Fig 2: Robo2 knockdown inhibited the development of liver fibrosis and the activation of Smad2/3 and PI3K/Akt signalling pathways in mouse model (A) The distribution and expression of α‐SMA and the GFP carried by the AAV vector were detected in the liver tissue sections of CCl4+sTREM‐1+AAV‐shNC and CCl4+sTREM‐1+AAV‐shRobo2‐treated mice (200X). (B) Liver histology (HE staining, 200X). (C) Liver fibrosis (Sirius Red staining, 100X). (D, E) α‐SMA and collagen I expressions (IHC staining, 200X). (F, G) Western blot analysis showed the protein expressions of Robo2, α‐SMA, collagen I, p‐Smad2, p‐Smad3, Samd2/3, p‐PI3K, PI3K, p‐Akt and Akt in CCl4+sTREM‐1+AAV‐shNC and CCl4+sTREM‐1+AAV‐shRobo2 groups of primary HSCs. * p < 0.05, ** p < 0.01

Fig 3: Liver fibrosis‐related protein expressions under Robo2 knockdown in LX‐2 cells (A, B) Western blot analysis showed the protein expressions of Robo2, α‐SMA, collagen I, p‐Smad2, p‐Smad3, Samd2/3, p‐PI3K, PI3K, p‐Akt and Akt in sTREM‐1+Lv‐shNC and sTREM‐1+Lv‐shRobo2 groups of LX‐2 cells. * p < 0.05

Fig 4: Multipolar horizontal cells use protrusions and navigate along repulsive Slit-Robo and attractive neurturin signaling(A) Chromobody (CB)-labelled F-actin (UAS:ActinCB-TagGFP2; ptf1a:Gal4, cyan) in apically migrating HCs at 54 hpf. Dashed line marks the PR layer. DAPI (gray) labels nuclei. Scale bar: 5 μm.(B) Migrating HCs (cyan, magnified white dashed box) with protrusions (white arrows) contacting PRs (magenta) at 54 hpf. Tg(atoh7:gap-RFP, yellow), Tg(ptf1a:Gal4; UAS:gap-YFP, cyan), and Tg(crx:gap-CFP, magenta). Scale bars: 20 and 10 μm respectively. See Figure 1B and Video S1.(C) Expansion microscopy of HC (Tg(ptf1a:Gal4; UAS:gap-YFP) protrusions. (i) 3D rendering showing an HC (cyan) protrusion touching the PR layer (white dashed line). (ii) HCs (fire) migrating between neighboring cells with a protrusion (magnification of boxed area) extending between two cells labeled with F-actin (white). Nuclei stained with DAPI (gray/blue). Scale bars: 20 μm. See Video S4 and Figure S4A.(D) prdm1b KO in a robo2 F1 background at 80 hpf. Areas with a more intact PR layer (solid line; i) reproduce the robo2 KO phenotype, whereas areas with absent or abnormal PR layer (dashed line; ii) show enhanced HC retention in the AC layer. Magnified areas (white boxes; i/ii); PR cell layer (white arrowheads). Tg(ptf1a:DsRed, yellow) labels ACs and HCs, and Tg(lhx1:GFP, cyan) labels HCs. DAPI (gray) marks nuclei. Scale bars: 20 μm. See Figure 1E.(E) Neurturin (nrtn+nrtn-like) KO in a robo2 F1 causes near-complete HC (cyan and yellow) retention in the AC layer (yellow) at 80 hpf. Other retinal layers are similar to controls. DAPI (gray) marks nuclei. Scale bars: 20 μm. See Figures S4B, S4C, and S4E and Video S4.(F) Ret KO in robo2 F1 phenocopies neurturin depletion, causing most HCs (cyan and yellow) to remain in the AC layer (yellow) at 80 hpf. Nuclei were stained with DAPI (gray). Scale bars: 20 μm. See Figure S4C.(G) Transplantation of control cells Tg(H2A.F/Z:GFP, cyan nuclei) into neurturin KO robo2 F1 Tg(lhx1:GFP, cyan), Tg(ptf1a:DsRed, yellow). Around areas with control cells (ii and iv, magnified from white boxes), more Robo2-lacking recipient HCs (cyan and yellow) reach control PRs (white arrows), compared to areas without donor cells (i and iii, magnified from orange boxes). Scale bars: 20 μm. See Figures 2C and S4F.(H) Quantification of HCs remaining in the AC layer (>5 μm away from PRs) after the depletion of attractive cues alone or combined with Robo2 loss. N = 31 (scrambled KO), 15 (ntf3 KO), 17 (nrtn+nrtn-like KO), 33 (ret KO), 43 (robo2 F1), 13 (prdm1b KO in Robo2 F1), 28 (nrtn+nrtn-like KO in robo2 F1), 32 (ntf3 KO in Robo2 F1), and 28 (ret KO in Robo2 F1). p values (Mann-Whitney test) = 0.3531 (ntf3 KO vs. scrambled KO) ns, 0.0684 (nrtn+nrtn-like KO vs. scrambled KO) ns, 0.4805 (ret KO vs. scrambled KO) ns, <0.0001 (prdm1b KO in robo2 F1 vs. robo2 F1) ∗∗∗∗<0.0001 (nrtn+nrtn-like KO in robo2 F1 vs. robo2 F1) ∗∗∗∗<0.0001 (ntf3 KO in robo2 F1 vs. robo2 F1) ∗∗∗∗<0.0001 (ret KO in robo2 F1 vs. robo2 F1) ∗∗∗∗0.7037 (prdm1b KO vs. nrtn+nrtn-like KO in robo2 F1) ns, and 0.9835 (prdm1b KO vs. ret KO in robo2 F1) ns. Boxes show interquartile range; center line = median; whiskers = min/max; dots = individual retinae.(I) Schematic of guidance cues coordinating multipolar HC navigation. At 48 hpf, ACs start to express Slit1b and Slit2 to repulse Robo2 expressing HCs toward the PRs. Concurrently, PRs initiate the expression of secreted neurturin to further attract HCs positive for the Gfrα1/2-Ret receptor complex toward the PR layer.

Fig 5: Slit-Robo signaling is required for HC exit from the amacrine cell layer(A) 72 hpf retinae from scrambled, slit1b+slit2 F0, and robo2 F0 as well as robo2 F1 KO embryos. In scrambled KO, HCs (cyan) laminate beneath the PR layer (dotted line with arrows), forming the HC layer (dense dotted line with arrows). In slit1b+slit2 or robo2 KOs, HCs remain within the AC layer (white line with arrows). Robo2 F1 mutants phenocopy robo2 F0. Tg(lhx1:GFP) labels the HCs. Scale bars: 20 μm. See Figure S3E.(B) Quantification of HCs remaining in the AC layer (>5 μm from PRs) in scrambled, slit1b+slit2, and robo2 KO and robo2 F1, as well as in robo2 F1 outcross with wild-type control. N = 31 (scrambled), 33 (slit1b+slit2), 9 (robo2 F0), 43 (robo2 F1), and 10 (robo2 F1 oX). See Figure 1F. p values (Mann-Whitney test vs. scrambled KO) = <0.0001 (slit1b+slit2ˆ∗∗∗∗); <0.0001 (robo2 F0 ∗∗∗∗), <0.0001 (robo2 F1∗∗∗∗), and 0.05 (robo2 F1 oX wild-type ns). Boxes show interquartile range; center line = median; whiskers = min/max; dots = individual retinae.(C) Ectopically placed HCs in the AC layer (cyan and yellow) of 5 dpf robo2 F1 larvae. The lower panel shows Airyscan images of the robo2 F1 retina. Gray arrows label ectopic HCs (cyan and yellow). HCs (gray arrows) show flattened nuclei compared to round AC nuclei (white arrows). Tg(ptf1a:dsRed) labels ACs and HCs; Tg(lhx1:GFP) labels HCs and DRAQ5 (gray) labels nuclei. Scale bars: 20 and 10 μm, respectively.(D) Time-lapse montage of HC (cyan and yellow) migration in a slit2+robo2 F0 embryo. Two dividing HCs are labeled (white and gray arrows), and their sister cells tracked after division. Scale bars: 5 μm. See Video S3 and Figure S3F.(E) Division positions of HCs relative to the PR cell layer (μm) in scrambled KO (blue) and in robo2 F1 (purple) embryos. N = 67 (scrambled KO) and 75 (robo2 F1) divisions from 6 scrambled and 6 robo2 F1 embryos. p value (Mann-Whitney test) = <0.001∗∗∗∗. Dashed line = median, dotted lines = quartiles, dots = individual measurements.(F) Division positions vs. final laminar outcome of sister HCs in scrambled KO (blue) and in robo2 F1 (purple) embryos; see Figure 4E. 0/2 = both sisters correctly laminate. 2/2 = both remain in the AC layer. N = 67 (scrambled KO) and 75 (robo2 F1) division events. Boxes show interquartile range; center line = median; whiskers = min/max; dots = individual retinae.