Fig 1.
3D and M-mode OCM imaging of post-embryonic Drosophila lifecycle.
(a) 3D OCM renderings of a 24B-GAL4/+ Drosophila flies at larva, pupa and adult stages. (b) Schematic representation of heart metamorphosis. Red arrows on larva and adult schematic denote the OCM M-mode imaging locations until PD1 24h and for subsequent time points, respectively. (c) Enface OCM projections showing heart metamorphosis. (d) Axial OCM sections showing heart remodelling during Drosophila lifecycle. * denotes the air bubble location during early hours of pupa development. (e) M-mode images at different developmental stages showing HR changes across lifecycle. (f) Examples demonstrating cardiac activity period (CAP) calculation. Scale bars in (c) and (d) represent 500 μm.
Fig 2.
Quantitative analysis of functional and structural cardiac parameters in 24B-GAL4/+ and UAS-dCry-RNAi; 24B-GAL4 flies at different developmental stages (a-d).
(a) Heart rate (HR), (b) Cardiac activity period (CAP), (c) End diastolic area (EDA) and (d) End systolic area (ESA). Both groups exhibit similar variations in HR and CAP at most of the early time points; however, differences in functional parameters became more prominent towards late pupa stages. Differences in structural parameters were more significant shortly after heart remodelling, i.e. PD2 40h, PD2 48h and PD3 56h. Red dotted line in (c) and (d) separates measurements obtained from A7 segment during early stages and those obtained from A1 segment during later stages. Black line in (a) represents lack of significant difference in HR between L2 and L3 (p = 0.37); all other time points showed significant difference compared to L2 (p < 0.001). * denote significant difference between 24B-GAL4/+ and UAS-dCry-RNAi; 24B-GAL4 flies at respective time points (*, p < 0.05; **, p < 0.01 and ***, p < 0.001). (e) Cardiac developmental diastasis duration. Comparison of (f) EDA and (g) ESA of 24B-GAL4/+ (n = 25) and UAS-dCry-RNAi; 24B-GAL4 (n = 23) flies that emerged as adult flies on adult day 1. Both EDA and ESA were significantly smaller in UAS-dCry-RNAi; 24B-GAL4 flies compared to control flies on adult day 1. Results are shown as mean ± s.e.m.
Fig 3.
Validation of the OCM observations indicating cardiac dysfunction and structural defects in UAS-dCry-RNAi; 24B-GAL4 flies through whole heart and cardiac ultrastructure analysis.
a) En face OCM projection of an adult UAS-dCry-RNAi; 24B-GAL4 fly. The red dotted line shows the imaging location corresponding to the axial OCM image shown in (c). (b, c) Representative axial OCM images of adult heart: (b) the cardiac tube of a control fly (24B-GAL4/+) appeared normal; however, (c) the cardiac tube of a UAS-dCry-RNAi; 24B-GAL4 fly on AD1 appeared slightly deformed with under-developed conical chamber (dotted circle) and folds along the dorsal surface (arrow). (d, e) Micrographs of whole adult heart by F-actin immuno-fluorescent staining: (d) Control fly (24B-GAL4/+) showed a normal cardiac tube. (e) Silencing of dCry in heart (UAS-dCry-RNAi; 24B-GAL4) resulted in a smaller, underdeveloped and irregular cardiac tube. Yellow dotted arrows point towards the cardiac tube. (f, g) Ultrastructure of adult heart longitudinal sections between A1 to A3 segments by TEM: (f) Control fly showed normal myofibril structure. (g) Silencing of dCry in heart resulted in immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria. Scale bars: 200 μm in (a), (b) and (c), 100 μm in (d) and (e), and 500 nm in (f) and (g).
Fig 4.
Silencing of dCry resulted in segment polarity phenotypes.
(a, c and d) Control larva (24B-GAL4/+) showed regular denticle belts in posterior A6 and A7 segments. (b, e and f) Silencing of dCry (UAS-dCry-RNAi; 24B-GAL4) results in disorganized cuticular morphologies in A6 denticle belt and significantly increased, enlarged and disorganized A7 denticle belt (denoted by arrows). (g, h) Control flies showed normal and organized notum bristles and A6 and A7 denticle belts. (i, j) The few emerged UAS-dCry-RNAi; 24B-GAL4 adult flies showed a smaller notum with disoriented and up-pointing bristles in notum (arrow in i), and disorganized and partially absent A6 and A7 denticle belts (arrow in j).
Fig 5.
Silencing of dCry led to abnormal wing vein distribution and Wg expression.
(a, b and c) Control fly with heterozygous En-GAL4; UAS-GFP alone (En-GAL4; UAS-GFP /+) exhibited normal wing. (d, e and f) Silencing of dCry in the wing (UAS-dCry-RNAi/UAS-GFP; En-GAL4) resulted in a marked increase in the acv, pcv (arrows in e), M, L3 and L4 wing veins. L4 vein was disorganized with extra veins in the distal part (arrow in f). (g) Control flies showed normal Wg expression pattern (a broad strip in the notum, a thinner strip in the prospective wing margin-dorsal/ventral (D/V) boundary, and a strip encircling the prospective wing blade). (h) Merged images of the expression of Wg and co-overexpression of GFP in the pattern of En in control flies. (i) In the dCry-RNAi wing discs, Wg expression level was markedly increased and Wg expression pattern was disorganized. (j) Merged images of the expression of Wg and co-overexpression of GFP in the pattern of En in UAS-dCry-RNAi/UAS-GFP; En-GAL4 flies.