Quantitative analysis of the results from D–E. p-Value for red cells in dome-GAL4, UAS-Fly-FUCCI; UAS-whd RNAi = 1.41×10−11, p-Value for green cells in dome-GAL4, UAS-Fly-FUCCI; UAS-whd RNAi = 2×10−4, p-Value for yellow cells in dome-GAL4, UAS-Fly-FUCCI; UAS-whd RNAi = 1.5×10−5 compared to control. ns.=not significant, Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 3—source data 1

Contains numerical data plotted in Figure 3C,Dʹ, Eʹ and F.

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Figure 3—figure supplement 1
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Loss of fatty acid β-oxidation caused higher redox levels and an increase in maintenance factor of hemocyte progenitors of the lymph gland.

Genotype of the larvae are mentioned in respective panels. (A–C) Elevated levels of Ci155 in the lymph gland of whd1 homozygous mutants (B) compared to control (A). (A'' and B''): Heat map is representing the intensity of Ci155 in the lymph gland of whd1 homozygous mutants (Bʹ) compared to control (Aʹ). (C). Quantitative analysis of the results from A–B reveals that FAO loss results in high Ci155 expression.p-Value for dome-GAL4, UAS-GFP; whd1/whd1=2.78×10−8 compared to control. (D–F) DHE (ROS) level is elevated in lymph gland in whd1 homozygous mutants (E) compared to control (D). (D'' and E''): Heat map is representing the intensity of DHE in the lymph gland of whd1 homozygous mutants (Eʹ) compared to control (Dʹ). (F). Quantitative analysis of the results from D–E. p-Value for dome-GAL4, UAS-GFP; whd1/whd1=1.64×10−7 compared to control. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 3—figure supplement 1—source data 1

Contains numerical data plotted in Figure 3—figure supplement 1C and F.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
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FAO upregulation results in precocious differentiation and G2 arrest in hemocyte progenitors.

Age and genotype of the larvae are mentioned in respective panels. (A–C) Comparison of differentiation (marked by P1) levels in dome > GFP lymph gland of control (A) and L-carnitine supplemented (B) larvae. (C) Quantitative analysis of results from A–B showing increased differentiation upon L-carnitine supplementation. p-Value for dome-GAL4, UAS-GFP = 2.37×10−10 supplemented with L-carnitine compared to control. (D–F) Comparison of differentiation (marked by P1) levels in dome > GFP lymph gland of control (D) and CRISPR-Cas9 mediated whd overexpression (E) in larval hemocyte progenitors. (F) Quantitative analysis of result from D–E depicting a significant increase in differentiation upon overexpression of whd. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-whd-OE = 5.82×10−12 compared to control. (G–H) Comparison of differentiation (marked by P1) levels in dome > GFP lymph gland secondary lobes (marked by the white dotted boundary) of control (G) and CRISPR-Cas mediated whd overexpression (H) in larval hemocyte progenitors. (I–K) Proliferation status (marked by EdU) in third early instar hemocyte progenitors (dome > GFP) of control (I–I') and L-carnitine supplemented (J–J') larvae. (K) Quantitative analysis of results from I–J' reveals a decline in the number of proliferating Dome+ progenitors upon FAO overexpression. p-Value for dome-GAL4, UAS-GFP fed with L-carnitine = 2.87×10−5 compared to control. (L–N) The decline in proliferation status (marked by EdU) in third early instar hemocyte progenitors of CRISPR-Cas9 mediated whd overexpression (M–M') compared to (dome > GFP) of control (L–L'). (N) Quantitative analysis of result from L–M'. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > whd-OE=2.28×10−9 compared to control. (O–P) Alteration in cell cycle status (reported by Fly-FUCCI) in the lymph gland of third late instar larvae grown in L-carnitine supplemented food (P) compared to control (dome > UAS-FUCCI) (O). (O'–P'): Pie chart depicting the fraction of G1 (green), S (red), and G2/M (yellow) progenitors in (O-P). (Q) Quantitative analysis of the results from O–P, illustrating the increase in G2-M upon FAO overexpression. p-Value for red cells in L-carnitine supplemented dome-GAL4, UAS-FUCCI = 1.78×10−7, p-Value for green cells in L-carnitine supplemented dome-GAL4, UAS-FUCCI; UAS-whd RNAi = 2.16×10−1. p-Value for yellow cells in L-carnitine supplemented dome-GAL4, UAS- FUCCI; UAS-whd RNAi = 1.71×10−5 compared to control. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 4—source data 1

Contains numerical data plotted in Figure 4C,F,K,N,Oʹ, Pʹ, and Q.

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Figure 4—figure supplement 1
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L-carnitine supplementation rescues differentiation defect.

Age and genotype of the larvae are mentioned in respective panels. (A–E) Status of progenitors (dome > GFP) and differentiated hemocytes P1 in control (A), whd1 homozygous (B), whd1 heterozygous (C) and whd1 hererozygous fed on L- carnitine (D). (E). Quantitative analysis of the results from A–D. p-Value for dome-GAL4, UAS-GFP; whd1/whd1 = 2.08×10−10 compared to control dome-GAL4, UAS-GFP. p-Value for dome-GAL4, UAS-GFP; whd1/+ = 1.18×10−9 compared to control dome-GAL4, UAS-GFP. p-Value for dome-GAL4, UAS-GFP; whd1/+ = 1.27×10−7 fed with L-carnitine compared to non-fed dome-GAL4, UAS-GFP; whd1/+. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 4—figure supplement 1—source data 1

Contains numerical data plotted in Figure 4—figure supplement 1E.

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Figure 5 with 1 supplement
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FAO loss in hemocyte progenitors led to sustained glycolysis.

(A) ATP levels in control and whd1/whd1 whole larvae. p-Value of whd1/whd1compared to control = 5.327×10−2. (B–D) Glucose incorporation (marked by 2-NBDG uptake) levels in control dome-MESO-EBFP2/+ (B–B'') and dome-MESO-EBFP2/+; whd1/whd1 (C–C'') lymph glands. (D). Quantitative analysis of results from B–C demonstrating a significant increase in glucose uptake in the whd1/whd1 progenitors. p-Value for dome-MESO-EBFP2/+; whd1/whd1 = 6.09×10-7 compared to control. (E–F) Increased lactate dehydrogenase in-vivo enzymatic staining assay of whd1/whd1 lymph gland (F) compared to control (E). (G) Fold change in the level of Hex-A mRNA expression in control w1118 and whd1/whd1 lymph glands. p-Value of whd1/whd1 = 6.379×10−3 compared to control. (H) Fold change in the level of Pfk mRNA expression in control w1118 and whd1/whd1 lymph glands. p-Value of whd1/whd1 = 3.739×10−3 compared to control. (I–M) Proliferation status (marked by EdU) in control dome > GFP (I–I'), dome > GFP; whd1/whd1 (J–J'), 2-DG fed dome > GFP; whd1/whd1 (K–K') and dome > GFP; whd1/whd1; UAS-Glut1 RNAi (L–L') lymph glands. (M). Quantitative analysis of results from I–L'. p-Value for dome > GFP; whd1/whd1 = 4.37×10−7 compared to control and p-value for dome > GFP; whd1/whd1 = 3.25×10−7 fed with 2-DG compared to non-fed dome > GFP; whd1/whd1. p-Value for dome > GFP; whd1/whd1; UAS-Glut1 RNAi = 4.53×10−7 compared to non-fed dome > GFP; whd1/whd1. (M–P) Comparison of differentiation (marked by P1) levels in control dome > GFP (M), dome > GFP; whd1/whd1 (N) and 2-DG fed dome > GFP; whd1/whd1 (O) lymph glands. (P). Quantitative analysis of results from M–O show decline in proliferation upon 2-DG feeding. p-Value for dome > GFP; whd1/whd1 = 4.43×10−11 compared to control and p-Value for dome > GFP; whd1/whd1 = 8.6×10−2 fed with 2-DG compared to non-fed dome > GFP; whd1/whd1. p-Value for dome > GFP; whd1/whd1; UAS-Glut1 RNAi = 5.9×10−2 compared to non-fed dome > GFP; whd1/whd1. n.s. = not significant. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 5—source data 1

Contains numerical data plotted in Figure 5A,D,G,H,M and R.

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Figure 5—figure supplement 1
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2-NBDG assay in the lymph gland primary lobe.

(A–B) Glucose incorporation (marked by 2-NBDG uptake) levels in control dome-MESO-EBFP2/+; Hml-DsRed lymph glands (A–A'''). (B) Quantitative analysis of results from A–A'''. p-Value for IPs = 3.64×10−4 compared to progenitors. p-Value for Cortical Zone Hml+=1.83×10−6 compared to progenitors. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 5—figure supplement 1—source data 1

Contains numerical data plotted in Figure 5—figure supplement 1B.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 1 supplement
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Hemocyte progenitors of HAT and FAO loss of function exhibits altered histone acetylation.

(A–H) Comparison of differentiation (marked by P1) levels in dome > GFP lymph gland of control (A) with progenitor-specific downregulation of (B) chm, (C) Gcn5, (D) AcCoAS and (E) whd1/whd1, (F) transheterozygote of whd and ATPCL (whd1/ATPCL01466) and transheterozygote of whd and sea (whd1/seaEPEP3364) (G). (H) Quantitative analyses of the results from A–G. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-chm RNAi = 2.267×10−15 compared to control. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-Gcn5 RNAi = 1.990×10−14 compared to control. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-AcCoAS RNAi = 2.601×10−15 compared to control. p-Value for dome-GAL4, UAS-GFP; whd1/whd1 = 1.400×10−15 compared to control. p-Value for dome-GAL4, UAS-GFP; whd1/ATPCL01466 = 6.835×10−3 compared to control dome-GAL4, UAS-GFP; whd1/whd1. p-Value for dome-GAL4, UAS-GFP; whd1/seaEP3364 = 2.974×10−2 compared to control dome-GAL4, UAS-GFP; whd1/whd1. (I–J) Western blot analysis of H3K9 acetylation level in control OreR and whd1/whd1 larvae with H3 as a loading control (I). (J). Quantitative analysis of H3K9 acetylation level in I. p-Value for whd1/whd1 = 8.056×10−3 compared to control OreR. (K–P) Clonal analysis of histone acetylation in the GFP-positive hs-Flp/Ay-GAL4 based clonal patches (GFP indicates cells where the whd function is knocked down). Immunostaining with H3 (K–L), H3K9 acetylation (M–N), and H4 pan acetylation (O–P) antibodies. (L). Quantitative analyses of H3 acetylation level in K–K'''. p-Value for hs-Flp/Ay-GAL4. UAS-GFP; UAS-whd RNAi = 8.188×10−1 compared to control. (N). Quantitative analysis of H3K9 acetylation level in (M–M'''). p-Value for hsFlp/+; Ay-GAL4. UAS-GFP; UAS-whd RNAi = 2.238×10−12 compared to control. (P). Quantitative analysis of H4 acetylation level in O–O'''. p-Value for hsFlp/Ay-GAL4. UAS-GFP, UAS-whd RNAi = 1.083×10−9 compared to control. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm n.s. = not significant.

Figure 6—source data 1

Contains numerical data plotted in Figure 6H,J,L,N and P.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
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Hemocyte progenitors of whd1 loss of function exhibit altered histone acetylation.

(A–D) Clonal analysis of histone acetylation in GFP-positive hsFlp/Ay-GAL4 based mock clonal patches and immunostaining with H3K9 acetylation (A–A''') and H4 pan acetylation (B–B''') antibodies. (B). Quantitative analysis of H3K9 acetylation level in A–A'''. p-Value for hsFlp/Ay-GAL4, UAS-GFP = 4.26×10−1 compared to control. (D). Quantitative analysis of H3K9 acetylation level in C–C'''. p-Value for hs-Flp/Ay-GAL4, UAS-GFP = 6.9×10−1 compared to control. (E–M) Progenitor-specific expression of UAS-whd RNAi and immunostaining with H3 (E–F'), H3K9 acetylation (H–I'), and H4 pan acetylation (K–L') antibodies. (G). Quantitative analysis of H3 acetylation level in E–F'. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-whd RNAi = 4.54×10−1 compared to control. (J). Quantitative analysis of H3K9 acetylation level in H–I'. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-whd RNAi = 7.87×10- 6 compared to control. (M). Quantitative analysis of H4 acetylation level in (K-Lʹ). p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-whd RNAi = 2.96×10−7 compared to control. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm. ns.=not significant.

Figure 6—figure supplement 1—source data 1

Contains numerical data plotted in Figure 6—figure supplement 1B,D,G,J and M.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig6-figsupp1-data1-v2.xlsx
Figure 7
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Acetate supplementation rescues differentiation defects of FAO mutant hemocyte progenitors.

(A–E) Comparison of differentiation (marked by P1) levels in dome > GFP lymph gland of control (A) dome > GFP supplemented with acetate (B) dome > GFP; whd1/whd1 (C) and dome > GFP; whd1/whd1 supplemented with acetate (D). (E). Quantitative analysis of results from A–D. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 = 2.718×10-1 fed with acetate compared to control dome-GAL4, UAS-GFP; tubGAL80ts20. p-Value for dome-GAL4, UAS-GFP; whd1/whd1 = 3.18×10−16 compared to control dome-GAL4, UAS-GFP; tubgal80ts20. p-Value for dome-GAL4, UAS-GFP; whd1/whd1 = 2.576×10−10 fed with acetate compared to control dome-GAL4, UAS-GFP; tubGAL80ts20. (F–G) Western blot analysis of H3K9 acetylation levels in control OreR and whd1/whd1 larvae supplemented with acetate and non-fed controls with H3 as a loading control. (G) Quantitative analysis of H3K9 acetylation levels in F. p-Value for OreR = 4.589×10−3 supplemented with acetate compared to non-fed control OreR. p-Value for non-fed whd1/whd1 = 8.001×10−3 compared to non-fed control OreR. p-Value for acetate supplemented whd1/whd1 = 3.582×10−2 compared to non-fed control whd1/whd1. (H–L) Acetate supplementation restores H3K9 acetylation status in the whd1/whd1 lymph gland (H-Iʹ). (L) Quantitative analysis of acetylation level in control, whd mutant, and whd mutant fed on acetate. p-Value for acetate supplemented dome-GAL4, UAS-GFP = 1.38×10−1 compared to non-fed control. p-Value for dome-GAL4, UAS-GFP; whd1/whd1 = 1.276×10−7 compared to dome-GAL4, UAS-GFP. p-Value for acetate supplemented dome-GAL4, UAS-GFP; whd1/whd1 = 1.31×10−6 compared to non-fed control dome-GAL4; UAS-GFP; whd1/whd1. (M–O) Comparison of H3K9 acetylation level in Dome+ progenitors of L-carnitine fed larvae (N–N') with non-fed control (M–M'). (O) Quantitative analysis of H3K9 acetylation levels in M–N'. p-Value for dome-GAL4, UAS-GFP = 1.079×10−8 supplemented with L-carnitine compared to non-fed control dome-GAL4, UAS-GFP. (P–Q) Western blot analysis of H3K9 acetylation levels in OreR larvae supplemented with L-carnitine and non-fed controls with H3 as a loading control. Quantitative analysis of H3K9 acetylation levels in N. p-Value for OreR = 3.17×10−4 supplemented with L-carnitine compared to non-fed control OreR. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm n.s. = not significant.

Figure 7—source data 1

Contains numerical data plotted in Figure 7E,G,L,O and Q.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig7-data1-v2.xlsx
Figure 8 with 1 supplement
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JNK regulates FAO in hemocyte progenitors of larval lymph gland.

(A–C) Comparison of differentiation (marked by P1) levels in dome > GFP lymph gland of control (A), and bsk/JNK knockdown in hemocyte progenitors by dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN (B). (C). Quantitative analysis of the differentiation level from A–B reveals a significant increase in the Dome+ progenitor zone and a decrease in differentiation. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=5.84×10−11 compared to control. (D–H) Differentiation levels (red, marked by Pxn) in overexpression of FOXO by dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-FOXO (E) is significantly increased compared to control (D). The increased differentiation in FOXO overexpression background is significantly rescued by one copy of the null allele of whd (G). (F). The differentiation level in one copy null allele of whd. (H). Quantitative analysis of the differentiation level from D–G reveals a significant increment in Pxn+ differentiated cell area in FOXO overexpression from Dome+ progenitors, which is significantly rescued by one copy null allele of whd. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-FOXO =5.77×10−11 compared to control. p-Value for dome-GAL4, UAS-GFP; whd1/+ = 2.11×10−5 compared to control. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-FOXO/whd1 = 3.84×10−9 compared to dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-FOXO. (I) Real-time expression analysis of fatty acid oxidation enzymes, whd, Mcad, Mtpα, scully, Mtpβ, and yip2 from dome > GFP and dome > GFP > UAS-bskDN lymph glands. The expression of whd shows a significant drop ~41% in dome > GFP > UAS-bskDN compared to control dome > GFP. p-Value for whd expression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=7.06×10−3 compared to control. p-Value for Mcad expression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=6.71×10−1 compared to control. p-Value for Mtpα expression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=8.95×10−1 compared to control. p-Value for scully expression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=9.73×10−1 compared to control. p-Value for Mtpβ expression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=7.7×10−1 compared to control. p-Value for yip2 expression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=2.42×10−2 compared to control. (J–O) Clonal analysis of histone acetylation in GFP-positive hsFlp/Ay-GAL4 based clonal patches expressing a dominant-negative form of bsk and immunostaining with H3 (J–J'''), H3K9 acetylation (L–L''') and H4 pan acetylation (N–N''') antibodies. (K). Quantitative analysis of H3 acetylation level in J–J'''. p-Value for hsFlp/Ay-GAL4. UAS-GFP, UAS-bskDN = 6.32×10−1 compared to control. (M). Quantitative analysis of H3K9 acetylation level in L–L'''. p-Value for hsFlp/Ay-GAL4. UAS-GFP; UAS-bskDN = 1.911×10−7 compared to control. (O). Quantitative analysis of H4 acetylation level in N–N'''. p-Value for hs-Flp/Ay-GAL4. UAS-GFP, UAS-bskDN = 8.22×10−9 compared to control. (P–T) Stalled differentiation levels (red, marked by Pxn) in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN (R) is significantly rescued in larvae reared in fly food supplemented with acetate (S). The differentiation level in control (P) dome-GAL4, UAS-GFP; tubGAL80ts20 remain unaltered upon acetate feeding (Q). (T). Quantitative analysis of the differentiation level from P–S reveals a significant rescue of differentiated cells upon acetate supplementation in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN lymph glands. p-Value for acetate supplemented dome-GAL4, UAS-GFP; tubGAL80ts20 = 5.655×10−1 compared to non-fed control. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=1.32×10−8 compared to control dome-GAL4, UAS-GFP; tubGAL80ts20. p-Value for acetate fed dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN=4.73×10−7 compared to non-fed dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS bskDN. (U–Y) The differentiation level (red, marked by Pxn) in control (U) dome-GAL4, UAS-GFP; tubGAL80ts20 increases upon L-carnitine feeding (V). Defect in differentiation levels (red, marked by Pxn) in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS bskDN (W) is significantly rescued in larvae reared in fly food supplemented with L-carnitine (X). (Y). Quantitative analysis of the differentiation level from U–X, reveals a significant rescue of differentiated cells upon L-carnitine supplementation in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN lymph glands. p-Value for L-carnitine supplemented dome-GAL4, UAS-GFP; tubGAL80ts20 = 1.69×10−8 compared to non-fed control. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN =4.5×10−10 compared to control dome-GAL4, UAS-GFP; tubGAL80ts20. p-Value for L-carnitine fed dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN =8.307×10−9 compared to non-fed dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS -bskDN. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 8—source data 1

Contains numerical data plotted in Figure 8C,H,I,K,M,O,T and Y.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
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JNK regulates FAO in hemocyte progenitors of the larval lymph gland.

(A–B''') Clonal analysis of histone acetylation in GFP-positive hsFlp/Ay-GAL4-based clonal patches expressing the dominant-negative form of bsk and immunostaining with H3K9 acetylation (A–A''') and H4 pan acetylation (B–B''') antibodies. A''' and B''' are heat map of A'' and B''. (C–K) Progenitor-specific expression of a dominant negative form of bsk and immunostaining with H3 (C-Dʹ), H3K9 acetylation (F-Gʹ), and H4 pan acetylation (I-Jʹ) antibodies. (E). Quantitative analysis of the H3 acetylation level in (C-Dʹ). p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN =6.6×10−1 compared to control. (H). Quantitative analysis of H3K9 acetylation level in (F-Gʹ). p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN =2.62×10−5 compared to control. (K). Quantitative analysis of H4 acetylation level in (I-J'). p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN =2.56×10−7 compared to control. (L–P) Stalled differentiation levels (red, marked by P1) in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN (M) is significantly rescued in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN, whd-OE (O). The differentiation level in control (L) dome-GAL4, UAS-GFP; tubGAL80ts20 and dome-GAL4, UAS-GFP; tubGAL80ts20 > whd-OE (N). (P). Quantitative analysis of the differentiation level from L–O reveals a significant rescue of differentiated cells upon whd overexpression in dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN,whd-OE lymph glands. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN =1.02×10−16 compared to control dome-GAL4, UAS-GFP; tubGAL80ts20. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > whd-OE =1.6×10−10 compared to control dome-GAL4, UAS-GFP; tubGAL80ts20. p-Value for dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN, whd-OE = 3.466×10−13 compared to dome-GAL4, UAS-GFP; tubGAL80ts20 > UAS-bskDN. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test). Scale bar: 20 µm.

Figure 8—figure supplement 1—source data 1

Contains numerical data plotted in Figure 8—figure supplement 1E,H,K and P.

https://cdn.elifesciences.org/articles/53247/elife-53247-fig8-figsupp1-data1-v2.xlsx
Figure 9
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The regulation of FAO by JNK is critical for differentiation.

ROS-JNK link has been previously shown to be essential for differentiation (Owusu-Ansah and Banerjee, 2009). The G2-M arrested hemocyte progenitors employ β-oxidation for their differentiation. ROS–JNK circuit impinges on FAO to facilitate progenitor differentiation. JNK signaling transcriptionally regulates whd, the rate-limiting enzyme of FAO leading to the production of acetyl-CoA. Acetyl-CoA leads to acetylation of histones in the hemocyte progenitors, which is critical for their differentiation.

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CG3902 YFP.

Five optical sections of 1µm thickness from the middle of the Z stack (No. 9-13) were merged into a single section for this panel.

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LipidTOX.

Five optical sections of 1µm thickness from the middle of the Z stack (No. 7-11) were merged into a single section for this panel.

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Nile Red.

Five optical sections of 1µm thickness from the middle of the Z stack (No. 10-14) were merged into a single section for this panel.

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Videos

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Mitochondrial distribution in the progenitors (red, Dome+) visualized by UAS-mito-HA-GFP.

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Gene (Drosophila melanogaster)domeFlybase:FB2020_01FLYB:FBgn
0043903
Gene (Drosophila melanogaster)HmlFlybase:FB2020_01FLYB:FBgn
0029167
Gene (Drosophila melanogaster)Tep4Flybase:FB2020_01FLYB:FBgn
0031888
Gene (Drosophila melanogaster)CG3902Flybase:FB2020_01FLYB:FBgn
0036824
Gene (Drosophila melanogaster)MtpαFlybase:FB2020_01FLYB:FBgn
0041180
Gene (Drosophila melanogaster)MtpβFlybase:FB2020_01FLYB:FBgn
0025352
Gene (Drosophila melanogaster)whdFlybase:FB2020_01FLYB:FBgn
0261862
Gene (Drosophila melanogaster)Hnf4Flybase:FB2020_01FLYB:FBgn
0041180
Gene (Drosophila melanogaster)chmFlybase:FB2020_01FLYB:FBgn
0028387
Gene (Drosophila melanogaster)Gcn5Flybase:FB2020_01FLYB:FBgn
0020388
Gene (Drosophila melanogaster)AcCoASFlybase:FB2020_01FLYB:FBgn
0012034
Gene (Drosophila melanogaster)Glut1Flybase:FB2020_01FLYB:FBgn
0264574
Gene (Drosophila melanogaster)ATPCLFlybase:FB2020_01FLYB:FBgn
0020236
Gene (Drosophila melanogaster)seaFlybase:FB2020_01FLYB:FBgn
0037912
Gene (Drosophila melanogaster)bskFlybase:FB2020_01FLYB:FBgn
0000229
Genetic reagent(Drosophila melanogaster)dome-GAL4BloomingtonDrosophilaStock CenterBDSC:81010; FLYB:FBti0022298; RRID:BDSC_81010FlyBase symbol: P{GawB}domePG14
Genetic reagent (Drosophila melanogaster)Hml-dsRed.ΔMakhijani et al., 2011FLYB:FBgn
0041180		FlyBase symbol: P{Hml-dsRed.Δ}
Genetic reagent (Drosophila melanogaster)HmlΔ-GAL4Sinenko and Mathey-Prevot, 2004FLYB: FBgn
0040877		FlyBase symbol: P{Hml-GAL4.Δ}
Genetic reagent (Drosophila melanogaster)Pvf2-lacZChoi et al., 2008FLYB:FBtp0052107FlyBase symbol: P{Pvf2-lacZ.C}
Genetic reagent (Drosophila melanogaster)TepIV-GAL4Kyoto Stock CenterDGGR:105442;
FLYB:FBti0037434;
RRID:DGGR_105442		FlyBase symbol: P{GawB}NP7379
Genetic reagent (Drosophila melanogaster)CG3902-YFPKyoto Stock CenterDGGR:115356;
FLYB:FBti0143519;
RRID:DGGR_115356		FlyBase symbol: PBac{566 .P.SVS-1}CG3902CPTI100004
Genetic reagent (Drosophila melanogaster)Mtpα[KO]Kyoto Stock CenterDGGR:116261;
FLYB:FBal0267653;
RRID:DGGR_116261		FlyBase symbol: MtpαKO
Genetic reagent (Drosophila melanogaster)Mtpβ[KO]Kyoto Stock CenterDGGR:116262;
FLYB:FBal0267654;
RRID:DGGR_116262		FlyBase symbol: MtpβKO
Genetic reagent (Drosophila melanogaster)UAS-whd RNAi [KK]ViennaDrosophila RNAi CenterVDRC:v105400;
FLYB:FBti0116709;
RRID:FlyBase_FBst0477227		FlyBase symbol: P{KK100935}VIE-260B
Genetic reagent (Drosophila melanogaster)OreRBloomingtonDrosophilaStock CenterBDSC:5; FLYB:FBsn0000277; RRID:BDSC_5FlyBase symbol: Oregon-R-C
Genetic reagent (Drosophila melanogaster)w[1118]BloomingtonDrosophilaStock CenterBDSC:3605; FLYB:FBal0018186;RRID:BDSC_3605FlyBase symbol: w1118
Genetic reagent (Drosophila melanogaster)UAS-Hnf4.miRNABloomingtonDrosophilaStock CenterBDSC:44398; FLYB:FBti0152533;RRID:BDSC_44398FlyBase symbol: P{UAS-Hnf4.miRNA}attP16
Genetic reagent (Drosophila melanogaster)UAS-whd RNAiBloomingtonDrosophilaStock CenterBDSC:34066; FLYB:FBal0263076; RRID:BDSC_34066FlyBase symbol: whdHMS00040
Genetic reagent (Drosophila melanogaster)UAS-FOXO.PBloomingtonDrosophilaStock CenterBDSC:9575; FLYB:FBtp0017636; RRID:BDSC_9575FlyBase symbol: P{UAS-foxo.P}
Genetic reagent (Drosophila melanogaster)Hnf4-GAL4BloomingtonDrosophilaStock CenterBDSC:47618; FLYB:FBti0136396; RRID:BDSC_47618FlyBase symbol: P{GMR50A12-GAL4}attP2
Genetic reagent (Drosophila melanogaster)UAS-FUCCIBloomingtonDrosophilaStock CenterBDSC:55121; RRID:BDSC_55121FlyBase symbol: P{UAS-GFP.E2f1.1–230}32; P{UAS-mRFP1.NLS.CycB.1–266}19
Genetic reagent (Drosophila melanogaster)UAS-mito-HA-GFPBloomingtonDrosophilaStock CenterBDSC:8442; FLYB:FBti0040803; RRID:BDSC_8442FlyBase symbol: P{UAS-mito-HA-GFP.AP}2
Genetic reagent (Drosophila melanogaster)UAS-chm RNAiBloomingtonDrosophilaStock CenterBDSC:27027; FLYB:FBal0220716; RRID:BDSC_27027FlyBase symbol: chmJF02348
Genetic reagent (Drosophila melanogaster)UAS-Gcn5 RNAiBloomingtonDrosophilaStock CenterBDSC:33981; FLYB:FBal0257611; RRID:BDSC_33981FlyBase symbol: Gcn5HMS00941
Genetic reagent (Drosophila melanogaster)UAS-AcCoAS RNAiBloomingtonDrosophilaStock CenterBDSC:41917; FLYB:FBal0279313; RRID:BDSC_41917FlyBase symbol: AcCoASHMS02314
Genetic reagent (Drosophila melanogaster)UAS-Glut1RNAiBloomingtonDrosophilaStock CenterBDSC:28645; FLYB:FBal0239561; RRID:BDSC_28645FlyBase symbol: Glut1JF03060
Genetic reagent (Drosophila melanogaster)ATPCL[01466]BloomingtonDrosophilaStock CenterBDSC:11055; FLYB:FBal0007976; RRID:BDSC_11055FlyBase symbol: ATPCL01466
Genetic reagent (Drosophila melanogaster)sea[EP3364]BloomingtonDrosophilaStock CenterBDSC:17118; FLYB:FBal0131420; RRID:BDSC_17118FlyBase symbol: seaEP3364
Genetic reagent (Drosophila melanogaster)UAS-bsk[DN]BloomingtonDrosophilaStock CenterBDSC:6409;
FLYB:FBti0021048; RRID:BDSC_6409		FlyBase symbol: P{UAS-bsk.DN}2
Genetic reagent (Drosophila melanogaster)UAS-mCD8::GFPBloomingtonDrosophilaStock CenterBDSC:5137; FLYB:FBti0180511; RRID:BDSC_5137FlyBase symbol: P{UAS-mCD8::GFP.L}2
Genetic reagent (Drosophila melanogaster)UAS-mCD8::RFPBloomingtonDrosophilaStock CenterBDSC:27400; FLYB:FBti0115747; RRID:BDSC_27400FlyBase symbol: P{UAS-mCD8.mRFP.LG}28a
Genetic reagent (Drosophila melanogaster)U-6;sgRNA-whd-KOBloomingtonDrosophilaStock CenterBDSC:77066; FLYB:FBal0335953; RRID:BDSC_77066FlyBase symbol: whdTKO.GS00854
Genetic reagent (Drosophila melanogaster)U-6;sgRNA-whd-OEBloomingtonDrosophilaStock CenterBDSC:68139; FLYB:FBal0337690; RRID:BDSC_68139FlyBase symbol: whdTOE.GS00536
Genetic reagent (Drosophila melanogaster)whd[1]BloomingtonDrosophilaStock CenterBDSC:441; FLYB:FBal0018515; RRID:BDSC_441FlyBase symbol: whd1
Genetic reagent (Drosophila melanogaster)Hnf4[Δ33]BloomingtonDrosophilaStock CenterBDSC:43634; FLYB:FBal0240651; RRID:BDSC_43634FlyBase symbol: Hnf4Δ33
Genetic reagent (Drosophila melanogaster)Hnf4[Δ17]BloomingtonDrosophilaStock CenterBDSC:44218; FLYB:FBal0240650; RRID:BDSC_44218FlyBase symbol: Hnf4Δ17
Genetic reagent (Drosophila melanogaster)tubGAL80[ts20]BloomingtonDrosophilaStock CenterBDSC:7109; FLYB:FBti0027796; RRID:BDSC_7109FlyBase symbol: P{tubP-GAL80ts}20
Genetic reagent (Drosophila melanogaster)hsFlpBloomingtonDrosophilaStock CenterBDSC:1929; FLYB:FBti0000784; RRID:BDSC_1929FlyBase symbol: P{hsFLP}12
Genetic reagent (Drosophila melanogaster)Ay-GAL4, UAS-GFPBloomingtonDrosophilaStock CenterBDSC:4411; FLYB:FBti0012290;FBti0003040RRID:BDSC_4411FlyBase symbol: P{AyGAL4}25; P{UAS-GFP.S65T}Myo31DFT2
Antibodyanti-P1 (Mouse monoclonal)Kurucz et al., 2007Cat# NimC1, RRID:AB_2568423IF(1:50)
Antibodyanti-Pxn (Mouse)Nelson et al., 1994IF(1:400)
Antibodyanti-proPO (Rabbit polyclonal)Jiang et al., 1997IF(1:1000)
Antibodyanti-DE-cadherin (Rat polyclonal)Developmental Studies Hybridoma BankCat# DE-cad, RRID:AB_2314298IF(1:50)
Antibodyanti-Ci155(Rat polyclonal)Developmental Studies Hybridoma BankCat# 2A1,
RRID:AB_2109711		IF(1:2)
Antibodyanti-GFP (Rabbit polyclonal)InvitrogenCat# A-11122,
RRID:AB_221569		IF(1:100)
Antibodyanti-H3 (Rabbit polyclonal)Cell Signaling TechnologiesCat# 9927,
RRID:AB_330200		IF(1:400), WB(1:1000)
Antibodyanti-H3K9 acetylation (Rabbit polyclonal)Cell Signaling TechnologiesCat# 9927,
RRID:AB_330200		IF(1:300), WB(1:1000)
Antibodyanti-H4 pan acetylation (Rabbit polyclonal)Cell Signaling TechnologiesCat# 06–598,
RRID:AB_2295074		IF(1:500)
Chemical compound, drugSodium butyrateEMD Millipore19–137
Chemical compound, drugNicotinamideSigma-Aldrich72345
Chemical compound, drugEtomoxirCayman ChemicalsCay119695 µM
Chemical compound, drugMildronateCayman ChemicalsCay15997100 µM
Chemical compound, drugL-carnitine hydrochlorideSigma-AldrichC0283100 mM
Chemical compound, drug2-DGSigma-AldrichD8375100 mM
Chemical compound, drugSodium acetateSigma-Aldrich7119650 mM
Chemical compound, drug2-NBDGInvitrogenN131950.25 mM
Chemical compound, drugLipidTOXMolecular ProbesH344771:1000
Chemical compound, drugStreptavidin-Cy3Molecular ProbesSA10101:200
Chemical compound, drugNile redMolecular ProbesN11420.5 ug/mL
Chemical compound, drugDHE (Dihydroxy Ethidium)Molecular ProbesD113470.3 µM
Sequence-based reagentPfk_FThis paperPCR primersATCGTATTTTGGCTTGCCGC
Sequence-based reagentPfk_RThis paperPCR primersCCAGAGAGATGACCACTGGC
Sequence-based reagentHex_FThis paperPCR primersCTGCTTCTAACGGACGAACAG
Sequence-based reagentHex_RThis paperPCR primersGCCTTGGGATGTGTATCCTTGG
Sequence-based reagentwhd_FThis paperPCR primersGGCCAATGTGATTTCCCTGC
Sequence-based reagentwhd_RThis paperPCR primersTGCCCTGAACCATGATAGGC
Sequence-based reagentAct5C_FThis paperPCR primersACACATTTTGTAAGATTTGGTGTGT
Sequence-based reagentAct5C_RThis paperPCR primersCCGTTTGAGTTGTGCTGT
Sequence-based reagentMcad_FThis paperPCR primersGGCCTGGATCTCGATGTGTT
Sequence-based reagentMcad_RThis paperPCR primersGATCACAGGAGTTTGGCCCAG
Sequence-based reagentMtpα_FThis paperPCR primersATCACTGTTGGTGACGGACC
Sequence-based reagentMtpα_RThis paperPCR primersCTGCAGCAGTCTGATGGCTT
Sequence-based reagentscully_FThis paperPCR primersGATCAAGAACGCCGTTTCCC
Sequence-based reagentscully_RThis paperPCR primersCAGATCGGCCAGGATCACG
Sequence-based reagentMtpβ_FThis paperPCR primersCAGGCACTCGCTTTTGTCAT
Sequence-based reagentMtpβ_RThis paperPCR primersCCTGGCAATGTTGGAGGTCT
Sequence-based reagentyip2_FThis paperPCR primersTCTGCCGCAACCAAAGGTAT
Sequence-based reagentyip2_RThis paperPCR primersTTAAGACCGGCAGCATCCAG
Software, algorithmFijiFijiRRID:SCR_002285
Software, algorithmPhotoshop CCAdobeRRID:SCR_014199
Software, algorithmImarisBitplaneRRID:SCR_007370
Commercial assay or kitClick-iT EdU plus (DNA replication kit)InvitrogenC10639
Commercial assay or kitATP bioluminescence kit HSIISigma11699709001
Commercial assay or kitHistone extraction kitAbcamab113476
Commercial assay or kitRNAeasy Mini KitQiagen74104

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  1. Satish Kumar Tiwari
  2. Ashish Ganeshlalji Toshniwal
  3. Sudip Mandal
  4. Lolitika Mandal
(2020)
Fatty acid β-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila
eLife 9:e53247.
https://doi.org/10.7554/eLife.53247