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

  1. Anne Sustar
  2. John C Tuthill  Is a corresponding author
  1. Department of Physiology and Biophysics, University of Washington, United States
3 figures, 2 tables and 1 additional file


Tibia amputation in wild-type (Canton-S) Drosophila legs.

(A) The experimental protocol. We amputated one hind leg per fly, at the midpoint of the tibia. After three weeks on control food or treated food, legs were fixed and analyzed. (B–E) Bright-field images of a control leg (B), four examples of freshly cut legs (C), and five examples of legs after three weeks on control food (D) or treated food (E). Insets showed magnified views of the cut site. Scale bar in B is the same for other panels.

Investigation of tissue identity in amputated legs.

Sensory neurons were labeled with ChAT-Gal4 >UAS GFP (A), muscles were labeled with phalloidin (B), and nuclei were labeled with DAPI (C) in uncut legs, freshly cut legs, and 3 weeks post-amputation with control food or treated food. Insets show magnified views of tissue within the femur and tibia. Scale bars in A are the same for all other panels except for insets in C.

The white blob on amputated tibia stumps is most likely composed of bacteria, not regenerating fly tissue.

(A) Bright-field (left) and confocal images (right) of leg stumps stained with EdU (red) and DAPI (white) to test for cell proliferation. Tibia stumps did not incorporate EdU. The positive control, fly gut (B) did stain for EdU. (C) Three additional examples of DAPI staining and one example showing lack of phalloidin staining (D) in white blobs (n=12). Note that cells in (C) are smaller and more densely packed than fly leg hemocytes (E, green = Hml > GFP), fly leg bristle sensory neurons (F, green = 39A11 Gal4>GFP), fly leg muscle cells, larval leg imaginal disc cells (H), or fly food yeast (I). The nuclei in (C and D) are consistent with small, densely packed bacteria, such as those observed in fly excrement (J).


Table 1
Summary of fly tibia amputation results.
Wild type
flies amputated240843
survival after 3 weeks117498
tibia stumpcuticle growth0%0/1170%0/498
white blob4%5/1173%16/498
phalloidin stain0%0/970%0/452
EdU stain0%0/200%0/46
sensory neuron GFP reporter
flies amputated100100
survival after 3 weeks6450
tibia stumpcuticle growth0%0/640%0/50
white blob0%0/644%2/50
Table 1—source data 1

Table of and details of amputation Experiments 1-5.
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)Canton-S wild typeCeleste Berg, UWN/AN/A
Genetic reagent (D. melanogaster)Mi{Trojan – Gal4}ChAT[MI04508-TG4.0] CG7715[MI04508-TG4.0-X]Bloomington 60317RRID:BDSC_60317; FBti0168134ChAT-GAL4
Genetic reagent (D. melanogaster)P{pJFRC7-020XUAS-IVS-mCD8::GFP}attP2Bloomington 32194RRID:BDSC_32194; FBti0131936UAS-mcd8::GFP
Genetic reagent (D. melanogaster)P{w[+mC]=Hml-GAL4.Delta}2, P{w[+mC]=UAS-2xEGFP}AH2Bloomington 30140RRID:BDSC_30140Hml >GFP
Genetic reagent (D. melanogaster)P{y[+t7.7] w[+mC]=GMR39 A11-GAL4}attP2Bloomington 50034RRID:BDSC_5003439A11-Gal4
Chemical compoundL-LeucineSigma-AldrichL80005 mM
Chemical compoundL-GlutamineSigma-AldrichG31265 mM
Chemical compoundInsulin (Human Recombinant)MP Biomedicals02193900800.1 mg/ml
Chemical compoundAlexa Fluor 647 PhalloidinThermoFisher ScientificA222871:50 in PBST
Chemical compoundEdUAbcam1461862 mg/mL in food
Commercial assay or kitClick-&-Go Plus EdU 555 Cell Proliferation Assay KitClick Chemistry Tools1351N/A
Chemical compoundVECTASHIELD Antifade Mounting MediumVector LaboratoriesH-1000–10
Chemical compoundVECTASHIELD Antifade Mounting Medium with DAPIVector LaboratoriesH-1200–10
Software, algorithmFIJIPMID:22743772RRID:SCR_002285

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  1. Anne Sustar
  2. John C Tuthill
Comment on 'A conserved strategy for inducing appendage regeneration in moon jellyfish, Drosophila, and mice'
eLife 12:e84435.