Comment on 'A conserved strategy for inducing appendage regeneration in moon jellyfish, Drosophila, and mice'
- Department of Physiology and Biophysics, University of Washington, United States
Figures
Figure 1
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.
Figure 2
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.
Figure 3
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).
Tables
Table 1
Summary of fly tibia amputation results.
| Control | n | Treated | n | |||
|---|---|---|---|---|---|---|
| Wild type (Canton-S) | flies amputated | 240 | 843 | |||
| survival after 3 weeks | 117 | 498 | ||||
| tibia stump | cuticle growth | 0% | 0/117 | 0% | 0/498 | |
| white blob | 4% | 5/117 | 3% | 16/498 | ||
| phalloidin stain | 0% | 0/97 | 0% | 0/452 | ||
| EdU stain | 0% | 0/20 | 0% | 0/46 | ||
| sensory neuron GFP reporter (ChAT >GFP) | flies amputated | 100 | 100 | |||
| survival after 3 weeks | 64 | 50 | ||||
| tibia stump | cuticle growth | 0% | 0/64 | 0% | 0/50 | |
| white blob | 0% | 0/64 | 4% | 2/50 | ||
| GFP | 0% | 0/64 | 0% | 0/50 | ||
-
Table 1—source data 1
Table of and details of amputation Experiments 1-5.
- https://cdn.elifesciences.org/articles/84435/elife-84435-table1-data1-v1.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | Canton-S wild type | Celeste Berg, UW | N/A | N/A |
| Genetic reagent (D. melanogaster) | Mi{Trojan – Gal4}ChAT[MI04508-TG4.0] CG7715[MI04508-TG4.0-X] | Bloomington 60317 | RRID:BDSC_60317; FBti0168134 | ChAT-GAL4 |
| Genetic reagent (D. melanogaster) | P{pJFRC7-020XUAS-IVS-mCD8::GFP}attP2 | Bloomington 32194 | RRID:BDSC_32194; FBti0131936 | UAS-mcd8::GFP |
| Genetic reagent (D. melanogaster) | P{w[+mC]=Hml-GAL4.Delta}2, P{w[+mC]=UAS-2xEGFP}AH2 | Bloomington 30140 | RRID:BDSC_30140 | Hml >GFP |
| Genetic reagent (D. melanogaster) | P{y[+t7.7] w[+mC]=GMR39 A11-GAL4}attP2 | Bloomington 50034 | RRID:BDSC_50034 | 39A11-Gal4 |
| Chemical compound | L-Leucine | Sigma-Aldrich | L8000 | 5 mM |
| Chemical compound | L-Glutamine | Sigma-Aldrich | G3126 | 5 mM |
| Chemical compound | Insulin (Human Recombinant) | MP Biomedicals | 0219390080 | 0.1 mg/ml |
| Chemical compound | Alexa Fluor 647 Phalloidin | ThermoFisher Scientific | A22287 | 1:50 in PBST |
| Chemical compound | EdU | Abcam | 146186 | 2 mg/mL in food |
| Commercial assay or kit | Click-&-Go Plus EdU 555 Cell Proliferation Assay Kit | Click Chemistry Tools | 1351 | N/A |
| Chemical compound | VECTASHIELD Antifade Mounting Medium | Vector Laboratories | H-1000–10 | |
| Chemical compound | VECTASHIELD Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200–10 | |
| Software, algorithm | FIJI | PMID:22743772 | RRID:SCR_002285 |
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Comment on 'A conserved strategy for inducing appendage regeneration in moon jellyfish, Drosophila, and mice'
eLife 12:e84435.
https://doi.org/10.7554/eLife.84435
