Zhang M , Chen Y , Xu H , Yang L , Yuan F , Li L , Xu Y , Chen Y , Zhang C , Lin G .

R01 HD084440 NICHD NIH HHS

	Graphical Abstract
	Figure 1. Hypothalamus Injury Inhibits Xenopus Tadpole Limb Regeneration (A–D) Hypothalamus injury by electrocautery in Xenopus tadpole. (A) is a diagram of a lateral view of a stage 53 tadpole brain. * shows the injury site of the hypothalamus (h). (B) is a ventral view of a stage 53 tadpole brain, showing the basal region of the hypothalamus (the black dotted line marks the expression domain of pomc and mc4r transcripts shown in Figure S1). (C) is a cross section of a hypothalamus-injured tadpole brain region. Black arrows indicate the damaged hypothalamus. (D) is an enlarged view of the injury site, showing disorganization of the tuberal (t) and the mammillary (m) region of the basal hypothalamus. (E–G) Examples of a hypothalamus-injured tadpole with reduced pigmentation (E) and inhibited limb regeneration at 3 and 7 days post amputation (dpa); (F) and (G), 16/20. (H) An example of a hypothalamus-injured tadpole limb, at 1 month post amputation (mpa), with one digit regenerated (5/20). More than half of the hypothalamus injured tadpoles failed to regenerate any digit (11/20); some regenerated only one digit (5/20) or two digits (4/20). (I–K) A sham-operated tadpole with normal pigmentation (I) and limb regeneration at 3 dpa (J) and 7 dpa (K). (L) An example of sham-operated control tadpole limb, at 1 mpa, with 5 digits regenerated (17/20). Some control tadpoles regenerated 4 digits (3/20). Red dotted lines demarcate regenerated regions. White arrowheads indicate amputation levels. Scale bars, 0.2 mm.
	Figure 2. Xenopus Tadpole Limb Regeneration Is Regulated by Mc4r Signaling (A) ELISA shows a decreased level of a-MSH in hypothalamus-injured tadpole limb stumps, 3 dpa. Error bars: standard derivation. **p < 0.01, Student’s t test, n = 3. (B) Expression of melanocortin receptors in Xenopus laevis by RT-PCR. (C) Detection of Mc4r transcripts by in situ hybridization in limb regenerate of a stage (st.) 53 tadpole. Mc4r mRNA is shown in purple blue. (D) Western blot detection of Mc4r in limb stumps of sham-operated or hypothalamus-injured stage 53 tadpole limb stumps, 3 dpa. Western blotting images shown are representatives for 3 independent experiments. (E) Western blot detection of Mc4r in st. 53 limb stumps, showing effective knockdown of Mc4r by mc4r-Mo1 and mc4r-Mo2. (F–I) Blastema formation in st. 53 tadpole limb is inhibited by mc4r-Mo2 or agrp and can be rescued by the overexpression of pomc DNA. Stump/regenerate of control (F), mc4r-Mo2-injected (G), agrp-injected (H), and agrp+pomc co-injected (I) limbs are shown. Red dotted lines indicate regeneration area. Insets in (F), (G), and (I) indicate efficient delivery of morpholino or DNA constructs. (J–M) Hematoxylin and eosin staining on sagittal sections of limb stump (5 dpa). (N–Q) Skeletal preparations of tadpole limbs injected with control-Mo, mc4r-Mo2, mc4r-Mo2 + mc4r DNA, and agrp + pomc DNA. White arrowheads (F–I) and black arrowheads (J–M) indicate amputation levels. Scale bars, 0.1 mm in (B)–(M); 1 mm in (N)–(Q). dpa, day post amputation. See also Table 1 and Figures S1 and S2.
	Figure 3. Elevation of Mc4r Signaling Stimulates Limb Regeneration in Stage 57 Regeneration-Defective Tadpoles (A and B) Non-regenerating st. 57 tadpole limbs (A) can be stimulated to regenerate by injection and electroporation of pomc DNA (B) 5 days post amputation (dpa). Insets in (A) and (B) are hematoxylin and eosin staining on sagittal sections of the respective limb stumps. (C and D) Detection of proliferating cells by PCNA immunofluorescence in control (C) and pomc DNA-injected (D) limb at 5 dpa. PCNA signal is shown in red. Nuclei are shown in blue. (E and F) Detection of pERK1/2 in the control limb (E) and pomc DNA-injected limb (F) at 5 dpa. pERK1/2 signal is shown in red. Nuclei are shown in blue. (G) Ventral view of a tadpole with pomc DNA injected in the left hindlimb at st. 57, and skeletal preparation of this tadpole’s limbs. 1 month post amputation. pomc DNA injected tadpole tend to regenerate mirrored digits I and/or II. (H) Skeletal preparation of an un-amputated tadpole hindlimb showing the pattern of digits (I to V). White arrowheads indicate amputation levels. Scale bars represent 100 mm in (A) and (B), 50 mm in (C)–(F), and 1 mm in (G) and (H). See also Table 1.
	Figure 4. Mc4r Is Expressed in the Mouse Digit and Is Required for Digit Tip Regeneration (A and B) Digit tip regeneration is inhibited in Mc4r-/- mouse and delayed in Mc4r+/- mouse. White dotted lines in (A) indicate the area of regeneration (r), which was measured (4 digits in each group) to obtain the data shown in (B). Error bars: standard derivation. ** indicates significant difference, Student’s t test. (C) Detection of the mc4r transcript by RT-PCR in wild-type mouse digit tip and the regenerates. dpa, day post amputation. (D and E) GFP expression in the digit tip regenerate of mc4r-gfp transgenic mice, 8 dpa. (E) is an enlarged area outline in (D). GFP+ cells are accumulating in the digit regeneration blastema. * indicates the scab, which will fall off around 12 dpa. (F–I) Detection of PDGFRa, Msx1, and b3-tubulin in the mouse digit blastema, 8 dpa. GFP shown in green, and PDGFRa, Msx1, and b3-tubulin are shown in red. (Fʹ–Iʹ) Enlarged view of areas marked by white dotted lines. Arrows indicate examples of PDGFRa+ GFP cells (Fʹ), Msx1+ GFP cells (Gʹ), and b3-tubulin+ GFP cells (Iʹ). White arrowheads indicate the digit amputation levels. Scale bars represent 0.5 mm in (A) and 0.1 mm in (D)–(I). See also Figure S4.
	Figure 5. a-MSH/Mc4r Has a Function that Substitutes for Effect of Nerve in Xenopus Limb Regeneration (A) Detection of a-MSH (shown in green) and b3-tubulin (shown in red) in stage 53 limb bud. (B) Western blot detection of b3-tubulin in control and spinal cord transected tadpole limb, 3 dpa. Similar results were observed in 3 independent experiments. (C) ELISA shows a reduced level of a-MSH in denervated tadpole limb stumps, 3 dpa. Error bars: standard derivation. *p < 0.05, Student’s t test, n = 3. (D) Western blot analysis shows decreased expression of Mc4r in denervated limb stumps, 3 dpa. Similar results were observed in 3 independent experiments. (E) Spinal cord transected tadpole limb stump, 15 dpa, with implantation of bead soaked in PBS (left) or 0.5 mMa-MSH (right). Black dotted lines demarcate limb regeneration regions. Insets in (E) are parasagittal sections of the respective limb stumps at 4dpa stained with hematoxylin and eosin. (F and G) Detection of proliferating cells in limb stumps by EdU incorporation (shown in green, F), and PCNA immunofluorescence (shown in red, G). (H and I) Detection of pERK1/2 (shown in red) in limb stump, 5dpa. (I) is an enlarged view of the area marked by white dotted lines in (H). (J) A denervated froglet regenerated a spike on the right forelimb after implantation of beads soaked with 0.5 mM a-MSH (right), 1 mpa. The left forelimb failed to regenerate when PBS-soaked beads were implanted. The white arrow in the inset shows an a-MSH bead, at an earlier stage of spike regeneration (10 days after bead implantation). White arrowheads in (E)–(J) indicate amputation plane. Scale bars represent 0.1 mm in (A) and (D)–(H) and 1 mm in (J). See also Figure S5.
	Figure 6. Mc4r Signaling Regulates Energy Metabolism in Xenopus Limb Cells (A) Relative expression of cAMP pathway components crem, c-fos, and pki in st. 53 tadpole limb regenerates, 3 dpa, normalized to st. 53 tadpole limb bud. Error bars: standard derivation, n = 3. (B) cAMP levels in control, mc4r-injected, and pomc-injected 3dpa tadpole limb regenerates, 3 dpa. Data from 3 independent luciferase assays on limb regenerate lysis, each with 12–15 limb regenerates. * p < 0.05, **** p < 0.001. Error bars: standard derivation. (C) The Agilent Seahorse XF Glycolysis stress test demonstrates an increase in glycolytic response in agrp DNA-injected and mc4-Mo2-injected limb cells, showing increased glycolysis stress in agrp- and mc4r-Mo2-injected cells. Serial injections of glucose (10 mM), oligomycin (1 mM), and 2-DG (100 mM) was performed in the Glycolysis stress test. * indicates p < 0.05, Student’s t test, data collected from 3 independent tests, each with 20 limbs. Error bars: standard derivation. (D) Agilent Seahorse XF Cell Mito Stress test of control, agrp DNA-injected, and mc4-Mo2-injected limb cells demonstrates changes of mitochondrial function and ATP production by agrp and mc4r-Mo2. * indicates p < 0.05, Student’s t test, data collected from 3 independent tests, each with 20 limbs. Error bars: standard derivation.
	Figure 7. Mc4r Signaling Regulates ROS Production in Xenopus Limb Cells (A) Quantitation of total ROS contents shows reduced ROS production in agrp- or mc4r-Mo2-injected tadpole limbs, 3 dpa. Co-expression of cyba DNA restores ROS production in mc4r-Mo2- and agrp-injected limb cells. Co-expression of pomc restores ROS contents in agrp-injected limb cells. Spinal cord transection inhibits ROS production, and a-MSH can restore ROS production in spinal cord transected (denervated) limb cells. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are from 3 independent experiments, with 12–15 tadpoles in each group. Error bars: standard derivation. (B) Real-time PCR and in situ hybridization of cyba in Xenopus tadpole limb regenerates. Error bars: standard derivation, n = 3. (C) cyba DNA injection rescues blastema formation inhibited by mc4r-Mo2 or agrp. White arrowheads indicate amputation levels; Dotted lines outline the regeneration regions. Scale bars, 0.2 mm. See also Table 1 and Figure S7.
	Fig. S1 Detection of agrp, pomc and mc4r transcripts in Xenopus brain and limbregenerates, Related to Figure 1. Whole mount in situ hybridization (WISH) of pomc, agrp, and mc4r in Xenopus tadpole brain and tadpole limb regenerates amputated at stage 53. Limb regenerates were collected at 3 and 7 days post amputation (dpa). Arrowheads indicate amputation levels. Related to Fig. 1.
	Fig. S2 Detection of Mc4r by immunofluorescence in tadpole spinal cord, and limb stumps, Related to Figure 2. (A-D) Immunofluorescence staining of Mc4r on cross sections of stage 53 tadpole spinal cord. Section incubated without primary antibody (-1Ëšcontrol, A) shows no signal after incubation with secondary antibody. (B,C) Section with both primary and secondary antibody incubation, Mc4r signals shown in red, nuclei counterstained with DAPI shown in blue. (D) Enlarged view of the area marked by dotted line in (C). (E-H) Mc4r immunofluorescence staining on sagittal sections of 5 dpa limb stump, injected with control (E,F) or mc4r-Mo2 (G,H) morpholino oligonucleotides, showing decreased expression of Mc4r by mc4r-Mo2 in the limb mesenchyme (blastema), but not in the epithelium. (F,H) Enlarged views of the areas marked by dotted lines in (E,G). Scale bars: 0.1mm. Related to Fig. 2.
	Fig. S3 AgRP inhibits limb regeneration in transgenic Xenopus, Related to Figure 2. (A) Body weight, length and food intake in HGEM-agrp transgenic and wild type (WT) young frogs, after heat shock induction of agrp. HGEM-agrp and wild type animals were heat treated daily in a 34ËšC water bath, and their body weight, length (snout-vent), width (hip girth) were measured weekly. Food pellets were provided, and the number of pellets consumed was recorded daily. Error bars: standard derivation, N = 3 (B) Localized light beam heated activation of agrp in transgenic tadpole limb leads to regeneration defects. Transgenic animals have green lens (green arrows). White dotted lines demarcate the edges of regenerating limbs. Scale bars: 0.2 mm. Related to Fig. 2.
	Fig. S4 Mc4r mutant mice, Related to Figure 4. (A,B) Mc4r-/- mice developed obesity, with significant gain of body weight after digit tip amputation. (C) Similar levels of blood glucose in mc4r-/-, mc4r+/- and wild type (WT) mice. Tail blood glucose was measured with a Roche Accu-check glucose monitor system. Error bars: standard derivation, N = 4 for each group. Scale bar in (A) 1cm. Related to Fig. 4.
	Fig. S5 Mc4r is regulated by innervation, Related to Figure 5. (A,B) Expression of a-MSH, Mc4r-GFP, and b3-tubulin on coronal section of E10.5 Mc4r-gfp mouse embryonic limb . a-MSH and b3-tubulin are shown in red, Mc4r-GFP reporter signals are shown in green. Counterstaining with DAPI is shown in blue. Scale bar: 0.1 mm. (C) Detection of a-MSH and b3-tubulin on sagittal section of transacted spinal cord shows the successful removal of spinal nerve in st.53 tadpoles. White arrowheads indicate the spinal cord transaction site. (D,E) Detection of b3-tubulin and Mc4R in control and denervated frog forelimb stumps shows diminished b3-tubulin in denervated limb, and reduced Mc4r in denervated limb stump. White arrowheads indicate amputation levels. (F,G) Detection of pERK1/2 on cross sections of limb stump implanted with a-MSH beads (marked by *). pERK1/2 is shown in red. Scale bars: 0.1mm. Related to Fig. 5.
	Fig. S6 Comparison of limb blastema and cultured limb blastema cells, Related to Figure 6. (A) Relative expression levels of genes known to function during limb growth and patterning, markers for hindlimb (hoxc10, pitx1, tbx4) and limb blastema (msx1, prx1, sall4). (B) Relative expression levels of genes of complex I-V in the electron transduction chain (ETC) for ATP generation. The compounds Rotenone targets complex I, Antimycin targets complex III, and Oligomycin targets complex V. Gene expression levels are analyzed with real-time PCR, and ratios are calculated, using blastemas as 1.0. Error bars: standard deviation. Data are from three independent experiments. Related to Fig. 6.
	Fig. S7 ROS production in Xenopus tadpole limb stumps, Related to Figure 7. (A) CellRox green staining shows diminished overall oxidative stress signals in mc4r-Mo injected tadpole limb stump, imaged at 5dpa. CellRox signals peak in 5dpa limb blastemas. (B) H2O2 production in agrp and mc4r-Mo injected HyPerYFP tadpole limbs were measured based on excitation ratio of HyPerYFP 490nm/HyPerYFP402 nm, as described in Love et al., Nat Cell Biol 15(2):222-228. Images shown represents typical observations from multiple specimens (N=6). Related to Fig. 7.

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