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Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection

  1. Parvathy Ramesh
  2. Nidhi Sharma Dey
  3. Aditya Kanwal
  4. Sudip Mandal
  5. Lolitika Mandal  Is a corresponding author
  1. Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, India
  2. Developmental Genetics Laboratory, IISER Mohali, India
  3. Molecular Cell and Developmental Biology Laboratory, IISER Mohali, India
Research Article
Cite this article as: eLife 2021;10:e67158 doi: 10.7554/eLife.67158
20 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Relish expression and its function in hematopoietic niche of Drosophila larval lymph gland.

Genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A) Schematic representation of Drosophila larval lymph gland with its different cell types. (B) Hematopoietic niche in larval lymph gland visualized by Antp-Gal4,UAS-GFP and Antennapedia (Antp) antibody. (C–D') Expression of Relish (antibody: red) in larval lymph gland. (C) Relish is expressed in the hematopoietic niche of lymph gland and in the progenitor population. (C') Zoomed in view of the niche showing the expression of Relish in control niche. (D–D') Relish expression is abrogated in the niche upon RNAi mediated downregulation. (E) Quantitation of Relish expression in the niche. Significant reduction in Relish expression was observed in niche (n=10, p-value=7.4 × 10−9, two-tailed unpaired Student’s t-test), whereas progenitor-specific expression remained unchanged (n=10, p-value=0.764 , two-tailed unpaired Student’s t-test). (F–G'') Effect of Relish loss from the niche on cell proliferation (F–F''), Antp expression marks the niche of wild-type lymph gland. (G–G'') Loss of Relish function from niche leads to increase in niche cell number. (H–I') Hematopoietic progenitors of larval lymph gland (red, reported by DE-Cadherin [Shg] immunostaining). Compared to control (H–H'), drastic reduction in progenitor pool was observed when Relish function was attenuated from niche (I–I'). (J) Quantitation of Shg-positive progenitor population upon Relish knockdown from the niche using Antp-GAL4 (n=10, p-value=8.47 × 10−6, two-tailed unpaired Student’s t-test). (K) Quantitation of niche cell number upon Relish knockdown from the niche using Antp-GAL4 (n=10, p-value=1.3 × 10−7, two-tailed unpaired Student’s t-test) and pcol85-GAL4 (n=11, p-value=1.2 × 10−12, two-tailed unpaired Student’s t-test). (L–M') Hematopoietic progenitors of larval lymph gland (red, reported by Ci155 immunostaining) (L–L'). Loss of Relish from the niche resulted in reduction in Ci155-positive progenitor pool (M–M'). (N–O') Compared to control (N–N'), increase in the amount of differentiated cell population (red, P1 immunostaining) was observed upon niche-specific downregulation of Relish (O–O'). (P) Quantitative analysis of (N–O') reveals significant increase in the amount of differentiated cells in comparison to control (n=10, p-value=2.3 × 10−9, two-tailed unpaired Student’s t-test). (Q–Q') Scheme based on our observation. The white dotted line mark whole of the lymph gland in all cases and niche in (F–G''). Yellow dotted lines mark the progenitor zone in (H–I') and (L–M'). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Error bar: standard deviation (SD). Individual dots represent biological replicates. Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 1—figure supplement 1
Relish negatively regulate niche cell proliferation.

Genotypes of the larvae are mentioned in respective panels. Scale bar: 20 µm. (A–B') Effect of Relish loss from the niche using an independent GAL4 line, pcol85-GAL4. Compared to control (A–A'), downregulation of Relish from the niche using pcol85-GAL4 (B–B') also leads to increased niche cell proliferation. (C–D') A substantial increase in niche number was observed in Relish mutant (RelE20) (D–D') when compared to control (C–C'). (E) Quantitation of niche cell number in RelE20 mutant in comparison to control (n=8, p-value=9.03 × 10−9, two-tailed unpaired Student’s t-test). (F– G'') In comparison to control (F–F''), overexpression of Relish in the niche resulted in a reduction in niche cell number (G–G''). (H) Quantitation of niche cell number in Relish overexpression in comparison to control (n=10, p-value=3.3 × 10−10, two-tailed unpaired Student’s t-test). (I–J') Lamellocytes were not observed in Relish loss scenario (red, integrin β-PS-immunostaining). Loss of β-PS-positive progenitor pool is further evident in Relish loss scenario compared to control (compare J- J' to I- I') (K–L') In comparison to the control (K–K'), no significant change in crystal cell index (number of crystal cells/total number of cells in the lobe) was observed in Relish downregulation scenario (L–L'). (M) Quantitative analysis of crystal cell index in both control and Relish loss condition (n=8, p-value=0.596, two-tailed unpaired Student’s t-test). The white dotted line mark whole of the lymph gland in all cases and niche in A' and B', C' and D', F'–F'', and G'–G''. Yellow dotted lines mark the progenitor zone in (I–J') The nuclei are marked with DAPI (blue). In all panels, the age of the larvae is 96 hr AEH. Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 2 with 1 supplement
Loss of Relish from the niche causes niche cell hyperplasia. Genotypes are mentioned in relevant panels.

Scale bar: 20 μm. Niche is visualized by Antp antibody expression. (A–H'') EdU or 5-ethynyl-2'-deoxyuridine marks the cells in S-phase of the cell cycle. EdU profiling at 54 hr AEH (A–B''), 64 hr AEH (C–D''), 86 hr AEH (E–F''), and 96 hr AEH (G–H'') displayed EdU incorporation in the niche (green) in control and upon Relish downregulation. Control niches showed scanty EdU incorporation beyond 84 hr (E–E'' and G–G''), whereas loss of Relish induced niche cells to proliferate more (F–F'' and H–H''). (I) Graph representing percentage of EdU incorporation in the niche during the course of development in control (black line) and Relish loss (red line). Significant increase in the niche cell number is observed with development in Relish loss scenario. (54 hr, n=6, p-value=0.294), (64 hr, n=6, p-value=1.3 × 10−3), (86 hr, n=6, p-value=2.9 × 10−2), (96 hr, n=6, p-value=5.9 × 10−3); two-tailed unpaired Student’s t-test. (J–K'') Significant increase in the number of mitotic cells (phospho-histone 3 [PH3], red) was observed upon Relish loss from the niche (K–K'') compared to the control (J–J''). (L) Quantitation of the mitotic index of wild-type and Relish loss niche (n=15, p-value=8.1 × 10−4; two-tailed unpaired Student’s t-test). The white dotted line marks whole of the lymph gland and the niches. In all panels, age of the larvae is 96 hr AEH, unless otherwise mentioned. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 2—figure supplement 1
Relish expression starts beyond the second-instar stage in the hematopoietic niche. The genotypes are mentioned in relevant panels.

Scale bar: 20 μm. (A–E') Expression of Relish (red, by antibody) at different developmental time points in the larval lymph gland (niche marked with AntpGAL4>UAS-GFP). Observations were made at 24 hr AEH (A–A'), 48 hr AEH (B–B'), 72 hr AEH (C–C'), 84 hr AEH (D–D'), and 96 hr AEH (E–E'). Relish expression in the niche can be detected around 48 hr AEH. (F–F'') Relish expression (yellow) in the progenitor cells co-localizes with prohemocyte markers Ance (green) and TepIV (red). (G–G') Relish expression (red) is restricted to progenitor cells, whereas it is downregulated in Pxn-YFP-positive differentiated cells (green). (H–I'''') Cell cycle status reported by Fly-FUCCI using niche-specific GAL4: Antp-Gal4. In control, niche cells are mostly in G1 (green, H''') and G2–M (yellow, H'''') phase, while few are in S phase (red, H''). Niche cells from where Relish function has been downregulated were mostly in S, (red, I'') and G2-M (yellow, I''''), and very less in G1 (green, I''') phase of the cell cycle. (J) Quantitative analyses of the cell cycle status of control and Relish loss niches (n=10, p-value for G1=7.3 × 10−5, p-value for S=4.2 × 10−4, p-value for G2-M = 0.657), two-tailed unpaired Student’s t-test. The white dotted line marks whole of the lymph gland and the niches in (H–I''''). Yellow dotted lines mark the progenitor zone in (F–G'). In all panels. age of the larvae is 96 hr AEH, unless otherwise mentioned. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 3 with 1 supplement
Upregulated Wingless signaling leads to increase in niche cell number. The genotypes are mentioned in relevant panels.

Scale bar: 20 μm. (A–B'') Expression of Wingless (antibody) in the lymph gland. The hematopoietic niche is visualized by Antp-GAL4>UAS-GFP. (A'–A'') and (B'–B'') are higher magnifications of (A) and (B), respectively. In comparison to the wild-type niche (A–A''), Wingless protein levels were substantially high in Relish loss of function (B–B''). (C) Statistical analysis reveals elevated wingless expression upon Relish knockdown in niche (n=15; p-value=5.8 × 10−9, two-tailed unpaired Student’s t-test.) (D–G') The increased niche number observed upon Relish loss (E–E') is rescued upon reducing Wingless level by the wg RNAi (F–F') in Relish loss genetic background (G–G'). The rescued niche cell number is comparable to control (D–D'). (H) Statistical analysis of the data in (D–G') (n=10, p-value=1.1 × 10−11 for control versus Rel RNAiKK, p-value=3.15 × 10−10 for Rel RNAiKK versus Rel RNAiKK; wg RNAi, n=10, p-value=0.10 for control versus wg RNAi, n=10, p-value=0.29 for control versus Rel RNAiKK; wg RNAi; two-tailed unpaired Student's t-test). (J–M) Hematopoietic progenitors of larval lymph gland (red, reported by DE-Cadherin [Shg] immunostaining). Knocking down wingless function from the niche resulted in loss of Shg-positive progenitors (L). Downregulating wingless using wg RNAi in Relish loss genetic background was unable to restore the reduction in prohemocyte pool (M) observed in Relish loss (K) scenario in comparison to control (J). (N) Statistical analysis of the data in (J–M) (n=10, p-value=6.74 × 10−6 for control versus Rel RNAiKK, p-value=4.03 × 10−7 for control versus wg RNAi; Rel RNAiKK, p-value=3.42 × 10−8 for control versus wg RNAi; two-tailed unpaired Student’s t-test). The white dotted line marks whole of the lymph gland and the niches in (A–G') Yellow dotted lines mark the progenitor zone in (J–M). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 3—figure supplement 1
Downregulating wingless in Relish loss condition rescues niche cell proliferation, but not differentiation.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–D) Increase in plasmatocyte population (marked by P1, red) was observed upon Relish (B) and wingless downregulation (C) from the niche compared to the control (A). Simultaneous downregulation of wingless function in Relish loss genetic background did not rescue the increased differentiation (D). (E) Statistical analysis of the data in (A–D) (n=10, p-value=2.97 × 10−9 for control versus Rel RNAiKK, p-value = 4.18 × 10−5 for control versus wg RNAi; Rel RNAiKK, p-value=2.8 × 10−4 for control versus wg RNAi; two-tailed unpaired Student's t-test). (F) Scheme depicting the temperature regime followed for the rescue experiments (G–U) for wingless mutant (wgts). (G–J) The increased niche number observed upon Relish loss (H) is rescued upon reducing Wingless level by the temperature-sensitive allele wgts (I) in Relish loss genetic background (J). The rescued niche cell number is comparable to control (G). (K) Statistical analysis of the data in (G–J) (n=10; p-value=2.4 × 10−7 for control versus Relish RNAi, p-value=4.3 × 10−4 for control versus wgts and p-value = 3.4 × 10−7 for wgts; Relish RNAi versus Relish RNAi; two-tailed unpaired Student's t-test). (L–O) Hematopoietic progenitors of larval lymph gland (red, reported by DE-Cadherin [Shg] immunostaining). Knocking down wingless function using wgts resulted in loss of Shg-positive progenitors (N). Downregulating wg function in Relish loss genetic background was unable to restore the reduction in prohemocyte pool (O) observed in Relish loss (M) scenario in comparison to control (L). (P) Statistical analysis of the data in (L–O) (n=10; p-value=4.80 × 10−6 for control versus Rel RNAi, p-value=3.8×10−4 for wgts; Rel RNAi versus control, p-value=2.18 × 10−7 for control versus wgts; two-tailed unpaired Student’s t-test). (Q–T) Increase in plasmatocyte population (marked by P1, red) was observed upon wingless (S) and Relish down regulation from the niche (R) compared with the control (Q). Simultaneous downregulation of wingless function using wgts in Relish loss genetic background did not rescue the increased differentiation (T). (U) Statistical analysis of the data in (Q–T) (n=10, p-value=2.1 × 10−6 for control versus Rel RNAi, p-value=5.9 × 10−6 for control versus wgts, p-value=6.8 × 10−8 for control versus wgts; Rel RNAi; two-tailed unpaired Student's t-test). The white dotted line marks whole of the lymph gland and the niches in (A–D) and (G–J). Yellow dotted lines mark the progenitor zone in (L–O) and (Q–T). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 4 with 3 supplements
Hedgehog release from the niche is affected in Relish loss of function.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–B'') Hedgehog (Hh) antibody staining in the lymph gland shows Hh enrichment in the niche. The hematopoietic niche in Relish loss of function (B–B'') exhibits higher level of Hh in comparison to the control (A–A''). (C) Statistical analysis of fluorescence intensity revealed more than 2.5-fold increase in Hh levels compared to control (n=15, p-value=2.5 × 10−17, two-tailed Students t-test). (D–E'') Progenitors in Relish loss of function exhibits lower level of Extracellular Hh (HhExtra) (E–E'') in comparison to those of control (D–D''). (E'' and D'') are zoomed in view of niche and the neighboring progenitor cells of (E' and D'), respectively. The yellow box denotes the area quantified in (F). (F) The intensity profile of HhExtra in progenitors (along the rectangle drawn from PSC to cortical zone housing differentiated cells in D' and E') reflects a stark decline in the level of HhExtra in Relish loss scenario compared to control. (G–I') Cellular filopodia emanating from the niche cells were stabilized by using untagged phalloidin. The filopodia in Relish loss of function niches were found to be smaller in length and fewer in number (H–H', I–I') as compared to control (G–G'). (J–K) Significant reduction in filopodial length (J, n=10, p-value=6.64 × 10−9, two-tailed Student’s t-test) and number (K, n=6, p-value=9.19 × 10−10, two-tailed Student’s t-test) were observed in Relish loss scenario compared to control. The white dotted line marks whole of the lymph gland and niches in A–B'', D-E'. In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 4—figure supplement 1
Loss of Diaphanous from the niche resulted in defect in filopodial formation and enhanced differentiation.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–B') The filopodia in dia loss of function niches were found to be smaller in length and fewer in number (B–B') as compared to control (A–A'). (C–D) Significant reduction in filopodial lengths (C, n=8, p-value=3.73 × 10−12, two-tailed Student’s t-test) and number (D, n=8, p-value=7.2 × 10−4, two-tailed Student’s t-test) was observed in dia loss scenario compared to control. (E–F') Progenitors in dia loss of function from niche exhibits lower level of extracellular Hh (HhExtra) (F–F') in comparison to those of control (E–E'). The yellow box denotes the area quantified in (G). (G) The intensity profile of HhExtra in progenitors (along the rectangle drawn from niche to Cortical zone housing differentiated cells in Figure 4E' and F') reflects a stark decline in the level of HhExtra in dia loss scenario compared to control. (H–I) Knocking down dia function resulted in loss of Shg-positive progenitors (I) compared to control (H). (L) Statistical analysis of the data in (H–I) (n = 10, p-value=1.8 × 10−5; two-tailed Student’s t-test). (J–K) Loss of dia, from the niche caused ectopic differentiation of progenitors (K) compared to control (J). (M) Differentiation index for dia loss niches compared to control (n=10, p-value=4.28 × 10−5; two-tailed Student’s t-test). The white dotted line mark whole of the lymph gland in all cases. Yellow dotted lines mark the progenitor zone in (H)–(I). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 4—figure supplement 2
Loss of Relish from the niche resulted in upregulation of actin remodelers.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–B'') F-actin (visualized by Phalloidin, red) highly enriched in the plasma membrane of niche cells where Relish function is downregulated (B–B'') in comparison to that of control (A–A''). (C) Statistical analysis of fluorescence intensity showed significant increase in F-actin in Relish loss niches compared to control (n=10, p-value=5.6 × 10−9, two-tailed Student’s t-test). (D–E'') Expression of Singed, an actin-bundling protein, is significantly upregulated in Relish loss niches (E–E'') compared to control (D–D''). (F) Statistical analysis of fluorescence intensity showed significant increase in Singed expression in Relish loss niches compared to control (n=15, p-value=7.0 × 10−13, two-tailed Student’s t-test). (G–H'') Enabled an actin polymerase, which is normally absent from the niche cells of control (G–G'') is upregulated upon Relish downregulation (H–H''). (I) Statistical analysis of fluorescence intensity showed significant increase in Ena expression in Relish loss niches compared to control (n=15, p-value=8.1 × 10−20, two-tailed Student’s t-test). The white dotted line mark whole of the lymph gland and the niches in all cases. In all panels age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 4—figure supplement 3
Downregulation of Ena in Rel loss genetic condition partially rescues the differentiation and HhExtra dispersal defects.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–C) Upon simultaneous knockdown of both Rel and Ena from the niche, the decrease in Shg-positive progenitors observed in Relish loss (B) was partially rescued (C) compared to control (A). (D) Statistical analysis of the data in (A–C) (n=10, p-value=6.8 × 10−5 for control versus Rel RNAi, p-value=3.4 × 10−2 for ena RNAiKK; Rel RNAi versus control; two-tailed unpaired Student's t-test). (E–G) Differentiation defects observed in Rel loss (F) was partially rescued when both Rel and Ena was simultaneously downregulated from the niche (G) compared to the control (E). (H) Statistical analysis of the data in (E–G) (n=10, p-value=5.5 × 10−5 for control versus Rel RNAi, p-value=1.1 × 10−2 for ena RNAiKK; Rel RNAi versus control; two-tailed unpaired Student's t-test). (I–K') Reduced extracellular Hh observed in the progenitors (HhExt) of Rel loss of function condition (J–J'), in comparison to those of control (I–I'), is partially rescued in simultaneous loss of both Rel and Ena from the niche (K–K'). The yellow box in I', J', and K' denotes the area quantified in L, M, and N, respectively. (L–N) The intensity profile of HhExtra in progenitors (along the rectangle drawn from niche to Cortical zone housing differentiated cells in I'–K') reflects a stark decline in the level of HhExtra in Rel loss scenario (M) compared to control (L) and a partial rescue when both Rel and Ena was downregulated simultaneously (N). The white dotted line mark whole of the lymph gland in all cases. Yellow dotted lines mark the progenitor zone in (A–C and E–G). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 5 with 3 supplements
Loss of Relish from the niche activated JNK causing niche hyperplasia.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–B') Upregulation of JNK signaling visualized by its reporter TRE-GFP (green) in Relish knockdown (B–B') compared with WT niche (A–A'). (C) Statistical analysis of fluorescence intensity (A–B') revealed a significant increase in TRE-GFP levels compared to control (n=15, p-value=4.2 × 10−19, two-tailed Student’s t-test). (D–G') Upon niche-specific simultaneous knockdown of Rel and JNK, the niche hyperplasia observed upon loss of Relish (E–E') is rescued (G–G') and is comparable to control (D–D') whereas loss of bsk from the niche does not alter niche cell number (F–F'). (H) Statistical analysis of the data in (D–G') (n=10, p-value=5.6 × 10−8 for control versus Rel RNAi, p-value=8.0 × 10−7 for bsk DN; Rel RNAi versus Rel RNAi, p-value=0.10 control versus for bsk DN; two-tailed unpaired Student's t-test). (I–N) Cellular filopodia from the niche cells in Rel loss of function is found to be smaller in length and fewer in numbers (J and M–N). Simultaneous loss of both JNK using bsk DN and Relish (L and M–N) rescued the stunted, scanty filopodia to control state (I and M–N), whereas loss of JNK did not affect filopodia formation (K and M–N). (M–N) Statistical analysis of the data in (I–L) (Filopodia number: n=10, p=6.96 × 10−8 for control versus Rel RNAi, p-value=8.11 × 10−7 for bsk DN; Rel RNAi versus Rel RNAi, p-value=0.153 for bsk DN versus control. Filopodia length: n=6, p-value=2.78 × 10−16 for control versus Rel RNAi, p-value=1.84 × 10−6 for bsk DN; Rel RNAi versus Rel RNAi, p-value=0.22 for bsk DN vs control; two-tailed unpaired Student’s t-test). (O–R) Knocking down JNK function from the niche did not have any effect on progenitors (visualized by Shg) (Q). Downregulating bsk function in Rel loss genetic background was able to restore the reduction in prohemocyte pool (R) observed in Relish loss (P) scenario in comparison to control (O). (S) Statistical analysis of the data in (O–R) (n=10, p-value=2.26 × 10−6 for control versus Rel RNAi, p-value=1.94 × 10−7 for bsk DN; Rel RNAi versus Rel RNAi, p-value=0.521 for control versus bsk DN; two-tailed unpaired Student's t-test) The white dotted line marks whole of the lymph gland in all cases and niches in (A–G'). Yellow dotted lines mark the progenitor zone in (O–R). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 5—figure supplement 1
Ectopic activation of JNK signaling in the niche affects niche cell proliferation and progenitor maintenance.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–B') An increase in niche cell numbers observed upon upregulating JNK signaling using Hepact in the niche (B–B') compared to control (A–A'). (C) Statistical analysis of the data in (A–B') (n=10; p-value=2.2 × 10−4 for control versus Hepact, two-tailed unpaired Student’s t-test). (D–E') A significant increase in differentiation observed upon JNK overexpression using Hepact in the niche (E–E') compared to control (D–D'). (F) Statistical analysis of the data in (D–E') (n=10, p-value=1.7 × 10−3 for control versus Hepact, two-tailed unpaired Student's t-test.) (G–H'') Robust increase in Enabled expression is observed when in Hepact (H–H'') compared to control (G–G''). (I) Statistical analysis of the data in (G–H'') (n=10; p-value=2.1 × 10−7 for control versus Hepact, two-tailed unpaired Student's t-test). (J–M) Increase in niche cell numbers observed upon overexpressing Hep in the niche (K) is rescued to control levels (J) in a simultaneous loss of both Hep and wingless function from the niche (M). Loss of wingless using wg RNAi had milder effect on niche cell number compared to control (compare L and J). (N) Statistical analysis of the data in (J–M) (n=10; p-value=2.20 × 10−5 for control versus Hepact, p-value=1.08 × 10−5 for Hepact versus Hepact; wg RNAi, p-value=0.178 for control versus wg RNAi; two-tailed unpaired Student's t-test). The white dotted line mark whole of the lymph gland in all cases and the niches in (A–B' and G'– H'' and J–M). In all panels age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (Blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 5—figure supplement 2
Downregulating JNK in Relish loss genetic background rescues progenitor loss and precocious differentiation.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–D) Differentiation defect observed in Relish loss (B) was reverted to control (A) in a simultaneous knockdown of both Relish and JNK (D) from the niche. Loss of JNK alone from the niche had no significant effect on differentiation (C). (E) Statistical analysis of the data in (A–D) (n = 10, p-value=1.5 × 10−9 for control versus Rel RNAi, p-value=1.79 × 10−8 for bsk DN; Rel RNAi versus Rel RNAi, p-value=0.392 for bsk DN versus control; two-tailed unpaired Student's t-test). (F–H') Reduced Extracellular Hh observed in the progenitors (HhExt) of Relish loss of function condition (G–G') in comparison to those of control (F–F'), is significantly rescued in simultaneous loss of both Rel and JNK from the niche (H–H'). The yellow box in (F', G', and H') denotes the area quantified in (I, J, and K) respectively. (I–K) The intensity profile of HhExtra in progenitors (along the rectangle drawn from niche to Cortical zone housing differentiated cells in F', G', and H') reflects a stark decline in the level of HhExtr in Rel loss scenario (J) compared to control (I) which is rescued upon simultaneous loss of both Rel and JNK from the niche (K). The white dotted line mark whole of the lymph gland in all cases. Yellow dotted line indicates the boundary between CZ and MZ in (A)–(D). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: SD. Data are mean ± (SD). *p<0.05, **p<0.01, and ***p<0.001.

Figure 5—figure supplement 3
Relish inhibits JNK signaling by restricting tak1 activity in the niche during development.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–D) Up regulation of JNK signaling visualized by its reporter TRE-GFP (green) in Rel knockdown (B) compared with WT niche (A) is rescued in simultaneous loss of both the function of tak1 and Rel (D) whereas JNK activation was not observed in tak1 loss (C). (E–H) Increase in niche cell numbers observed upon loss of Rel from the niche (F) is rescued to control levels (E) in a simultaneous loss of both Rel and tak1 function from the niche (H) whereas no significant change in niche cell number was observed in tak1 loss (G). (I) Statistical analysis of the data in (E–H) (n=10, p-value=6.9×10−10 for control versus Rel RNAi, p-value=1.9×10−9 for tak12; Rel RNAi versus Rel RNAi, p-value=0.201 for control versus tak12; two-tailed unpaired Student's t-test). (J–M) Loss of tak1 function from the niche did not have any effect on progenitors (Shg) (L). Downregulating tak1 function in Rel loss genetic background could restore the reduction in prohemocyte pool (M) observed in Relish loss (K) scenario in comparison to control (J). (N) Statistical analysis of the data in (J–M) (n = 10, p-value=2.26×10−6 for control versus Rel RNAi, p-value = 3.1×10−4 for tak12; Rel RNAi versus Rel RNAi, p-value=0.891 for control versus tak12; two-tailed unpaired Student's t-test). (O–R) Differentiation defects observed in Rel loss (P) was comparable to control (O) in simultaneous loss of both Rel and tak1 function (R) from the niche. No significant change in differentiation was observed in tak1 loss from the niche (Q). (S) Statistical analysis of the data in (O–R) (n=10; p-value=1.5×10−4 for control versus Relish RNAi, p-value = 4.7×10−5 for; Rel RNAi versus tak12; Rel RNAi, p-value=0.115 for control versus tak12; two-tailed unpaired Student’s t-test). The white dotted line mark whole of the lymph gland in all cases and niches in (A–D and E–H). Yellow dotted lines marks the progenitor zone in (J–M). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 6 with 2 supplements
Ecdysone regulates Relish expression and functionality in the niche.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–C') Niche number remains comparable to control (A–A') both in axenic larval lymph gland (B–B') and in PGRP-LB mutant where there is upregulation in systemic peptidoglycan levels (C–C'). (D) Statistical analysis of the data in (A–C') (n=9; p-value = 0.262 for control versus germ free and 0.392 for control versus PGRP-LB mutant; two-tailed unpaired Student's t-test). (E–G') Compared to that of control (E–E') Rel expression is significantly downregulated both in EcR loss (G–G') as well as in Rel loss from the niche (F–F'). (H) Statistical analysis of the data in (E–G') (n=10, p-value=7.81 × 10−12 for control versus Rel RNAi loss and p-value = 3.76 × 10−10 for control versus EcR-DN; two-tailed unpaired Student's t-test). (I–K') Similar to Rel loss from the niche (J–J'), EcR loss also results in increase in niche cell numbers (K–K') compared to that of control (I–I'). (L) Statistical analysis of the data in I-K' (n=10, p-value=6.6 × 10−5 for control versus EcR-DN and p-value = 3.1x10−5 for control versus Rel RNAi; two-tailed unpaired Student’s t-test). (M–O') Compared to control (M–M'), both loss of Rel (N–N') and EcR (O–O') from the niche results in increase in differentiation. (P) Statistical analysis of the data in (M–O') (n=10, p-value=4.3 × 10−5 for control versus Rel RNAi and p-value=2.2 × 10−6 for control versus EcR-DN; two-tailed unpaired Student’s t-test). (Q–T') Increase in niche cell numbers observed upon EcR loss from the niche (R–R') is rescued to control levels (Q–Q') when Relish was overexpressed in an EcR loss genetic background (T–T'). Overexpression of Relish in the niche reduced the cell number compared to control (compare S–S' and Q–Q'). (U) Statistical analysis of the data in (Q–T') (n=10; p-value=1.7×10−9 for control versus EcR-DN, p-value=7.8 × 10−11 for Ecr-DN versus UAS-Rel 68kD; EcR-DN, p-value=3.63 × 10−6 for control versus UAS-Rel 68kD; two-tailed unpaired Student’s t-test). The white dotted line marks whole of the lymph gland and niches in all the cases. In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 6—figure supplement 1
Ecdysone signaling is active in the hematopoietic niche.

Genotypes of the larvae are mentioned in respective panels. Scale bar: 20 µm (A–A') Larval homogenates were spread on LB Agar plates to check the presence of commensal gut microbiota. In control scenario (A) bacterial colonies were visible post incubation whereas in axenic condition no growth was observed on the plates (A'). (B) The efficacy of removal of gut microflora was further checked by performing PCR analysis on DNA isolated from larval guts using 16S rDNA primers. Drosophila actin was used as control. Significant reduction in the amount of both Lactobacillus (compare lane 1 (axenic) with 4 (control)) and Acetobacter (compare lane 2 [axenic] with 5 [control]) species was observed in axenic condition compared to control scenario (compare lane 3 [axenic] and 6 [control]). (C–C') TRE-GFP expression in the hematopoietic niche (visualized by Antp, red) in axenic condition (C') is comparable to that of control (C). (D–D') Differentiation status (visualized by Hml>GFP, pan plasmatocyte marker) in axenic condition (D’) is comparable to control (D). (E–E'') Nuclear expression of Ecdysone receptor (red, EcR common) in the hematopoietic niche (green). The white dotted line marks whole of the lymph gland and the niches in (E–E''). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue).

Figure 6—figure supplement 2
Relish expression is transcriptionally regulated by ecdysone signaling in the hematopoietic niche.

Genotypes of the larvae are mentioned in respective panels. Scale bar: 20 µm (A–C') Fluorescent in situ hybridization (FISH) analysis showing the expression of Rel transcript in the lymph gland of the control larvae (A–A'). Loss of EcR from the niche resulted in loss of Re-positive progenitors (B–B'). Rel transcripts were also detected in salivary gland of the control larvae (C–C'). (D–E) Sense probe (negative control) showing nonspecific background expression in the control lymph gland (D) and salivary gland (E). (F–G'') Whole-mount immunofluorescence (IF) and FISH on third-instar lymph gland. Compared to control (F–F''), drastic reduction of the Rel transcript was observed in the niche from where EcR levels were downregulated (G–G''). Please note the smaller size of the LG in G–G' reflects the peeling off of the cortical zone due to excessive differentiation around 96 hr AEH in EcR loss from the niche. The increased differentiation renders fragility to the LG, which is unable to withstand harsh in situ process. (H) Statistical analysis of the data in (F'–G'') (n=10, p-value=1.56 x 10−10 for control versus EcR-DN; two-tailed unpaired Student’s t-test). (I–L) Differentiation defects observed in EcR loss (J) was reverted to control (I) when Relish was overexpressed in EcR loss genetic background (L). Slight decrease in differentiation of progenitors were observed upon Relish overexpression in the niche (compare I and K). (M) Statistical analysis of the data in (I–L) (n=10; p=3.8 × 10−7 for control versus EcR-DN, p=3.3 × 10−6 for Ecr-DN versus UAS-Rel 68kD; EcR-DN, p=7.2 × 10−2 for control versus UAS-Rel 68kD; two-tailed unpaired Student's t-test). (N) Model depicting the developmental role of Relish in hematopoietic niche maintenance. Downregulation of Relish affects the proliferation and primary function of the niche by upregulated JNK signaling. Upregulated JNK disturbs niche homeostasis through wingless and cytoskeletal remodeling, thereby affecting progenitor maintenance. The white dotted line mark whole of the lymph gland in all cases. Yellow dotted line marks the niche in (F–G'') and the boundary between CZ and MZ in (I–L). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 7 with 1 supplement
Niche-specific expression and function of Relish is susceptible to pathophysiological state of the organism.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–C') Compare to uninfected conditions (A–A') and sham (B–B'), significant reduction in Relish expression was observed in the hematopoietic niche 4 hr post-infection (C–C'). (D) Statistical analysis of the data in (A–C') (n=15; p=6.62×10−18 for unpricked versus infected, p=2.5×10−7 for sham versus infected, two-tailed unpaired Student’s t-test). (E–G) Nuclear expression of Relish was observed in infected (G) fat body cells 4 hr post in contrast to uninfected (E) and sham (F) larval fat body. (H–H') Overexpressing Relish N-terminus (UAS-Rel-68kD) could not rescue loss of Relish expression post-infection. (I–J) Compared to sham (I), significant reduction in Shg-positive progenitors (red) were observed in infected lymph glands (J). (K) Statistical analysis of the data in (I–J) (n=10; p-value=5.2 × 10−6 for sham versus infected, two-tailed unpaired Student's t-test). (L–M) Drastic increase in differentiation (visualized by Pxn-YFP, green) was observed in infected lymph glands (M) compared to sham (L). (N) Statistical analysis of the data in (L-M) (n=10; p-value = 4.65×10−6 for sham versus infected, two-tailed unpaired Student's t-test). The white dotted line mark whole of the lymph gland in all cases. Yellow dotted line marks the niche in (A– C' and H–H') and the boundary between CZ and MZ in (L–M). In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (Blue). Individual dots represent biological replicates. Error bar: standard deviation (SD). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Figure 7—figure supplement 1
Upregulation in JNK signaling and increase in cell proliferation was observed in the niche during infection.

The genotypes are mentioned in relevant panels. Scale bar: 20 μm. (A–B') An overall up regulation in JNK signaling (visualized by its reporter TRE-GFP [green] was observed in infected lymph glands (B–B') compared to sham (A–A')). (C–D) Significant increase in niche proliferation was observed in infected lymph gland niches (D) compared to sham infected (C). (E) Statistical analysis of the data in (C–D) (n=10; p-value=1.1×10−4 for sham versus infected, two-tailed unpaired Student's t-test). (F) Model based on current results depicting how upon bacterial challenge Relish expression is differentially modulated in the niche to bolster the cellular immune response by eliciting precocious differentiation of the lymph gland hemocytes. The white dotted line mark whole of the lymph gland and yellow doted lines marks the niches in all cases. In all panels, age of the larvae is 96 hr AEH. The nuclei are marked with DAPI (blue). Individual dots represent biological replicates. Error bar: standard deviation (S.D). Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001.

Developmental requirement of Relish in the niche for progenitor maintenance.

Scheme describing how loss of Relish from the niche alters cytoskeletal elements of the cells. The change in cytoskeletal architecture affects cytoneme-like filopodial formation thereby trapping Hedgehog within the niche. The failure of Hh delivery in turn interferes with progenitor maintenances and pushes them toward differentiation.

Author response image 1
A-C: No significant change in Relish expression in the niche was observed at 72 hours (AEH) (B-B') compared to 60 hours (AEH) ( A-A').

Statistical analysis of the data from A-B' (n=29 P-value =.297 ; two tailed Students t-test).

Author response image 2
A-C': Post 20E incubation, slight increase in Relish expression was observed compared (B-C') to mock incubated samples (A-A').

D. Statistical analysis of the data from A-B' (n=24 P-value=6.19 x10-7; two tailed Students t-test). E-F. JNK expression remained unaltered in 20E incubated (F) and mock (E) incubated samples.

Author response image 3
A-B: No significant change in EcR expression in the niche was observed upon infection and sham.

C. Statistical analysis of the data from A-B (n= 12 P-value=.364).

Author response image 4
A-D.

Slight decrease in Crystal cell index was observed in wg-RNAi (C) compared to control (C) whereas no significant change was observed in Rel RNAi(KK) (B) and Rel RNAi(KK), wg RNAi (D). E. Statistical analysis of the data from A-D (n=11 P–value=3.4x10-2 for control versus wg-RNAi).

Author response image 5
Significant decrease in LG area was observed in wgts compared to control (n=10, P-value = 2.

3x10-4 two tailed students t-test).

Author response image 6
Crystal cells marked by Lz>GFP also expresses Hh (red).
Author response image 7
Slight decrease in Crystal cell index was observed in tak12 (C) and tak12; Rel RNAi (D) compared to control (A) whereas no significant change was observed in Rel RNAi (B).

E. Statistical analysis of the data from A-D (n=10 P-value=1.5 x10-2 for control versus tak12 and P-value=7.4x10-2 control versus tak12; Rel RNAi, two tailed students t-test).

Author response image 8
Slight decrease in Crystal cell index was observed in bskDN (C) and bskDN; Rel RNAi (D) compared to control (A) whereas no significant change was observed in Rel RNAi (B).

E. Statistical analysis of the data from A-D (n=10 P-value=8.5 x10-2 for control versus bskDN and P-value=6.5x10-2 for control versus bskDN; Rel RNAi, two tailed students t-test).

Author response image 9
A-D.

Compared to control (A) ectopic differentiation and peeling off of lymph glands was observed in infected (B) as well as Rel loss samples (C).

Author response image 10
A-B: Rel expression in the niche was observed in control as well as in UAS-Rel68KD tissues.
Author response image 11
Significant increase in crystal cell index was observed in infected samples compared to sham (n=9, P-value =1.

8x10-3, two tailed students t-test).

Author response image 12
A-B'.

Loss of Relish function from the niche resulted in upregulation of hh transcription (A- A') (marked by hh-F4f GFP) compared to control niches (B-B')C. Statistical analysis of the data provided in A-B' (n=15, P-value= 2.2x10-11, two tailed Students t-test).

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Drosophila melanogaster)AntpFlybase:FB2020_01FLYB:FBgn0260642
Gene (Drosophila melanogaster)HmlFlybase:FB2020_01FLYB:FBgn
0029167
Gene (Drosophila melanogaster)Collier/knFlybase:FB2020_01FLYB:FBgn0001319
Gene (Drosophila melanogaster)wgFlybase:FB2020_01FLYB:
FBgn0284084
Gene (Drosophila melanogaster)hepFlybase:FB2020_01FLYB:FBgn0010303
Gene (Drosophila melanogaster)EcRFlybase:FB2020_01FLYB:FBgn0000546
Gene (Drosophila melanogaster)PGRP-LBFlybase:FB2020_01FLYB:FBgn0037906
Gene (Drosophila melanogaster)Tak1Flybase:FB2020_01FLYB:FBgn0026323
Gene (Drosophila melanogaster)bskFlybase:FB2020_01FLYB:FBgn
0000229
Gene (Drosophila melanogaster)EnaFlybase:FB2020_01FBgn0000578
Gene (Drosophila melanogaster)HhFlybase:FB2020_01FBgn0004644
Gene (Drosophila melanogaster)DiaFlybase:FB2020_01FBgn0011202
Genetic reagent (D. melanogaster)Antp-Gal4Emerald and Cohen, 2004FLYB:FBal0155891FlyBase symbol: GAL4Antp-21
Genetic reagent (D. melanogaster)P(col5-cDNA)/CyO-TM6B, TbKrzemień et al., 2007FLYB:FBti0077825FlyBase symbol: P{GAL4}col85
Genetic reagent (D. melanogaster)Hml-GAL4.ΔSinenko and Mathey-Prevot, 2004FLYB:FBtp0040877FlyBase symbol:P{Hml-GAL4.Δ}
Genetic reagent (D. melanogaster)UAS-Rel RNAiKKVienna Drosophila Resource CenterVDRC:v108469;
FLYB:FBti0116709;
RRID:FlyBase_FBst0477227
FlyBase symbol: P{KK100935}VIE-260B
Genetic reagent (D. melanogaster)w[1118]Bloomington Drosophila Stock CenterBDSC:3605; FLYB:FBal0018186;RRID:BDSC_3605FlyBase symbol: w1118
Genetic reagent (D. melanogaster)UAS-Rel RNAiBloomington Drosophila Stock CenterBDSC:33661; FLYB:FBti0140134;RRID:BDSC33661FlyBase symbol: P{TRiP.HMS00070}attP
Genetic reagent (D. melanogaster)UAS-wg RNAiBloomington Drosophila Stock CenterBDSC:33902; FLYB:FBal0263076; RRID:BDSC_33902FlyBase symbol: P{TRiP.HMS00844}attP2
Genetic reagent (D. melanogaster)UAS-dia RNAiBloomington Drosophila Stock CenterBDSC:35479;
FLYB:FBtp0068562; RRID:BDSC_35479
FlyBase symbol: P{TRiP.GL00408}
Genetic reagent (D. melanogaster)UAS-hep.ActBloomington Drosophila Stock CenterBDSC:9305; FLYB:FBti0074410; RRID:BDSC_9305FlyBase symbol: P{UAS-Hep.Act}1
Genetic reagent (D. melanogaster)UAS-FUCCIBloomington Drosophila Stock CenterBDSC:55121; RRID:BDSC_55121FlyBase symbol: P{UAS-GFP.E2f1.1–230}32; P{UAS-mRFP1.NLS.CycB.1–266}19
Genetic reagent (D. melanogaster)TRE-GFPBloomington Drosophila Stock CenterBDSC:59010; FLYB:FBti0147634; RRID:BDSC_59010FlyBase symbol: P{TRE-EGFP}attP16
Genetic reagent (D. melanogaster)Pxn-YFPKyoto Stock Centerkyoto:115452;
FLYB: FBti0143571;
RRID:FlyBase_FBst0325439
FlyBase symbol: PBac{802 .P.SVS-2}PxnCPTI003897
Genetic reagent (D. melanogaster)hhF4f-GFPTokusumi et al., 2012FBtp0070210FlyBase symbol:P{hhF4f-GFP}
Genetic reagent (D. melanogaster)UAS-GMABloomington Drosophila Stock CenterBDSC:31774; FLYB:FBti0131130; RRID:BDSC_31774FlyBase symbol:P{UAS-GMA}1
Genetic reagent (D. melanogaster)UAS-Rel 68kDBloomington Drosophila Stock CenterBDSC:55778; FLYB:FBti0160486; RRID:BDSC_55778FlyBase symbol: P{UAS-FLAG-Rel.68}i21-B
Genetic reagent (D. melanogaster)UAS-Rel 68kDBloomington Drosophila Stock CenterBDSC:55777;
FLYB:FBti0160484
RRID:BDSC_55777
FlyBase symbol:
P{UAS-FLAG-Rel.68}
Genetic reagent (D. melanogaster)UAS-EcR.B1ΔBloomington Drosophila Stock CenterBDSC:6872; FLYB:FBti0026963; RRID:BDSC_6872FlyBase symbol: P{UAS-EcR.B1-ΔC655.W650A}TP1-9
Genetic reagent (D. melanogaster)PGRP-LB[Delta]Bloomington Drosophila Stock CenterBDSC:55715; FLYB:FBti0180381; RRID:BDSC_55715FlyBase symbol: TI{TI}PGRP-LBΔ
Genetic reagent (D. melanogaster)wgl-12 cn1 bw1/CyOBloomington Drosophila Stock CenterBDSC:7000; FLYB:FBal0018504; RRID:BDSC_7000FlyBase symbol: wgl-12
Genetic reagent (D. melanogaster)Tak1(2)Bloomington Drosophila Stock CenterBDSC:26272; FLYB:FBal0131420; RRID:BDSC_26272FlyBase symbol: dTak12
Gene (Drosophila melanogaster)RelE20Flybase:FB2020_01FLYB:FBgn0014018
Genetic reagent (D. melanogaster)UAS-bsk[DN]Bloomington Drosophila Stock CenterBDSC:6409; FLYB:FBti0021048; RRID:BDSC_6409FlyBase symbol: P{UAS-bsk.DN}2
Genetic reagent (D. melanogaster)UAS-ena RNAiKKVienna Drosophila Resource CenterVDRC: v106484
FBst0478308;
RRID:v106484
FlyBase symbol: P{KK107752}VIE-260B
Genetic reagent (D. melanogaster)UAS-mCD8: RFPBloomington Drosophila Stock CenterBDSC:27400; FLYB:FBti0115747; RRID:BDSC_27400FlyBase symbol: P{UAS-mCD8.mRFP.LG}28a
Genetic reagent (D. melanogaster)tubGAL80[ts20]Bloomington Drosophila Stock CenterBDSC:7109; FLYB:FBti0027796; RRID:BDSC_7109FlyBase symbol: P{tubP-GAL80ts}20
AntibodyAnti-P1 (Mouse monoclonal)Kurucz et al., 2007Cat# NimC1, RRID:AB_2568423IF(1:50)
AntibodyAnti-c Rel (Mouse monoclonal)Stöven et al., 2000Cat#21F3,
RRID:AB_1552772
IF (1:50)
AntibodyAnti-Ci155 (Rat polyclonal)Developmental Studies Hybridoma BankCat# 2A1,
RRID:AB_2109711
IF(1:2)
AntibodyAnti-Wg (Mouse monoclonal)Developmental Studies Hybridoma BankCat#4D4
RRID:AB_528512
IF(1:3)
AntibodyAnti-Singed
(Mouse monoclonal)
Developmental Studies Hybridoma BankCat# sn 7C
RRID:AB_528239
IF(1:20)
AntibodyAnti-Enabled
(Mouse monoclonal)
Developmental Studies Hybridoma BankCat#5G2
RRID:AB_528220
IF(1:30)
AntibodyAnti-PH3(Rabbit monoclonal)Cell signaling
Technology
Cat# 3642S
RRID:AB_10694226
IF(1:150)
AntibodyAnti-Hh (Rabbit monoclonal)Forbes et al., 1993IF(1:500)
AntibodyAnti-Hnt
(Mouse monoclonal)
Developmental Studies Hybridoma BankCat#1G9
RRID:AB_528278
IF(1:5)
AntibodyAnti-EcR common
(Mouse monoclonal)
Developmental Studies Hybridoma BankCat#DDA2.7
RRID:AB_10683834
IF(1:20)
AntibodyAnti-Ance (rabbit monoclonal)Hurst et al., 2003IF(1:500)
AntibodyAnti-GFP
(rabbit polyclonal)
Cell signaling
Technology
Cat#2555IF(1:100)
AntibodyAnti-shg
(rat monoclonal)
Developmental Studies Hybridoma BankCat#DCAD2
RRID:AB_528120
IF(1:50)
AntibodyAnti-β-PS
(mouse monoclonal)
Developmental Studies Hybridoma BankCat#CF.6G11
RRID:AB_528310
IF(1:3)
AntibodyAnti-DIG-POD (sheep polyclonal)Sigma-AldrichCat#11207733910IF(1:1000)
Chemical compound, drugPhalloidin from Amanita phalloidesSigma-AldrichCat#P2141IF(1:500)
Chemical compound, drugRhodamine PhalloidinThermo ScientificCat# R415
RRID:AB_2572408
IF(1:500)
Sequence-based reagentRelish cDNA cloneDGRCClone id: GH01881
FLYB: FBcl0110737
Sequence-based reagentActin_FElgart et al., 2016PCR primersGGAAACCACGCAAATTCTCAGT
Sequence-based reagentActin_RElgart et al., 2016PCR primersCGACAACCAGAGCAGCAACTT
sequence-based reagentAceto_FElgart et al., 2016PCR primersTAGTGGCGGACGGGTGAGTA
Sequence-based reagentAceto_RElgart et al., 2016PCR primersAATCAAACGCAGGCTCCTCC
Sequence-based reagentLacto_FElgart et al., 2016PCR primersAGGTAACGGCTCACCATGGC
Sequence-based reagentLacto_RElgart et al., 2016PCR primersATTCCCTACTGCTGCCTCCC
Software, algorithmFijiFijiRRID:SCR_002285
Software, algorithmPhotoshop CCAdobeRRID:SCR_014199
Software, algorithmImarisBitplaneRRID:SCR_007370
Commercial assay or kitClick-iTEdU plus (DNA replication kit)InvitrogenCat# C10639
Commercial assay or kitAlexa Fluor 594 Tyramide ReagentThermo FischerCat# B40957

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