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. 2020 Jun 12;9:e53247. doi: 10.7554/eLife.53247

Figure 2. 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.

Figure 2.

Figure 2—figure supplement 1. Fatty acid β-oxidation is essential for lymph gland progenitor differentiation.

Figure 2—figure supplement 1.

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

Figure 2—figure supplement 2.

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.