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MARCKS-like protein is an initiating molecule in axolotl appendage regeneration

Abstract

Identifying key molecules that launch regeneration has been a long-sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a considerable portion of the body part has been removed1,2,3. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals4,5. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Here, using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS-like protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. The identification of a regeneration-initiating molecule opens the possibility of understanding how to elicit regeneration in other animals.

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Figure 1: Extracellular AxMLP identified by expression cloning is necessary and sufficient for cell cycle re-entry in vitro.
Figure 2: AxMLP is sufficient to induce cell cycle entry in vivo.
Figure 3: AxMLP induces cell cycle re-entry of muscle-derived cells in the newt limb.
Figure 4: AxMLP is necessary for cell proliferation during early tail regeneration.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The sequence of AxMLP has been deposited in the NCBI GenBank database under accession number KT367888.

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Acknowledgements

We thank A. Tazaki, Y. Taniguchi, I. Wagner, A. Rodrigo-Albors, D. Knapp, P. Murawala, B. Borgonovo and D. Drechsel for technical advice and important discussions; M. Schuez, A. Telzerow and Y. Taniguchi for assistance; B. Gruhl, A. Wagner, S. Mögel for animal care; and J. Currie, T. Sandoval-Guzman and E. Nacu for comments on the manuscript. E.M.T. was supported by a German Federal Ministry of Education and Research (BMBF) Biofutures grant, German Research Foundation (DFG) grant TA274/5-1, European Research Council Advanced Grant, Institutional funding from the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), and the DFG Research Center for Regenerative Therapies Dresden (CRTD) and A.S. by the Swedish Research Council and Cancerfonden.

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Affiliations

Authors

Contributions

R.B. performed oocyte injection assay and established expression cloning. T.S. designed and performed expression cloning, in vitro cell assays, biochemical experiments, in vivo axolotl experiments, analysed experiments and data, and wrote the manuscript. E.M.T. conceived of the project, analysed experiments and data, wrote the manuscript and secured funding. H.W. designed and performed in vivo newt experiments, analysed the data and wrote the corresponding parts of the manuscript. A.S. supervised and designed in vivo newt experiments, analysed data, edited the manuscript and secured funding.

Corresponding authors

Correspondence to Takuji Sugiura or Elly M. Tanaka.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Schematic illustration of the expression cloning approach.

a, 110,592 clones from a 6-day tail blastema library were arrayed on 288 × 384-well plates. b, One 384-well plate was pooled into one conical tube and called a ‘pool’. In total, 288 pools were prepared from the library. c, Twenty-four pools were combined in one conical tube and called a ‘superpool’ (SP) containing 9,216 clones. In total 12 superpools were prepared. d, Bacteria of each superpool was cultured and plasmid was prepared. e, The superpool plasmids were transfected into HEK293 cells. f, Individual supernatants were tested on A1 myotubes for cell cycle re-entry activity (myotube assay) (see Fig. 1b). Positive superpools were successively subfractionated and the assay process was repeated back from the positive superpool (first screen) to come to a single clone (fourth screen) (ac, right) (see Extended Data Fig. 2a–c).

Extended Data Figure 2 Expression cloning of AxMLP as a myotube cell cycle inducer.

a, The results of the second-round screen of superpool 9 (see Fig. 1b and Extended Data Fig. 1) and its sub-pooling diagram (right). Sub-pool D and sub-pool 2 showed higher BrdU incorporation activity than the others, identifying pool number 212 as positive (n = 12: 4 biological, 3 technical replicates each; mean ± s.d.). b, The result of the third-round screen of pool number 212 from superpool 9 and its sub-pooling diagram (right). Sub-pool A1 showed activity (n = 6: 2 biological, 3 technical replicates each; mean ± s.d.). c, Fourth-round screen of SP9 identified a single active clone (c1), AxMlp (n = 12: 4 biological, 3 technical replicates each; mean ± s.d.). The pooling diagram is shown on the right side. d, AxMLP supernatant induces an S-phase response in a dose-dependent manner in the newt myotube assay. Different amounts of AxMLP-containing supernatant (30 μl, 20 μl, 10 μl and 5.0 μl, respectively) were provided to the myotube cell culture medium. The myotube BrdU incorporation correlated with the amount of supernatant provided, whereas pCMV-SPORT6 supernatant did not provoke cell cycle entry at any dose (n = 6: 2 biological, 3 technical replicates each; mean ± s.d.). e, f, Newt myotubes treated with purified AxMLP (e) or flow-through (f) were immunostained for BrdU and MHC. More BrdU-incorporated nuclei (red) in myotubes (green) were observed in culture supplied with purified AxMLP compared with flow-through-treated cultures. Scale bar, 1 mm.

Extended Data Figure 3 AxMLP is classified as a member of the MARCKS family and characterization of its extracellular release in HEK293 cells.

a, Amino-acid sequence alignment of AxMLP with sequences from other vertebrates, human, mouse, rat, chick, newt, Xenopus and zebrafish. AxMLP contains three conserved domains: (1) myristoylated N terminus domain; (2) MARCKS homology domain; and (3) effector domain. b, A phylogenetic tree of vertebrate MARCKS family proteins. The tree was constructed by the neighbour-joining method with the ClustalW program. The percentage beside the nodes shows that a node was supported in 1,000 bootstrap pseudo replications. The scale bar indicates evolutionary distance. c, Schematic illustration of His-tagged AxMlp (left) and eGFP-fused AxMlp (right). 3C protease PreScission site was inserted between AxMlp and the tag for both constructs. d, e, AxMLP does not induce significant cell death. The percentage of GFP-expressing HEK293 cells (d) and absolute number of the cells (e) (n = 16: 4 biological, 4 technical replicates each; mean ± s.d.; centre values as median; whiskers as maximum and minimum, respectively) at the indicated time points of culture. There was no significant difference with Student’s t-test between AxMlp-transfected cells and the control in any time points. f, Characterization of anti-AxMLP antibodies by western blot. Cell lysates from HEK293 cells transfected with the indicated plasmids were tested for the full-length AxMLP polyclonal antibody (left) and C-terminal AxMLP polyclonal antibody (right). g, Silver staining of the fractions from AxMLP–His purification. Bovine serum albumin (BSA) was added to purified fraction as a carrier protein. h, AxMLP–His purification analysed by anti-His-tag western blotting. NS, not significant.

Extended Data Figure 4 AxMLP is sufficient to induce cell cycle entry in axolotl tail and limb.

ad, Sections from AxMLP-injected tails immunostained for BrdU/PAX7 (a, b) and BrdU/MEF2C (c, d) (refers to data in Fig. 2). Scale bars, 100 μm. e, Schematic illustration of the protein injection into axolotl limb. f, Quantification of BrdU+ cells in the limbs injected with PBS, flow-through or purified AxMLP (n = 4: biological replicates; centre values as median; points represent each sample). gn, Transverse sections from purified AxMLP-injected (gj) or flow-through-injected limbs (kn). Scale bars: lower-magnification images, 200 μm; higher-magnification images, 50 μm. Sections were immunostained for BrdU (g, k), BrdU/myelin basic protein (MBP) (h, l), BrdU/PAX7 (i, m) and BrdU/GFP (j, n). GFP+ cells represent connective tissues in lateral plate mesoderm (LPM)-GFP transplanted axolotls. All molecular markers used except MBP had nuclear expression, and therefore allowed one-to-one colocalization of nuclear BrdU with nuclear staining of the marker. Therefore, we refer to the MBP data as ‘MBP-associated’. White boxes highlight the magnified images. Yellow circles indicate two bones in the lower limb. NS, not significant; *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005 with Student’s t-test. White arrowheads indicate marker+/BrdU+ cells.

Extended Data Figure 5 Upregulation of AxMlp transcript during early regeneration and alteration of AxMLP protein localization in wound epidermis cells.

a, e, Measurement of AxMlp expression by qPCR at the indicated time points during tail (a) and limb (e) regeneration (n = 3: biological replicates; mean ± s.d.). To obtain the values of fold-change for each time point, the relative concentrations of the PCR products were calculated by the standard curve method. The concentration of AxMlp was normalized to that of large ribosomal protein 4 (Rpl4). bh, Immunostaining with anti-AxMLP antibody (white) on tails (bd) and limbs (fh) of intact (b, f: transverse sections), 1 dpa (c: sagittal; g: horizontal) and 6 dpa (d: sagittal; h: horizontal) samples. By 6 dpa, the epithelial organization and AxMLP expression appeared to be returning to a less tightly adherent, less membrane-associated appearance (d, h). Scale bars: left, 200 μm; right 50 μm. Red arrowheads indicate spinal cord; green arrowheads indicate wound epidermis; yellow arrowheads indicate normal epidermis; yellow circles indicate two bones in the lower limb.

Extended Data Figure 6 AxMLP morpholinos specifically and efficiently reduce AxMLP translation in cultured cells.

a, Schematic illustration of wild-type (WT) AxMlp (top) and N-terminal deletion AxMlp (bottom) constructs used to characterize AxMlp morpholino 1. The N-terminally deleted AxMlp lacks the morpholino-binding site. Both constructs have a His-tag on their C terminus (a). ED, effector domain; M, myristoylated N terminus domain; MH, MARCKS homology domain. bi, Electroporated A1 myoblasts were stained with the indicated markers. be, Wild-type AxMlp plasmid was co-electroporated with the control morpholino (c) or the AxMlp-specific morpholino 1 (d), whereas wild-type AxMlp plasmid only (b) or wild-type AxMlp only without any primary antibody staining were used as negative controls (e). fh, ΔN-AxMlp plasmid was co-electroporated with the control morpholino (g) or the AxMlp-specific morpholino 1 (h), whereas ΔN-AxMlp plasmid only (f) or pCMV-SPORT6-3C-His (empty vector) plasmid only served as negative controls (i). j, Western blotting for the cell lysates from the experiment above. AxMLP morpholino 1 specifically reduced AxMLP protein expression. k, Schematic illustration of the constructs used to characterize AxMlp morpholino 2. The original AxMlp expression clone from the cDNA library (BL212a101: top) was used as it included the 5′ untranslated region (UTR) target site for AxMlp morpholino 2. The subcloned AxMlp-His construct lacks the binding site for AxMlp morpholino 2 and was used as the control construct. lr, Electroporated A1 myoblasts were stained with the indicated markers. ln, BL212a101 plasmid was co-electroporated with the five-mismatch control morpholino (m) or the AxMLP-specific morpholino 2 (n), or BL212a101 plasmid only (l) or pCMV-SPORT6-3C-His (empty vector) plasmid only served as negative controls (o). AxMLP was detected using an anti-AxMLP antibody (red), and morpholinos were detected via FITC conjugation (green). pr, AxMlp-3C-His plasmid was co-electroporated with the five-mismatch control morpholino (q) or the AxMlp-specific morpholino 2 (r) or AxMlp-3C-His plasmid only (p). AxMLP was detected using an anti-His-tag antibody (red), and morpholinos were detected via FITC conjugation (green). s, Western blotting for the cell lysates from the experiment above. AxMlp morpholino 2 specifically reduced AxMLP protein expression. Scale bars, 100 μm.

Extended Data Figure 7 AxMLP morpholinos knockdown endogenous AxMLP in vivo.

aj, The morpholinos shown in were used in Fig. 4 and Extended Data Figs 6a–j,8a–f. kt, The morpholinos shown were used in Extended Data Figs 6k–s, 8g–j. Transverse sections from AxMLP-specific morpholino 1 (ae) or control morpholino (fj) electroporated tail. b, The spinal cord (SC) boxed in a. c, The higher-magnification images of the spinal cord boxed in b. AxMLP expression was detected in morpholino-negative cells (red asterisks), whereas it was reduced in morpholino-positive cells (yellow asterisks). d, The epidermis boxed in a. e, AxMLP expression was unaffected in morpholino-negative cells (red asterisks), whereas it was reduced in morpholino-positive cells (yellow arrowheads). In the control morpholino-electroporated tail (f) there was no morpholino-specific knockdown phenotype in either spinal cord (g, h) or epidermis (i, j). The same experiments were performed with AxMLP-specific morpholino 2 (ko) and the corresponding five-mismatch control morpholino (pt). kt, The data sets were the same as aj. Scale bars, 200 μm (a, f, k, p); 50 μm (be, gj, lo, qt).

Extended Data Figure 8 AxMLP is necessary for initial cell proliferation during tail regeneration.

ad, Representative transverse sections of the morpholino-electroporated/protein-injected blastemas that were used for quantification of BrdU incorporation in Fig. 4d. Rhodamine was co-injected with the protein samples. e, Quantification of BrdU+ cells in blastema sections of morpholino-electroporated/protein-injected tails at 3 dpa (n = 4: biological replicates; centre values as median; points represent each sample). f, The length of the blastema during tail regeneration. The data at 6 dpa were plotted in Fig. 4c. By 14 days the difference in total regenerate length among the samples was not statistically significant. gj, The same experimental scheme (shown in Fig. 4a) as was used for AxMLP morpholino 1 was implemented for a second specific morpholino (AxMLP-specific morpholino 2). g, Bright-field images of the morpholino-2-electroporated/protein-injected tails at 6 dpa. Red bars indicate amputation planes. Dashed lines delineate the shape of the mesenchymal blastema. h, Blastema length at 6 dpa (n = 4: biological replicates; centre values as median; points represent each sample). i, The length of the blastema during tail regeneration. The data at 6dpa were plotted in h. j, Transverse sections immunostained for BrdU from morpholino-electroporated/protein-injected tails at 3 dpa. AxMLP-specific morpholino 2 combined with flow-through (FT) injection shows reduction of BrdU incorporation, whereas AxMLP protein injection rescues the phenotype. The corresponding five-mismatch control morpholino does not affect BrdU incorporation. Yellow circles indicate spinal cord (top) and notochord/cartilage (bottom). NS, not significant; **P < 0.005, ***P < 0.0005, ****P < 0.00005 with Student’s t-test. Scale bars, 200 μm (ad, j); 500 μm (g).

Extended Data Figure 9 Anti-AxMLP antibody significantly blocks BrdU incorporation during tail regeneration.

a, Schematic illustration of antibody injection into axolotl tail. b, Quantification of BrdU+ cells in blastema sections of antibody-injected tails at 3 dpa (n = 4: biological replicates; centre values as median; points represent each sample). NS, not significant; **P < 0.005, ***P < 0.0005, ****P < 0.00005 with Student’s t-test.

Extended Data Figure 10 Exogenous AxMLP accelerates normal tail regeneration.

a, Schematic illustration of the protein injection into axolotl tail and blastema. b, Bright-field images of the protein-injected tails at 4 dpa. c, Blastema length at 4 dpa (n = 6: PBS, FT; n = 8: AxMLP, biological replicates; centre values as median; points represent each sample). The blastema from purified AxMLP injected tails significantly increased the regenerate length. Scale bar, 500 μm. Red bars indicate amputation planes; dashed lines delineate the shape of the mesenchymal blastema. NS, not significant; ***P < 0.0005 with Student’s t-test.

Supplementary information

Supplementary Table 1

This table contains raw values of qPCR in tail regeneration time course (a, ExFig. 5a) and limb (b, ExFig. 5e). (XLSX 48 kb)

Supplementary Table 2

A list of oligos used in this manuscript, see “Plasmid construction” section in the Online Methods. (XLSX 51 kb)

AxMLP transfected HEK293 cells proliferate and did not show additional cell death.

Time-lapse videos of HEK293 cells transfected with AxMLP-3C-His plasmid between 5 and 72 hours after transfection. The culture medium was changed at 24 hours post transfection from 10% serum medium to the serum free, FreeStyle 293 medium. (MOV 4861 kb)

pCMV-SPORT6-3C-His transfected HEK293 cell proliferation during culture

Time-lapse videos of pCMV-SPORT6-3C-His as a negative control to compare to AxMLP transfected HEK293 cells (Video 1) between 5 and 72 hours after transfection. The culture medium was changed at 24 hours post transfection from 10% serum medium to the serum free, FreeStyle 293 medium. (MOV 4859 kb)

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Sugiura, T., Wang, H., Barsacchi, R. et al. MARCKS-like protein is an initiating molecule in axolotl appendage regeneration. Nature 531, 237–240 (2016). https://doi.org/10.1038/nature16974

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