A Circadian Clock Gene, <i>Cry</i>, Affects Heart Morphogenesis and Function in <i>Drosophila</i> as Revealed by Optical Coherence Microscopy

<div><p>Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal <i>in vivo</i> are poorly understood. The <i>Drosophila</i> cryptochrome <i>(dCry)</i> is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of <i>dCry</i> showed a significant increase (>100-fold) in <i>Drosophila</i> during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the <i>Drosophila</i> heart throughout its post-embryonic lifecycle for the first time. The <i>Drosophila</i> heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of <i>Drosophila</i> showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of <i>Drosophila</i> at different developmental stages were quantitatively determined. In order to study the functional role of <i>dCry</i> on <i>Drosophila</i> heart development, we silenced <i>dCry</i> by RNAi in the <i>Drosophila</i> heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of <i>dCry</i> resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, <i>dCry</i>, plays an essential role in heart morphogenesis and function.</p></div

Silencing of <i>dCry</i> resulted in segment polarity phenotypes.

<p>(<b>a, c and d</b>) Control larva (24B-GAL4/+) showed regular denticle belts in posterior A6 and A7 segments. (<b>b, e and f</b>) Silencing of <i>dCry</i> (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). (<b>g, h</b>) Control flies showed normal and organized notum bristles and A6 and A7 denticle belts. (<b>i, j</b>) The few emerged UAS-dCry-RNAi; 24B-GAL4 adult flies showed a smaller notum with disoriented and up-pointing bristles in notum (arrow in <b>i</b>), and disorganized and partially absent A6 and A7 denticle belts (arrow in <b>j</b>).</p

Silencing of <i>dCry</i> resulted in segment polarity phenotypes.

<p>(<b>a, c and d</b>) Control larva (24B-GAL4/+) showed regular denticle belts in posterior A6 and A7 segments. (<b>b, e and f</b>) Silencing of <i>dCry</i> (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). (<b>g, h</b>) Control flies showed normal and organized notum bristles and A6 and A7 denticle belts. (<b>i, j</b>) The few emerged UAS-dCry-RNAi; 24B-GAL4 adult flies showed a smaller notum with disoriented and up-pointing bristles in notum (arrow in <b>i</b>), and disorganized and partially absent A6 and A7 denticle belts (arrow in <b>j</b>).</p

3D and M-mode OCM imaging of post-embryonic <i>Drosophila</i> lifecycle.

<p>(<b>a</b>) 3D OCM renderings of a 24B-GAL4/+ <i>Drosophila</i> flies at larva, pupa and adult stages. (<b>b</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. (<b>c</b>) Enface OCM projections showing heart metamorphosis. (<b>d</b>) Axial OCM sections showing heart remodelling during Drosophila lifecycle. * denotes the air bubble location during early hours of pupa development. (<b>e</b>) M-mode images at different developmental stages showing HR changes across lifecycle. (<b>f</b>) Examples demonstrating cardiac activity period (CAP) calculation. Scale bars in (c) and (d) represent 500 μm.</p

Silencing of <i>dCry</i> led to abnormal wing vein distribution and Wg expression.

<p>(<b>a, b and c</b>) Control fly with heterozygous <i>En-GAL4; UAS-GFP</i> alone (En-GAL4; UAS-GFP /+) exhibited normal wing. (<b>d, e and f</b>) Silencing of <i>dCry</i> in the wing (UAS-dCry-RNAi/UAS-GFP; En-GAL4) resulted in a marked increase in the acv, pcv (arrows in <b>e</b>), M, L3 and L4 wing veins. L4 vein was disorganized with extra veins in the distal part (arrow in <b>f</b>). (<b>g)</b> 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). (<b>h)</b> Merged images of the expression of <i>Wg</i> and co-overexpression of GFP in the pattern of <i>En</i> in control flies. (<b>i)</b> In the dCry-RNAi wing discs, Wg expression level was markedly increased and Wg expression pattern was disorganized. (<b>j)</b> Merged images of the expression of Wg and co-overexpression of GFP in the pattern of <i>En</i> in UAS-dCry-RNAi/UAS-GFP; En-GAL4 flies.</p

Quantitative analysis of functional and structural cardiac parameters in 24B-GAL4/+ and UAS-dCry-RNAi; 24B-GAL4 flies at different developmental stages (a-d).

<p>(<b>a</b>) Heart rate (HR), (<b>b</b>) Cardiac activity period (CAP), (<b>c</b>) End diastolic area (EDA) and (<b>d</b>) 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 <b>(c)</b> and <b>(d)</b> separates measurements obtained from A7 segment during early stages and those obtained from A1 segment during later stages. Black line in (<b>a</b>) 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). (<b>e</b>) Cardiac developmental diastasis duration. Comparison of (<b>f</b>) EDA and (<b>g</b>) 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.</p