Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium
- University of California, San Francisco, United States
Figures
Figure 1
The close proximity of wing disc myoblasts and the ASP.
(A–C) Confocal images show the trachea and myoblasts at early (A), mid (B), and late (C) L3 stages. (Genotype: 1151-Gal4/+;btl-LHG/+;UAS-CD8GFP/lexO-mCherry-CAAX). Scale bar: 50 μm. (D) Sagittal cross-section shows myoblasts adjacent to the proximal portion of the ASP. Genotype as in (A–C). Scale bar: 50 μm. (E) Electron microscopic image shows a sagittal view of the wing disc columnar epithelium, associated myoblasts (white arrows), and the ASP. Scale bar: 5 μm. (F) Cartoon showing a cross-section of the ASP (red) and wing disc epithelium (blue). In the left drawing, dotted lines represent the approximate positions of upper and lower layers of ASP and the vertical dashed line corresponds to the location of the transverse optical section on the right and in Figure 2B. Vertical dashed line in transverse section corresponds to plane imaged in Figure 2B′.
Figure 2
Wing disc myoblasts contact the lower layer of the ASP.
(A) Medial optical section, (B) sagittal plane and (B′) transverse plane of the ASP (red, Cherry fluorescence) with green fluorescence indicating reconstituted GFP at regions of contact between myoblasts that expressed GFP1–10 and the ASP that expressed GFP11. Cytonemes (arrows in [A]) extend from the ASP; green fluorescence marks cytonemes that contact myoblasts. (Genotype: 1151-Gal4/+;btl-LHG, lexO-mCherry-CAAX/lexO-CD4-GFP11;UAS-CD4-GFP1–10/+). (C) Medial optical section and (D) sagittal plane of a preparation similar to (A and B), but with the myoblasts marked with Cherry fluorescence. Myoblast cytonemes extend to the ASP; green fluorescence marks cytonemes that contact the ASP. (Genotype: 1151-Gal4/+;UAS-CD8-mCherry/btl-LHG;UAS-CD4-GFP1–10 lexO-CD4-GFP11/+). (E–H′) Upper (left panels) and lower (right panels) optical sections (see Figure 1F) show GFP reconstitution across synaptic partners (GRASP) fluorescence (E and E′), staining with α-Hnt antibody (F–G′), and staining with α-ß-galactosidase antibody (H and H′). GRASP fluorescence, Hnt levels and Su(H)lacZ levels were higher in the lower than in the upper layer of the ASP. Scale bars: 25 μm.
Figure 3
Notch signaling in the ASP depends upon myoblast-produced Delta.
(A–F) Confocal images show that compared to controls (A and D), the ASP morphology was abnormal under conditions that either knockdown (B) or stimulate (C) Notch signaling, or that perturb myoblasts by expression of Rpr (E) or DlRNAi (F). Animals were reared at 18°C for 4 days (to L2 stage) and shifted to 29°C for >48 hr (to late L3 stage). (Genotypes: (A) btl-Gal4 UAS-CD8:GFP/+;tub-Gal80ts/+; (B) btl-Gal4, UAS-CD8:GFP/UAS-NotchDN;tub-Gal80ts/+; (C) btl-Gal4, UAS-CD8:GFP/UAS-NotchCA;tub-Gal80ts/+; (D) 1151-Gal4/+;btl-LHG lexO-Cherry:CAAX/+; (E) 1151-Gal4/+;btl-LHG lexO-Cherry:CAAX/UAS-rpr; and (F) 1151-Gal4/+;btl-LHG lexO-Cherry:CAAX/+;UAS-DlRNAi/+). (G–I) Compared to controls (G and J), levels of Hnt and Su(H)lacZ expression were lower under conditions that reduce Notch activity in ASP cells (H and K) or reduce Dl expression in myoblasts (I and L). Staining was with α-Hnt (G–I) and α-ß-galactosidase (J–L) antibodies and Alexa Fluor 555 secondary antibodies. ASPs are outlined by dotted lines. (M and N) The number of myoblasts (marked with CD8:GFP) and intensity of GFP fluorescence were reduced by expression of Rpr (N) relative to control (M). (O) Expression of DlRNAi in the wing disc had no apparent effect on ASP development. (Genotypes: (G) 1151-Gal4/+; (H) btl-Gal4/UAS-NotchDN;tub-Gal80ts/+; (I) 1151-Gal4/+;UAS-DlRNAi/+; (J) Su(H)lacZ/+;btl-Gal4/+, (K) Su(H)lacZ/+;UAS-NotchDN/+;btl-Gal4/tub-Gal80ts; (L) Su(H)lacZ/1151-Gal4;UAS-DlRNAi/+; (M) 1151-Gal4/+;UAS-CD8:GFP/+; (N) 1151-Gal4/+;UAS-rpr/+;UAS-CD8:GFP/+; (O) ap-Gal4/+;btl-LHG lexO-CD8:GFP/DlRNAi). Scale bars: 50 μm.
Figure 4
Delta localizes to myoblast cytonemes.
Confocal image of a late L3 ASP marked with CD2:GFP (btl-LHG lexO-CD2:GFP) and myoblasts marked with CD4:IFP (1151-Gal4 UAS-CD4:IFP2.0-HO1) and that express Dl:RFP (1151-Gal4 UAS-Dl:RFP). Dl:RFP puncta in IFP-marked myoblast cytonemes and CD2:GFP-containing ASP cytonemes are visible extending across the basal surface of the lower layer of the ASP. Scale bar: 50 μm.
Figure 5
Delta is motile in myoblast cytonemes and activates Notch signaling in the ASP.
(A–C) Confocal images of a late L3 ASP marked with CD2:GFP (btl-LHG lexO-CD2:GFP) and myoblasts marked with CD4:IFP (1151-Gal4 UAS-CD4:IFP2.0-HO1) and that express Dl:RFP (1151-Gal4 UAS-Dl:RFP). Dl:RFP puncta are visible in myoblasts and myoblast cytonemes. Images taken 5 s apart captured the motion of Dl-containing puncta (arrowheads in A, B, C). Scale bar: 10 μm. (D–D′) Projection images in control (1151-Gal4/+;UAS-CD8:GFP/+) and diaRNAi (1151-Gal4/+;UAS-CD8:GFP/UAS-diaRNAi) flies show that myoblasts with reduced diaRNA had fewer cytonemes. (E–F′) ASPs with the same genotypes as (D and D′) stained with α-Hnt (E and E′) and α-ß-galactosidase antibodies (F and F′) and with Alexa Fluor 555 secondary antibodies show that both Hnt and Su(H)lacZ were reduced under conditions of diaRNAi. (G and H) Bar graphs showing the dependence on dia and nrg function of myoblast cytoneme numbers per unit length of the imaged circumference of the ASP (G), and (I) Hnt and Su(H)lacZ levels (measured as fluorescence intensity in arbitrary units). p values, (G) 7.17E-12 and 8.475E-08 for diaRNAi and nrgRNAi, respectively; (H) 3.48E-05 and 1.79E-03 for diaRNAi and nrgRNAi levels of Hnt, respectively, and 5.9E-04 for diaRNAi levels of Su(H)lacZ; error bars: standard deviation. (I–L) Myoblast cytonemes (marked with CD8:GFP) that extend across the ASP were reduced in the presence of nrgRNAi (I and J); and in the ASP, the level of α-Hnt staining (a readout of Delta-Notch signaling) was also reduced (K and L). Genotypes: control (1151-Gal4/+;UAS-CD8:GFP/+) and nrgRNAi (1151-Gal4/+;UAS-nrgRNAi/+;UAS-CD8:GFP/+). (M and M′) Sagittal views of ASPs (red, Cherry fluorescence) show that contacts between myoblasts and the ASP marked by GRASP fluorescence were reduced in dia-RNAi (M′; 1151-Gal4/+;UAS-CD4:GFP1–10 lexO-CD4:GFP11/btl-LHG, lexO-mCherry-CAAX;UAS-diaRNAi/+) flies compared to controls (M; 1151-Gal4/+;UAS-CD4:GFP1–10 lexO-CD4:GFP11/btl-LHG lexO-Cherry:CAAX). Scale bars: 50 μm.
Figure 6
Myoblast functions necessary for Notch signaling in the ASP.
Compared to controls (A, D, G, J), reduction of Rab5 function (B, E, H, K) or of Dynamin function (C, F, I, L) perturbed ASP development (B, C, E, F) and reduced Hnt (H and I) and Su(H)lacZ (K and L) expression. (G–L) Staining was with α-Hnt (G–I) and α-ß-galactosidase antibodies (J–L) and Alexa Fluor 555 secondary antibodies. (Genotypes: (A) 1151-Gal4/+;btl-LHG, lexO-mCherry-CAAX/+; (B) 1151-Gal4/+;btl-LHG, lexO-Cherry:CAAX/+;UAS-Rab5DN/+; (C) 1151-Gal4/+;btl-LHG, lexO-Cherry:CAAX/+;UAS-ShiDN/+; (D) 1151-Gal4/+;UAS-CD8:GFP/+; (E) 1151-Gal4/+;UAS-CD8:GFP/UAS-Rab5DN; (F) 1151-Gal4/+;UAS-CD8:GFP/UAS-Shi5DN; (G) 1151-Gal4/+; (H) 1151-Gal4/+;UAS-Rab5DN/+; (I) 1151-Gal4/+;UAS-ShiDN/+; (J) Su(H)lacZ/1151-Gal4; (K) Su(H)lacZ/1151-Gal4;UAS-Rab5DN/+; (L) Su(H)lacZ/1151-Gal4;UAS-ShiDN/+). Scale bars: 50 μm.
Figure 7
Wg expression in the wing disc affects ASP development and Notch signaling.
Compared to controls (A, D, G), ectopic Wg expression (B, E, H) and wgRNAi expression (C, F, I) in the wing disc perturbed ASP development (B and C). Ectopic Wg expression reduced levels of Hnt (E) and Su(H)lacZ (H); wgRNAi increased Hnt and Su(H)lacZ (F and I). (D–I) Staining was with α-Hnt (D–F) and α-ß-galactosidase antibodies (G–I) and Alexa Fluor 555 secondary antibodies. (J) Drawings showing (left) areas of sal expression (red) in the wing disc (Grieder et al., 2009) and (right) Tr2 tracheal branches (Rao et al., 2015). Dashed white line indicates approximate location of sagittal sections shown in Figure 11E–H. (K) Over-expression of Wg in the sal domain reduced ASP development; (L) Expression of TCFDN in trachea had no apparent effect on ASP development. (Genotypes: (A) btl-LHG, lexO-Cherry:CAAX/+; (B) btl-LHG, lexO-Cherry:CAAX/ap-Gal4; UAS-wg/tub-Gal80ts; (C) wg-Gal4/UAS-wgRNAi; btl-LHG, lexO-Cherry:CAAX/UAS-wgRNAi; (D) wg-Gal4/+; (E) ap-Gal4/+;UAS-wg/tub-Gal80ts; (F) wg-Gal4/UAS-wgRNAi;UAS-wgRNAi/+; (G) Su(H)lacZ/+; (H) Su(H)lacZ/+;ap-Gal4/tub-Gal80ts;UAS-wg/+; (I) Su(H)lacZ/+;wgGal4/UAS-wgRNAi;UAS-wgRNAi/+; (K) sal-Gal4/+;UAS-wg/tub-Gal80ts; (L) btl-Gal4 UAS-CD8:GFP/UAS-TCFDN). Scale bars: 50 μm.
Figure 8 with 1 supplement
Wg signaling regulates the abundance of Delta in wing disc myoblasts.
(A–C) A MARCM clone (Lee et al., 2000) of dishevelled mutant cells (outlined with dashed white line) expressed GFP (B) and up-regulated Dl (C). α-Apontic antibody staining identified myoblasts (A). Clone was induced by 1 hr heat shock 3–4 days after egg laying. (D–F) The ASP developed abnormally in the presence of TCFDN in wing disc myoblasts (E) compared to control (D); the phenotype was suppressed by expression of DeltaRNAi in the myoblasts. (G–L) TCFDN expression in the wing disc myoblasts increased Hnt (H) and Su(H)lacZ (K) relative to controls (G and J); DlRNAi reduced Hnt and Su(H)lacZ expression to control levels (I and L). (Genotypes: (A–C) dsh3 FRT19A/hsFLP tub-Gal80ts,FRT19A;act-Gal4 UAS-GFP/+; (D) 1151-Gal4/+;btl-LHG, lexO-Cherry:CAAX/+, (E) 1151-Gal4/+;btl-LHG lexO-Cherry:CAAX/UAS-TCFDN and (F) 1151-Gal4/+;btl-LHG, lexO-Cherry:CAAX/UAS-TCFDN;UAS-DeltaRNAi/+). Scale bars: 50 μm.
Figure 8—figure supplement 1
disheveled mutant clones up-regulate Delta expression.
MARCM clones mutant for dsh3 in myoblasts (identified by expression of Apontic, left panels) that were induced 3–4 days after egg laying expressed GFP (middle panels) and increased Dl expression (right panels). Staining was with α-Apontic and α-Hnt antibodies. (Genotype: dsh3 FRT19A/hsFLP tub-Gal80ts,FRT19A; act-Gal4 UAS-GFP/+).
Figure 9
Proximity of Wg-expressing cells, myoblasts and ASP in the wing disc.
(A–C) Images show the presumptive notum region of the wing disc during the third instar; the Wg domain (red) labeled by α-Wg antibody staining and myoblasts by GFP fluorescence (Genotype: 1151-Gal4 UAS-CD8:GFP). (D–F) Images of the presumptive notum region of the wing disc show the ASP (red) and Wg-expressing disc cells (green) in early (D), mid (E) and late (F) L3 stage discs. (Genotype: btl-LHG/wg-Gal4 UAS-CD8:GFP; lexO-Cherry:CAAX/+). (G–I) Images show contacts (green fluorescence) between Wg-expressing disc cells and myoblasts generated by reconstituted GFP (GRASP). Auto-fluorescence of air-filled tracheal lumen was detected at 405 nm; perimeter of ASP is indicated by dashed white lines. (Genotype: 15B03-lexA/wg-Gal4;UAS-CD4-GFP1–10 lexO-CD4-GFP11/+). Scale bars: 50 μm.
Figure 10
ASP development depends on Wg expression at the early third instar stage.
(A) Image shows ASP (highlighted by α-Discs large antibody staining and outlined with dashed white line) from a GRASP experiment in which the fragments of GFP were expressed in the Wg domain of the disc and in the trachea. The absence of GFP fluorescence indicates that Wg-expressing disc cells did not directly contact the ASP. (Genotype: wg-Gal4/lexO-CD4-GFP11;btl-LHG/UAS-CD4-GFP1–10). (B–I) Expression of wgRNAi in the wing disc during the early L3 was induced as depicted in the line drawings above the images—either as a 24 hr temperature pulse at the non-permissive temperature for Gal80ts (29°C) 7 days after egg laying (early L3, B and D), continuously at 29°C after 7 days (C and E), 8.5 days (mid L3, F and H) or after 10 days (late L3, G and I). wgRNAi perturbed ASP development (D and E) and increased Hnt expression (B and C) when expressed at early L3, but had no apparent effect on either Hnt expression (F and G) or ASP development (H and I) when expressed only at mid or late L3. (F, G, H, I) Staining with α-Hnt antibody was detected with Alexa Fluor 555 secondary antibody. (J) Quantitation of Hnt staining in (B, C, F, G) normalized to control (wg > wgRNAi, Gal80ts at 18°C). N = 5 for each time point and control; error bars: standard deviation. (Genotypes: (B, C, F, G) wg-Gal4/UAS-wgRNAi;UAS-wgRNAi/tub-Gal80ts; (D, E, H, I) btl-LHG lexO-Cherry:CAAX/wg-Gal4;UAS-wgRNAi/tub-Gal80ts). (K–P) Expression of TCFDN in myoblasts (with the 1151-Gal4 driver) was induced as depicted in the line drawings above the images—either at the non-permissive temperature for Gal80ts (29°C) 7 days after egg laying (early L3), 8.5 days (mid L3) or 10 days (late L3). ASP development was abnormal and Hnt staining increased with expression of TCFDN at early L3, but had no apparent effect on either Hnt or ASP development when expressed only at mid or late L3. Scale bars: 50 μm.
Figure 11 with 1 supplement
Delta and Wg localize to myoblast cytonemes.
(A and B) Myoblast cytonemes (marked with CD8:GFP) at early L3. Images show Wg-expressing disc cells (red), the ASP (outlined by white dashed line in (A)), trachea (autofluorescence at 405 nm in (A)), and myoblasts (green, and indicated with black stars in (A)). Myoblast cytonemes extended toward the ASP (white arrows) and toward Wg-expressing cells (white arrowheads). Yellow arrow in (A) indicates a myoblast with both types of cytonemes. (C) Fz:Cherry puncta (white arrowhead points to one) visualized in myoblasts and myoblast cytonemes (marked with CD8:GFP). (D) Images obtained at 5 s intervals visualizing Wg:Cherry expressed in the Wg domain of the wing disc. Fluorescent puncta associated with disc cells (red background area), with myoblasts (green area) and with myoblast cytonemes (white arrowhead indicates a motile puncta). (E–H) Sagittal images showing wing disc and myoblasts (green; encircled by dotted lines) and stained with α-Delta (E and F) and α-β-catenin (G and H) antibodies (red) in the absence ((E and G) or presence (F and H) of diaRNAi expressed in myoblasts. Fluorescence intensity in the indicated boxes was measured using ImageJ and the levels in experimental samples relative to controls were calculated by comparing the ratio of myoblast to disc fluorescence. (Genotypes: (A and B) UAS-CD8:RFP lexO-CD8:GFP/+;wg-Gal4, 15B03-LexA/+; (C) 15B03-LexA lexO-Fz:Cherry/lexO-CD2:GFP; (D) 1151-Gal4/+;wg:Cherry/+; UAS-CD8:GFP/+; (E and G) 1151-Gal4/+;UAS-CD8:GFP/+; (F and H) 1151-Gal4/+;UAS-CD8:GFP/UAS-diaRNAi). Scale bars: 30 μm (A–D), 25 μm (E–H).)
Figure 11—figure supplement 1
Expression of Neuralized in the wing disc and associated myoblasts.
Staining with α-Neuralized antibody (red) detected elevated levels in the myoblasts relative to the disc epithelium.
Videos
Video 1
Motile Delta-RFP in myoblast cytonemes.
Dl:RFP expressed in myoblasts was detected in fluorescent puncta that moved along dynamic myoblast cytonemes (labeled by CD4:IFP2.0–HO1); the ASP was labeled by CD2:GFP. (Genotype: 1151-Gal4/+;btl-LHG lexO-CD2:GFP/+; UAS-Dl:RFP UAS-CD4:IFP2.0-HO1/+.)
Video 2
Motile Frizzled receptors in the cytonemes.
Fz:Cherry expressed in myoblasts was detected in fluorescent puncta that move along dynamic myoblast cytonemes (labeled with CD2:GFP). (Gentoype: 15B03-LexA lexO-Fz:Cherry/lexO-CD2:GFP.)
Video 3
Wg puncta is transported in myoblast cytonemes.
Wg:Cherry expressed in the wg domain of the wing disc was detected in fluorescent puncta that move along myoblast cytonemes (labeled with CD8:GFP). (Genotype: 1151-Gal4/+;wg–wg:Cherry/+;UAS-CD8:GFP/+.)
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium
eLife 4:e06114.
https://doi.org/10.7554/eLife.06114
