Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

Abstract

Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Niche-specific AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

Fig. 1. Correlation of the AMP position and polarity relative to the disc.

A Drawing of an L3 wing disc showing the spatial organization of AMPs, wing disc notum and hinge areas, and ASP (air-sac primordium) and TC (transverse connective); dashed box (left), ROI used for all subsequent YZ cross-sectional images. B–D TEM sections of wing disc (w¹¹¹⁸) showing YZ views of different wing disc notum areas; double-sided dashed arrows, long axes of elliptical AMPs; p-d dashed arrow, proximo (p)-distal (d) axis relative to the disc plane (dashed line); white arrow, cytoneme-like disc-invading projections from AMPs (D see Supplementary Fig. 1A,A’); BM basement membrane. E–E” Spatial organization of nls:GFP-marked AMP nuclei, orthogonal to the wing disc notum (E, E”) and hinge (E, E’) as illustrated in A. F–H Cross-sections of wing disc regions (indicated in ROI box in A) harboring nls:GFP-marked AMPs; double-sided arrows, nuclear orientation; F’ green channel of F; dashed and solid arrows, distal and proximal layer cells, respectively; G drawing illustrating the strategy to measure nuclear orientation as angles (Theta, θ) between AMP nuclei and the disc plane; H graph showing quantitative analyses of AMP nuclear orientation at different disc-relative locations; p: proximal (125 nuclei), d: distal (58 nuclei), p⁻¹: one layer above p (119 nuclei), d⁻¹: one layer below d (84 nuclei); also see Supplementary Table 1 and see “Methods” section for statistics; source data are provided as a “Source Data” file. I–K Single XY optical sections of the discs, showing diverse morphologies of distal AMPs; dashed arrows, elongated syncytial cells; arrowhead, small nonpolar cells (also see Supplementary Fig. 1B–D’). E, F” red, phalloidin, marking tissue outlines (also indicated by dashed line). Genotype: UAS-nls:GFP/+; htl-Gal4/+ (E–K). Scale bars: 20 μm; 10 μm (B, C, K); 5 μm (D).

Fig. 2. Disc-specific AMP polarity and adhesion are linked to polarized AMP cytonemes.

A Schematic depiction of htl>FRT>stop>FRT>Gal4 construct and its application to generate FLIP-out clones exclusively in AMPs (also see Supplementary Fig. 1E, F). B-B” Wing disc harboring random CD8:GFP-marked AMP clones (hs-Flp; UAS-CD8:GFP; htl>FRT>stop>FRT>Gal4) showing orthogonal and lateral polarity of AMPs and AMP cytonemes relative to disc plane; arrow and dashed arrow, proximal and distal AMPs/AMP cytonemes, respectively; arrowhead, distal small non-polar cells; *, adherent distal AMPs; phalloidin (red), actin-rich disc-AMP junction and cell-cortex (also see Supplementary Fig. 1G–H”); B’ GFP channel of B; B”’ Violin plots showing angles (Theta, θ; see Fig. 1G) between proximal and distal AMPs and their cytonemes relative to their underlying disc plane (see Supplementary Table 2 for statistical analyses). C–E Comparison of AMP cytonemes (arrows) marked by various fluorescent proteins driven by different transcription drivers, in fixed and live tissues, as indicated; arrowhead, actin-rich (phalloidin-marked) apical-junction of disc epithelium; E Graphs comparing length and numbers (count/100 μm length of AMP-disc interface) of orthogonal cytonemes (n = >125 cytonemes for each genotype/condition, imaged from >5 wing disc/genotype under fixed condition and four discs under live condition; see “Source Data” for statistics). F Actin-rich cytonemes (arrow) from wing disc cells expressing mCherryCAAX and Lifeact:GFP (fixed tissue). G–J Live dynamics of AMP cytonemes; G 3D-rendered image showing live cytonemes captured from p-to-d direction of the tissue; H dynamics of cytonemes (arrow) (2 min time-lapse, also see Supplementary Movies 1–4); I Graphs showing numbers of niche-occupying cytonemes over time within selected ROIs; three graph colors, three discs; J Graph showing the distribution of cytonemes lifetimes (n = 77 cytonemes; also see Supplementary Fig. 2D). Source data for B”‘, I, and J are provided as a “Source Data” file. C–J Genetic crosses: enhancer-Gal4/-LexA x UAS-/LexO-fluorescent protein (FP), as indicated. Scale bars: 20 μm; 5 μm (H).

Fig. 3. AMP cytonemes anchor AMPs to the wing disc adherens junctions.

A, B Schematic depictions of the genetic strategy (A) used to simultaneously mark AMPs and the disc notum, and imaging strategy (B) using multi-view microscopy for deep-tissue imaging. C, D”’ Triple-view confocal imaging showing CD2:GFP-marked orthogonally polarized cytonemes (arrow) emanating from disc-proximal AMPs (*, in C’) and invading through the intercellular space between nls:mCherry-marked disc cells (C, D, D’); D”-D”’ single XY cross-sections of disc, as illustrated in D”’, showing multiple cytonemes sharing the same intercellular space. E–H’ CD2:GFP-marked AMP cytonemes at the intercellular space of mCherryCAAX-marked wing discs approaching apical adherens junctions (Dlg stain, blue); E’, E”, dashed box area in E; F XY cross-section of disc showing niche sharing by multiple cytonemes; G Airyscan image of cytoneme tip approaching adherens junction (arrowhead), H, H’ AMP cytonemes (arrow) in both ths-Gal4 expressing (red) and non-expressing areas (dashed line). I, J Tip regions of AMP cytonemes contacting disc adherence junction that is marked with DCAD2 (I, I’) and Arm (J); *, helical twists in cytonemes; arrowhead, contact sites; I’ zoomed-in image from ROI in I. K, K’ Synaptic cytoneme-disc contact sites mapped by syb-GRASP (see “Methods” section) between sybGFP^1–10- and mCherryCAAX-expressing AMP cytonemes and the actin-rich (phalloidin, blue) apical junction of CD4:GFP¹¹-expressing wing-disc cells. All images are YZ cross sections unless noted. All panels, Gal4/UAS or LexA/LexO or genetic combinations of both used, as indicated (see Methods). Scale bars: 20 μm; 5 μm (E, E’, I’, F, G); 2 μm (J).

Fig. 4. Disc-cytoneme adhesion determines AMP position and fates.

A–D AMP cytoneme formation depends on Dia; A, B mCherryCAAX-marked AMP cytonemes localizing Dia:GFP (A UAS-mCherryCAAX/UAS-Dia:GFP; htl-Gal4/+) and Dia^act:GFP (B hsflp/+; UAS-mCherryCAAX/+; htl>FRT>stop>FRT>Gal4/UAS-Dia^act:GFP). C, D Loss of cytonemes (arrow) in Lifeact:GFP-marked AMPs expressing dia-i; average numbers of orthogonal cytonemes/100 μm of AMP-disc interface (± standard deviation (SD)): control (htl-Gal4>Lifeact:GFP) = 36.9 ± 3.8, and dia-i condition (htl-Gal4>Lifeact:GFP; dia-i) = 6 ± 4.1; Source data are provided as a “Source Data” file. E–I’ Comparison of control disc (E, H, H’) and disc expressing dia-i in AMPs under htl-Gal4, showing changes in the number of AMPs and AMP nuclei, morphologies, and orientations relative to the disc; G, G’ DAPI and Twi-stained; E–G’ and H–I’ AMPs expressing Lifeact:GFP and nls:GFP, respectively; red, phalloidin; arrowhead, actin-rich cell outline; * examples of giant nuclei within a large chamber; arrow in G’ shows the cytoplasmic space and thin Lifeact:GFP-marked membrane cortex surrounding each giant nucleus indicating hemifusion; dashed line in H and H’, tracheal outline and AMP-disc junction, respectively. J–O Comparison between control (J–K’) and dia-i-expressing (L–O’) AMP clones for their proximo-distal localization, polarity, and morphology; dashed arrow, distal cell/cytonemes, solid arrow, proximal cell/cytonemes, dashed line, AMP-disc junction, dashed double-sided arrow, space between basal disc surfaces and distal clones; M–O’ arrowhead, multi-nucleated cells (M), actin-rich (phalloidin stained and Lifeact:GFP-marked) fusogenic synapse (N–O’). XY or YZ views are indicated. Genotypes: UAS-X/+, htl-Gal4/UAS-dia-i (D, F–G’, I, I’). UAS-FP/+; htl-Gal4/+ (C, E, H, H’) HS-Flp/+; UAS-X/+; htl>FRT>stop>FRT>Gal4/+ (J–K’). HS-Flp/+; UAS-X/+; htl>FRT>stop>FRT>Gal4/UAS-dia-i (L–O’). X = FP, as indicated. Scale bars: 20 μm; 10 μm (A, B, M–O).

Fig. 5. AMP cytonemes localize Htl and require FGF signaling for disc-adherence.

A CD2:GFP-marked AMP cytonemes (arrows) localize Htl:mCherry (LexO-Htl:mCherry/+; htl-LexA, LexO-CD2:GFP/+). B Orthogonal AMP cytonemes localize Htl:GFP^fTRG. C, C” nls:GFP-marked AMPs stained with anti-dpERK. D–F’ CD8:RFP-marked clones (green, pseudo-colored) of control and dia-i-expressing AMPs showing the FGF signaling state (nuclear dpERK, red); D drawing depicting optical sections in E and E’, showing differences in numbers of dpERK positive clones between proximal (95.77% ±6.63 (±SD); 52 clones) and distal (3.19% ±3.62; 92 clones) AMP layers (p < 0.0001)). G–L Effects of htl-i expression under either htl-Gal4 (G, G”, J, K) or htl>FRT>Gal4 (single cell clones; H, I, L, L’); G, G”, J, K Discs harboring either Lifeact:GFP-marked (G, G”) or nls:GFP-marked (J, K) htl-i-expressing AMPs showing selective loss of-orthogonal cytonemes (G, G”, cytoneme numbers/100 μm of AMP-disc interface: control = 36.9 ± 3.8 and htl-i = 0; p < 0.0001), cell polarity (H-K), AMP number and layers (J, J”), and induction of fusogenic responses (G, G”, H, J’, J”). I Graphs comparing orthogonal and lateral cytoneme numbers per single-cell AMP clone; Control proximal layer had only orthogonal cytonemes (average ± SD: 2.6 ± 0.9/cell; total n = 64 cytonemes/25 clones) and distal layer had only lateral cytonemes (5.6 ± 1.8/cell; total n = 105 cytonemes/19 clones); htl-i-expressing clones were distal and had only lateral cytonemes (6.1 ± 1.7/cell; total n = 146 cytonemes/24 clones); error bars: SD; also see Supplementary Table 2. K Graphs comparing AMP nuclear angles (Theta, θ) in control (n = 125 proximal/58 distal nuclei) and htl-i-expressing AMPs (n = 33 nuclei; p < 0.0001). L, L’ Discs with DAPI and PH3 staining showing relative location and orientation of nls:GFP-marked control (L) and htl-i-expressing (L’) AMP clones. C, C’, E–L’ dashed arrow, distal cells/cytonemes; solid arrow, proximal cell/cytonemes; dashed line, AMP-disc junction; dashed double-sided arrow, space between the basal disc surface and the distal layer. Source data are provided as a “Source Data” file; p-values, unpaired two-tailed t-test. Genotypes: HS-Flp/+;UAS-X/+;htl>FRT>stop>FRT>Gal4/+ (E, E”, L). HS-Flp/+;UAS-X/+;htl>FRT>stop>FRT>Gal4/UAS-diaRNAi (F, F’); HS-Flp/+;UAS-X/UAS-htlRNAi;htl>FRT>stop>FRT>Gal4/+ (H, I, L’). X = FP as indicated. Scale bars: 20 μm; 30 μm (C, C”); 10 μm (E, E’).

Fig. 6. Wing discs express Pyr and Ths in distinct zones to support different AMP subtypes.

A Spatial patterns of ths-Gal4-driven mCherryCAAX expression in the wing disc notum and the distribution of CD2:GFP-marked AMPs; arrow, ths expression-free hinge area; right panel, red channel from the left panel. B Scheme depicting the genome editing strategy to generate a pyr-Gal4 enhancer trap construct (see Supplementary Fig. 3). C, D Wing disc expression patterns of pyr-Gal4-driven CD8:GFP (C) and mCherryCAAX (D) and its spatial correlation to the AMP distribution (D arrow). E–I Images showing CD8:GFP-marked ths-Gal4 and pyr-Gal4 expression zones in wing discs and their correlation with the localization of IFM-specific (high Vg, blue; low Cut, red; dashed arrow) and DFM-specific (high Cut, red; dashed arrow) progenitors; I schematic depicting the results of E–H. J–M” Images, showing effects of ths-i expression from ths source (ths-Gal4; mCherryCAAX-marked, blue) on the resident AMPs (AMP number, cytonemes, polarity, multi-layered organization, and dpERK signaling (red)) and non-resident AMPs (over the unmarked pyr-zone); dotted line (M”), ASP. N–P’ Images showing effects of pyr-i expression from pyr source (pyr-Gal4, blue area) on the resident AMPs and non-resident AMPs (unmarked ths-expression zone). J–P’ solid arrows, pyr expression zone; dashed arrow, ths-expression zone; dashed box, ROIs used to produce YZ views. Scale bars: 20 μm.

Fig. 7. Cytoneme-mediated FGF exchange generates niche-specific asymmetric signaling.

A, B’ XY and YZ views (as indicated) of wing discs expressing mCherryCAAX and either Ths:GFP under ths-Gal4 (A, A”) or Pyr:GFP under pyr-Gal4 (B, B’). C, D’ Images of wing discs harboring mCherryCAAX-marked AMPs; wing discs expressing either Ths:GFP or Pyr:GFP as indicated. A–D’ dashed arrow, signal in the source; arrow, non-autonomous punctate distribution in the AMP; *, non-expressing areas of the lacking signal distribution. E, F’ Wing discs expressing either Ths:GFP or Pyr:GFP as indicated, showing niche-specific effects of signal overexpression on proliferation of niche-resident and non-resident AMPs (nls:GFP marked); thick and thin dashed line, interface between AMPs and ths-expression and pyr-expression zones, respectively; dashed and solid arrows, effects on ths-expression and pyr-expression zones, respectively. G–J YZ views of wing discs expressing either Ths:GFP or Pyr:GFP as indicated and harboring mCherryCAAX-marked AMPs, showing disc-invading cytonemes receiving GFP-tagged signal (arrow) from the disc cells; arrowheads, localized signal enrichment in source cells. Scale bars: 20μm; 5 μm (H, H’).

Fig. 8. Cytoneme-mediated FGF-specific organizations in ectopic niches.

A–F Asymmetric FGF signaling and ligand-specific organization of DFM-specific (αCut, blue) and IFM-specific (αVg, blue) AMPs when dpp-Gal4 ectopically expressed either Pyr:GFP or Ths:GFP (dpp-Gal4>UAS-Pyr:GFP or Ths:GFP,>UAS-mCherryCAAX) in the wing disc pouch as illustrated in A; A Drawing depicting the experiments and results in B–F; dashed line, AMP-disc junctions; B’ XZ section from ROI in B; dashed and solid arrows, Cut and Vg expressing AMPs, respectively; double-sided arrow (D), proximal zone of Pyr:GFP expressing niche lacking Vg-positive AMPs. G, G”’ Wing disc pouch expressing Ths:GFP under dpp-Gal4 and showing mCherryCAAX-marked AMP and AMP cytonemes; *, proximal AMPs with orthogonal polarity and cytonemes; G”, G”’, single optical sections showing Ths:GFP on cytonemes (arrowheads); dashed lines, disc epithelium. H, H’ Wing disc pouch expressing Pyr:GFP under dpp-Gal4 and showing mCherryCAAX-marked AMP and AMP cytonemes; arrowhead, Pyr:GFP on cytonemes. I, J Models for the niche-specific asymmetric signaling and organization via cytonemes-mediated Pyr and Ths signaling (I) and signaling feedbacks reinforcing the cytoneme polarity and adhesion (J). Scale bars: 20 μm.