1. Cell Biology
  2. Developmental Biology

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
https://doi.org/10.7554/eLife.67158
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Cite this article
  1. Parvathy Ramesh
  2. Nidhi Sharma Dey
  3. Aditya Kanwal
  4. Sudip Mandal
  5. Lolitika Mandal
(2021)
Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection
eLife 10:e67158.
https://doi.org/10.7554/eLife.67158
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20 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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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 …

Figure 1—source data 1

Contains numerical data plotted in Figure 1C–D', Figure 1F–G'', Figure 1H–I', Figure 1N–O' and Figure 1—figure supplement 1C–D', Figure 1—figure supplement 1F–G'' and Figure 1—figure supplement 1K–L'.

https://cdn.elifesciences.org/articles/67158/elife-67158-fig1-data1-v2.xlsx.xlsx
Figure 1—figure supplement 1
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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'), …

Figure 2 with 1 supplement
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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 …

Figure 2—source data 1

Contains numerical data plotted in Figure 2A–B'', Figure 2C–D'', Figure 2E–F'', Figure 2G–H'', Figure 2J–K'' and Figure 2—figure supplement 1H–I''''.

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Figure 2—figure supplement 1
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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 …

Figure 3 with 1 supplement
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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 …

Figure 3—source data 1

Contains numerical data plotted in Figure 3A–B'', Figure 3D–G', Figure 3J–M and Figure 3—figure supplement 1A–D, Figure 3—figure supplement 1G–J, Figure 3—figure supplement 1L–O and Figure 3—figure supplement 1Q–T.

https://cdn.elifesciences.org/articles/67158/elife-67158-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
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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 …

Figure 4 with 3 supplements
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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 …

Figure 4—source data 1

Contains numerical data plotted in Figure 4A–B'', Figure 4D–E'', Figure 4G–I', Figure 4—figure supplement 1A–B', Figure 4—figure supplement 1E–F, Figure 4—figure supplement 1H–I, Figure 4—figure supplement 1J–K, Figure 4—figure supplement 2A–B'', Figure 4—figure supplement 2D–E'', Figure 4—figure supplement 2G–H'', Figure 4—figure supplement 3A–C, Figure 4—figure supplement 3E–G and Figure 4—figure supplement 3I–K'.

https://cdn.elifesciences.org/articles/67158/elife-67158-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
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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 …

Figure 4—figure supplement 2
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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 …

Figure 4—figure supplement 3
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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 …

Figure 5 with 3 supplements
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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–…

Figure 5—source data 1

Contains numerical data plotted in Figure 5A–B', Figure 5D–G', Figure 5I–L, Figure 5O–R, Figure 5—figure supplement 1A–B', Figure 5—figure supplement 1D–E', Figure 5—figure supplement 1G–H'', Figure 5—figure supplement 1J–M, Figure 5—figure supplement 2A–D, Figure 5—figure supplement 2F–H', Figure 5—figure supplement 3E–H, Figure 5—figure supplement 3J–M and Figure 5—figure supplement 3O–R.

https://cdn.elifesciences.org/articles/67158/elife-67158-fig5-data1-v2.xlsx.xlsx
Figure 5—figure supplement 1
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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 …

Figure 5—figure supplement 2
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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 …

Figure 5—figure supplement 3
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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 …

Figure 6 with 2 supplements
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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 …

Figure 6—source data 1

Contains numerical data plotted in Figure 6A–C', Figure 6E–G', Figure 6I–K', and Figure 6M–O', Figure 6Q–T', Figure 6—figure supplement 2F–G'', Figure 6—figure supplement 2I–L.

https://cdn.elifesciences.org/articles/67158/elife-67158-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
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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 …

Figure 6—figure supplement 2
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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 …

Figure 7 with 1 supplement
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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 …

Figure 7—source data 1

Contains numerical data plotted in Figure 7A–C', Figure 7I–J, Figure 7L–M, Figure 7—figure supplement 1C–D.

https://cdn.elifesciences.org/articles/67158/elife-67158-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
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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…

Figure 8
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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 …

Author response image 1
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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
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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
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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
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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 …

Author response image 5
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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
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Crystal cells marked by Lz>GFP also expresses Hh (red).
Author response image 7
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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
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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
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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
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A-B: Rel expression in the niche was observed in control as well as in UAS-Rel68KD tissues.
Author response image 11
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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
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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 …

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

Additional files

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