Fig 1.
The Bellymount platform enables intravital imaging of the adult Drosophila abdomen.
(A) Cartoons of Bellymounted animal. Top and side views are shown. The cuticle of the ventrolateral abdomen is glued to the bottom coverslip. To maximize contact between the cuticle and the glue, a second, smaller coverslip gently compresses the abdomen. (B) Underside view of a Bellymounted adult female. Gluing caused the ventral cuticle to become light transparent. The edge of the glue patch appeared as a refractive line around the abdomen. Right panels are close-ups of boxed region in left panel. (C) Bellymounted animals ingest nutrients, undergo GI transit, and defecate. As a mounted animal ingested blue-colored sugar water, its midgut gradually became colored blue. Image shows a single time point from S3 Movie. (D) Cartoon of Bellymount apparatus for delivery of CO2 anesthesia during confocal imaging. Isometric drawing shows assembled (D) and exploded (D′) configurations. The coverslip with the glued animal sits inside the base of the apparatus. CO2 flows through the indicated ports. CAD files for 3D printing of the lid and base are provided as S1 and S2 Files. See also S2 Fig. (E) Bellymount apparatus positioned for imaging on an upright microscope. Red box shows the location of the glued animal. (F) CO2 anesthesia minimizes tissue movements during acquisition of confocal z-stacks. Without CO2 (left), tissue movements during z-stack acquisition caused individual cells to be represented multiple times. With CO2 (right), tissue movements were inhibited, and z-stack images were accurate. Bottom panels are close-ups of boxed regions in top panels. Red asterisks label the identical cell in images taken without and with CO2. Blue, nuclei; yellow, midgut stem cells and enteroblasts. Images are projections of confocal stacks. Genotype: esg>LifeActGFP; ubi-his2av::mRFP. See S4 Movie. CAD, computer-aided design; EB, enteroblast; esg, escargot-Gal4; GFP, green fluorescent protein; GI, gastrointestinal; his2av, histone variant His2av; mRFP, monomeric red fluorescent protein; RFP, red fluorescent protein; SC, stem cell.
Fig 2.
Whole-abdomen, micron-resolution imaging of organs, tissues, and cells in live, intact animals.
(A) Native arrangement of abdominal organs. Visible organs include the crop (cr), midgut (mg), ovary (ov), oviduct (ovd), fat body (fb), uterus (ut), Malphigian tubules (mp), and terminalia (term). Inverted grayscale, nuclei. Genotype: esg>his2b::CFP, GBE-Su(H)-GFP::nls; ubi-his2av::mRFP (only RFP is shown). See S5 Movie. (B) Nuclear morphologies of individual midgut cells. Immature diploid cells (SCs [magenta nuclei] and terminal EBs [green nuclei]) were dispersed among mature, polyploid enterocytes (blue nuclei). Right panel is a close-up of boxed region in left panel. Arrowheads indicate nuclei of a stem cell (magenta) and two enteroblasts (green). Genotype: esg>his2b::CFP, GBE-Su(H)-GFP::nls; ubi-his2av::mRFP. (C) The abdominal tracheal network. Whole-abdomen imaging showed connectivity of tracheal branches from cuticular spiracles to internal organs. Ingested food in the midgut lumen exhibited autofluorescence (yellow asterisk). Boxed areas are shown as close-ups in D and E. (D) An extensive network of secondary and tertiary trachea wrap around the midgut tube. (E) Primary trachea exhibit branching near their origin at the cuticular spiracle. Genotype in C–E: breathless>GFP; ubi-his2av::mRFP (only GFP is shown). (F) Whole-abdomen distribution of hemocytes. Individual hemocytes tended to localize to pigmented regions of abdominal tergites. (G) Morphology of individual hemocytes. Image is a close-up of boxed region in F. Scale bar, 30 μm. Genotype in F–G: hml>GFP; ubi-his2av::mRFP (only the GFP channel is shown in F). (H) Developing egg chambers. Each chamber included the immature oocyte and polyploid nurse cells that are surrounded by diploid follicle cells. Four chambers, arranged by developmental stage, are aligned within one ovariole at the bottom. One chamber from a different ovariole is at the top. Nuclei, inverted grayscale. Genotype: ubi-his2av::mRFP. See S6 Movie. All images are projections of confocal stacks. CFP, cyan fluorescent protein; EB, enteroblast; esg, escargot-Gal4; GBE, Grainyhead binding element; GFP, green fluorescent protein; hml, hemolectin; his2av, histone variant His2av; mRFP, monomeric red fluorescent protein; nls, nuclear localization sequence; RFP, red fluorescent protein; SC, stem cell; Su(H), Suppressor of Hairless.
Fig 3.
Serial imaging reveals longitudinal dynamics of midgut stem cell lineages.
(A) Time points for serial imaging of GFP-marked midgut stem cell clones. Clones arose spontaneously during adult days 0–4. The first imaging time point, which was performed at adult day 4, is designated as day 0 in the experiment schematic. (B) Clones form organ-level patterns that enable their reidentification across imaging sessions. Blue boxes outline 3 midgut clones in Fly 3 that were tracked for the duration of the experiment. Green, GFP-labeled clones; grayscale, nuclei. See S4 Fig. (C) Longitudinal dynamics of clone cell addition and loss. Cells were counted at each imaging time point for 8 clones from the 3 animals in A. Between time points, numbers of cells per clone either increased, decreased, or remained constant. The data underlying this figure are included in S1 Data. (D,G,J) Time-series images of individual clones. Clones A, B, and C from Fly 3 are shown. See S4 Fig and S7 Movie. (E,H,K) Some clone cells differentiate into enterocytes during the imaging time course. Enterocyte differentiation was characterized by increased nuclear volume (green-to-blue shading; see S5 Fig). Over time, cells in clones became more likely to exhibit nuclear volumes typical of enterocytes (>300 μm3). The data underlying this figure are included in S1 Data. (F,I,L) Cartoons of clones. Nuclei in cartoons are color-coded to represent the measured nuclear volumes of individual clone cells. Volumes correspond to the white-green-blue color scale in E, H, and K. Genotype for all panels: UAS-CD8-GFP, hs-flp12; tubGal4; FRT82, tubGal80/FRT82. All images are projections of confocal stacks. CD8, membrane protein CD8; FRT, Flippase Recognition Target; GFP, green fluorescent protein; hs-flp, heat-shock flippase; tub, αTubulin84b promoter; UAS, Upstream Activation Sequence.
Fig 4.
Serial imaging reveals regional dynamics of gut bacterial colonization.
(A) Time points for serial imaging of gut colonization by L. plantarum. Animals were administered a 1-day pulse of L. plantarum-mCherry. L. plantarum was removed, and individual animals were imaged as indicated. The first imaging session is designated day 0. (B) Whole-animal CFUs following a 1-day pulse of L. plantarum-mCherry. Single flies were homogenized at the indicated times, and L. plantarum CFUs were measured. Each point represents CFUs from one animal. CFUs plateaued at 3 days, indicating stable colonization. The data underlying this figure are included in S2 Data. (C–E) Longitudinal imaging of L. plantarum in the crop of a single animal. (C) Whole abdomen on day 0. L. plantarum-mCherry (inverted grayscale) filled the crop and midgut. The boxed region is shown as a close-up in D. (D) Time series of L. plantarum in the crop of the animal in C. The distribution of L. plantarum was initially dense and became sparser over time. (E) Detailed images of crop from day 2. Sagittal (left) and orthogonal (right) sections are shown. L. plantarum accumulated along the crop’s inner wall (blue arrows) and formed prominent clumps (red asterisk). Ingested yeast cells (orange arrows) were also visible due to autofluorescence. See S8 Movie. (F–G) Longitudinal imaging of L. plantarum in the midgut of a single animal. (F) Whole abdomen on day 0. Inverted grayscale, L. plantarum-mCherry. Boxed region is shown as a close-up in G. (G) Time series of L. plantarum in midgut of animal in F. Only one midgut loop was visible on day 0; 2 loops (proximal/R2 and distal/R5; see S7 Fig) were visible on days 2 and 4. Over time, L. plantarum became clumped in the lumen of the proximal/R2 loop and individually dispersed in the lumen of the distal/R5 loop. See S6 Fig and S9 and S10 Movies. (H) Heterogenous dynamics of L. plantarum colonization and dispersal in distinct gut regions. Crop, proximal/R2 midgut, and distal/R5 midgut each exhibited distinct spatial patterns of bacterial localization over time. Genotype for all panels: ubi-his2avD::YFP. Images in C–F are single optical sections. Images in G are maximum projections of confocal stacks. CFU, colony-forming unit; his2av, histone variant His2av; Lp, L. plantarum; YFP, yellow fluorescent protein.