Biophysical characterization of liposomes/lipoplexes and their fusogenicity with membranes. (A) Structures of symmetric and asymmetric cationic lipids. (B) Biophysical parameters of liposomes prepared using various ratios of lipids. (C) Fluorescence anisotropy measurement of diphenylhexatriene (DPH) in three types of liposomes, showing higher membrane fluidity of liposomes prepared using either asymmetric lipid or a 1:1 ratio of Sym-Lip and Asym-Lip. Error bars indicate s.d. (D) Schematic of lipoplex formation with anti-miRs and liposomes. (E) Scheme for reconstitution of the membrane using planarian lipids and fluorophore-tagged lipids (FRET pair of NBD-PE and Rho-PE). The proximity of the two fluorescent lipids in reconstituted planarian membrane enables FRET. Fusion of liposomes leads to separation of the fluorophores and inhibits FRET. Thus, emission of NBD-PE can be recovered. (F) Recovery of emission fluorescence at 530 nm indicates the higher fusogenic ability of cationic lipids over Lipofectamine. (G) TEM images of liposomes. (H) Confocal microscopy of planarians that were transfected with fluorescent dye-tagged anti-miRs using either the commercial agent Lipofectamine LTX or cationic liposomes. The bar chart shows the fluorescence intensity of Lipofectamine LTX and cationic liposomes (calculated using ImageJ). Error bars indicate s.d. Scale bar: 200 μm.

Validation of liposome-mediated ovo and miR-124 KD. (A) ovo(RNAi) animals (15/15) showed an eyeless regeneration phenotype in the first round of anterior regeneration after 7 dpa. (B) WISH showing the expression of miR-124c at 7 dpa during the second round of regeneration in KD animals (n=5). (C) Animals treated with 100 μM scrambled miRs rarely showed regeneration defects (4/90, 4.4%), whereas 10 μM (7/33, 21.2%), 50 μM (12/33, 36%) or 100 μM (91/112, 81%) anti-miR-treated animals frequently showed eye regeneration defects. Arrows indicate eye in the scrambled-treated or anti-miR-treated animals. Scale bars: 100 µm. (D) The percentage of animals that show various categories of photoreceptor (PR) defects observed in regenerated animals following 100 µM anti-miR treatment.

miR-124 KD disrupts regeneration of the eyes and brain. (A) Immunostaining of the optic chiasm (anti-ARRESTIN) in scrambled-treated versus anti-miR-124-treated animals (100 μM each) at 7 dpa after the second round of regeneration. 13/16 KD animals showed diverse eye defects. Arrowheads indicate defects in KD animals. (B) FISH showing the expression of ovo in the eye and immunostaining for photosensory neurons (anti-ARRESTIN) in miR-124 KD (n=10) and scrambled-treated (n=7) animals. (C) Quantification of the size of the eyes determined by the ratio of the area of ovo staining to the total body area in scrambled-treated (n=6) versus anti-miR-124-treated (n=5) animals. (D) FISH showing the expression of ovo in the eye and trail cells posterior to the eye in scrambled-treated and miR-124 KD animals at 4 dpa (n=6 animals per treatment condition). ovo⁺ trail cells were quantified in each condition. (E) Quantification of anti-SYNORF-1 staining of anteriorly regenerating animals normalized to body area in scrambled-treated versus anti-miR-124-treated (100 μM) animals (n=5 each). (F) Organization of the brain, ventral nerve cords, (anti-SYNORF-1, green) and photosensory neurons (anti-ARRESTIN, red) in scrambled (10/10) versus miR-124 KD animals regenerating either a head (10/10; top) or tail (13/16; bottom). Illustrations show the amputation plane (dashed line) and lighter colored areas indicate regenerated tissue. (G) FISH for neuronal subtypes in the brain of scrambled-treated versus miR-124 KD animals, including dopaminergic (th, red; n=6), GABAergic (gad, red; n=6) and cholinergic neurons (chat, green; n=6). Nuclei are stained with Hoechst (blue). Arrowheads indicate the neural subtypes. **P<0.01, ***P<0.001 (Student's t-test). Error bars indicate s.d. Scale bars: 50 µm in A,B,D; 100 µm in F,G.

miR-124 is required for eye-specific regeneration. (A) Methodology of eye-specific regeneration experiments. ovo dsRNA was administered by injection for 5 days, and on the sixth day the animals were amputated pre-pharyngeally. LNA anti-miRs were administered on alternative days for 8 days after 25 days of head regeneration. (B) Live images and anti-SYNORF-1 staining of ovo KD animals after 25 days of RNAi (n=15 animals). (C) Live images of ovo KD animals showing recovery of eyes after scrambled treatment (n=15/15 recovery) but not after anti-miR-124 treatment (n=12/15 failure). (D) Molecular characterization of eye phenotypes using anti-ARRESTIN (photosensory neurons and axons), opsin (photosensory neuron cell bodies) and tyrosinase (pigment cup cells). miR-124 KD animals (10/12) showed drastic reduction in opsin and tyrosinase staining, along with mispatterned photoreceptor neurons. Scale bars: 100 µm in B; 50 µm in C,D.

Target prediction to identify miR-124 targets. (A) Luciferase assay was carried out to validate miRNA targets. *P<0.05, ***P<0.001 (Student's t-test). Error bars indicate s.d. (B) FISH for porcupine-1 revealed that the organization of the intestine is indistinguishable between scrambled-treated and anti-miR-124-treated animals. (C) WISH for notum in scrambled-treated and anti-miR-124-treated animals (D) Colorimetric WISH showing slit-1 expression in scrambled-treated and anti-miR-124-treated animals at 7 dpa from the second round of regeneration. Arrows indicate the expression of slit-1 in the midline region. Scale bars: 100 µm.

Regulation of notch-2 and slit-1 by miR-124. (A) Colorimetric WISH shows broad expression of notch-2 in uninjured planarian. (B-C″) notch-2 KD leads to cyclopia (B′ versus C′; 14 dpa) and fusion of posterior gut branches (B″ versus C″; mat, green; DAPI, blue; 17 dpa). (D) Colorimetric WISH for slit-1 after long-term notch-2 RNAi. slit-1 is significantly reduced in notch-2(RNAi) planarians compared with controls. (E) Colorimetric WISH shows broad expression of numb-1 in uninjured planarian. (F) Colorimetric WISH for slit-1 in control versus numb-1(RNAi) planarians at 23 dpa. Higher magnification images of the head show ectopic slit-1⁺ cells in numb-1(RNAi) planarians. (G,G′) Double FISH showing colocalization (arrows) of miR-124c (red) and notch-2 (green). (H,H′) Double FISH for slit-1 and miR-124c during anterior regeneration. miR-124c colocalizes with slit-1 (arrows) at 3 dpa, but not at 7 dpa. Scale bars: 100 µm.