Response to comment on 'A conserved strategy for inducing appendage regeneration in moon jellyfish, Drosophila, and mice'

Abstract

Previously we reported evidence that a regenerative response in the appendages of moon jellyfish, fruit flies, and mice can be promoted by nutrient modulation (Abrams et al., 2021). Sustar and Tuthill subsequently reported that they had not been able to reproduce the induced regenerative response in flies (Sustar and Tuthill, 2023). Here we discuss that differences in the amputation method, treatment concentrations, age of the animals, and stress management explain why they did not observe a regenerative response in flies. Typically, 30-50% of treated flies showed response in our assay.

Keywords: D. melanogaster; appendage; evolutionary biology; insulin; leucine; moon jellyfish; mouse; regeneration; regenerative medicine; stem cells.

Figure 1.. Sustar and Tuthill did not replicate our protocol.

(A) In our amputation method, muscle bundles in the residual tibia remain in place (see Figure 2). By contrast, in the method used by Sustar and Tuthil, muscle bundles collapsed (see their Figure 2B). (B) The lack of stress management in the protocol of Sustar and Tuthill is reflected in the much higher mortality rate in their experiment (as reported in their Table 1).

Figure 1—figure supplement 1.. Duration of CO₂ exposure and housing density can alter the experimental outcomes.

In these experiments, adult flies were amputated across the tibia, and placed on food supplemented with leucine, glutamine, and insulin. The effects of the treatment were assessed three days after amputation by the absence of scab formation over the amputation site. (A) To perform the amputation, we normally anesthetize the flies using the lowest possible CO₂ level, and limit anesthesia duration to one to three minutes. Even at thissub-anesthetic CO₂ level, increasing the CO₂ exposure time to five minutes is enough to halving the frequency of flies responding to the treatment. (B) After amputation, we normally place up to 6 flies per vial. Increasing the housing density reduces the frequency of flies responding to the treatment.

Figure 2.. Muscle structure in the limb.

To assess the muscle structure in the limb, we analyzed the Mhc-GFP flies, in which promoter of the muscle-specific myosin heavy chain (Mhc) gene drives GFP expression. In these images, limbs were dissected and imaged using laser-scanning confocal microscopy.