Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Abstract

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Figure 1:

OCT imaging system integrated with 617 nm LED module for optogenetic control of Drosophila heart function. Please click here to view a larger version of this figure.

Figure 2:. Generating D. melanogaster animals expressing opsin in the heart.

(A) Genetic cross diagram. Females Hand-GAL4/TM6 SbTb were crossed to males carrying eNpHR2.0. The resulting Hand-GAL4/eNpHR2.0 progeny (marked by the red star) were collected for OCT imaging, and Hand-GAL4/TM6 Sb Tb were discarded based on their phenotypic appearance. (B) Genetic cross diagram. Females Hand-GAL4/TM6 SbTb were crossed to males carrying ReaChR. The resulting Hand-GAL4/ReaChR progeny (marked by the red star) were collected for OCT imaging, and Hand-GAL4/TM6 Sb Tb were discarded based on their phenotypic appearance. (C) Phenotypic differences between Hand-GAL4/opsin (red star) and Hand-GAL4/TM6 Tb progeny. Animals carrying the Tb gene mutation on the TM6 chromosome have a “tubby” body shape compared to normal, non-Tb larva or pupa. The left panel shows larvae; the right panel shows early pupae. Images also include a ruler with 1 mm marks. Please click here to view a larger version of this figure.

Figure 3:. Schematic presentation and timeline of the imaging preparation procedures.

Parental stocks are kept in fly bottles; virgin females and males are crossed in narrow vials filled with regular food (indicated by yellow color). Actively egg-laying flies are transferred to ATR-containing media (shown in brown) vials. Vials with developing progeny need to be kept in the dark from this step. 3rd instar larvae and early pupa are collected from the vial walls for imaging. Please click here to view a larger version of this figure.

Figure 4:. D. melanogaster early pupa expressing UAS-GFP (BDSC 6658) driven by Hand-GAL4 (BDSC 48396).

The fluorescence pattern confirms the heart specificity of the Hand-GAL4 driver. Please click here to view a larger version of this figure.

Figure 5:. Simulation of heart arrest, bradycardia, and tachycardia in D. melanogaster larva.

(A) OCT image of a larval body cross-section. The heart appears as a circle below the body’s surface. (B) Graphic presentation of the restorable heart arrest. The upper panel shows the timing (X-axis) of the red-light illumination (Y-axis, light source power level percentage). The middle panel indicates the change in heart area (Y-axis, square micrometers) over-time (X-axis). The lower panel shows the heart rate change (Y-axis, hertz) over-time (X-axis). (C) Graphic presentation of eNpHR2.0-mediated restorable bradycardia. The upper panel shows pulses of the red-light illumination, inducing two periods of bradycardia: 50% of the RHR and 25% of the RHR. Heart area and heart rate changes are shown on the middle and lower panels, respectively. (D) Graphic presentation of the heart pacing by activated ReaChR. The upper panel shows a series of 20 ms red light pulses occurring at RHR + 0.5 Hz, RHR + 1 Hz, and RHR + 1.5 Hz frequencies. The heart contractions follow the light pulse frequencies, as shown on the middle and lower panels. Please click here to view a larger version of this figure.

Figure 6:. Simulation of heart arrest, bradycardia, and tachycardia in D. melanogaster pupa.

(A) OCT image of pupal body cross-section. The heart appears as a circle below the body’s surface. (B) Graphic presentation of the restorable heart arrest. The upper panel shows the timing (X-axis) of the red-light illumination (Y-axis, light source power level percentage). The middle panel indicates the change in heart area (Y-axis, square micrometers) over-time (X-axis). The lower panel shows the heart rate change (Y-axis, hertz) over-time (X-axis). (C) Graphic presentation of eNpHR2.0-mediated restorable bradycardia. The upper panel shows pulses of the red-light illumination, inducing two periods of bradycardia: 50% of the RHR and 25% of the RHR. The middle and lower panels show heart area and heart rate changes, respectively. (D) Graphic presentation of the heart pacing by activated ReaChR. The upper panel shows a series of 20 ms red light pulses at RHR + 0.5 Hz, RHR + 1 Hz, and RHR + 1.5 Hz frequencies. The heart contractions follow the frequencies of the light pulse, as shown on the middle and lower panels. Please click here to view a larger version of this figure.