BUSCADOR RECOLECTA

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(A) Schematic of a 5-dpf liver, highlighting the biliary network. (B-B’) Maximum projection (200 μm z-stack) of a 120-hpf liver expressing tp1:H2B-mCherry (BEC) and stained for Hnf4ɑ (hepatocyte). Autofluorescent blood cells appear in bright white. (N = 4, n ≥ 12 livers) (C) Relative distribution of BECs and hepatocytes at 120 hpf (N = 4, n ≥ 12 livers). (D-F) Mathematical models simulating hepatoblast differentiation employing different parameter combinations: proliferation rates of differentiated cell types is equal (D, F) or slower in BECs (E). Hepatoblasts either are all bipotent (D, E) or represent a heterogeneous population with mixed probabilities for uni-or bipotent differentiation (F). Plots showing the simulated cell proportions over simulation time (n = 10) and the final cell type ratio in bar graphs. The numerical values that were used to generate the graphs in (C-F) can be found in S1 Data. BEC, biliary epithelial cell; dpf, day postfertilization; Hb, hepatoblast; Hc, hepatocyte; hpf, hours post fertilization., Peer reviewed

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(A, B) Confocal images of the same liver showing the embryonic left liver lobe at 5 dpf with a 3-cell mKate2+ clone (A) and at juvenile stage (SL = 14.4 mm) including a continuous Kate2+ clone in the ventral lobe (N = 1, n = 1 liver). (C, D) Juvenile livers (C–SL = 8.46 mm and D–SL = 10.93 mm) with connected clusters that are oriented along the tissue edge and spread through the left and the ventral lobe. Arrows indicate cluster growth direction (N = 4, n = 14 livers). (E-P) Brightfield images of stages I-VI livers in loco within the fish (E, G, I, K, M, O) or dissected out (F, H, J, L, N, P). In (M), the liver is removed and the gut bend is visible. A, anterior; P, posterior; R, right; L, left; RL, right lobe; LL, left lobe; VL, ventral lobe., Peer reviewed

(A) Fish standard length (SL) plotted against fish age. (B, C) Fish weight (B) and liver weight (C) increases with SL represented in a semi-log plot. (D) Liver-to-body weight ratio during postembryonic growth is constant in adult fish. (N > 10, n ≥ 300 fish). Gender of the corresponding samples is colour coded: male (blue), female (pink), and ND (green). The numerical values that were used to generate the graphs can be found in S1 Data., Peer reviewed

Supplementary methods, tables (1-4) and figures legends (1-10)., Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS−/−) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS−/− mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS−/− livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease., This work was supported by the MICINN Retos RTI2018-095673-B-I00, PID2020-11782RB-I00, PID2020-117941RB-I00, all of which were co-funded with FEDER funds, AMMF 2018/117, COST Action CA17112 and Comunidad de Madrid S2022/BMD-7409. This project has received funding from the European Horizon’s research and innovation program HORIZON-HLTH-2022-STAYHLTH-02 under agreement No. 101095679. The research group belongs to the validated Research Groups Ref. 970935 Liver Pathophysiology, 920631 Lymphocyte Immunobiology and IBL-6 (imas12-associated). KZ was supported by the China Scholarship Council. SM-G was supported by a predoctoral scholarship from Complutense University., Peer reviewed

(A, B) Distribution of nuclear distance to the liver surface displayed for hepatocytes and EdU+ hepatocyte (A), and BECs and EdU+ BECs (B) (N = 2, n ≥ 6 livers). (C, D) Distribution of nuclear distance to the nearest neighbour (NN) shown for hepatocytes and EdU+ hepatocytes (C) and BECs and EdU+ BECs (D) (N = 2, n ≥ 8 livers). The numerical values that were used to generate the graphs can be found in S1 Data., Peer reviewed

(A) Adult liver displaying clones along the central vein; recombination was induced in hepatoblasts at 26 hpf (n = 9 livers). (B, C) Adult livers exhibiting giant clusters in the ventral lobe (n = 3 livers). For (A-C) total numbers: N = 9, n = 79 livers. (D) Adult liver with a cluster along a central vein (n = 5 livers) and (E) clusters oriented in lateral stripes (n = 10 livers) upon recombination induced in hepatocytes. For (D, E) total numbers N = 4, n = 31 livers). (F) Giant clusters in the ventral lobe are also apparent in juvenile livers when labelling was induced at 4 dpf in hepatocytes (n = 1; total N = 5, n = 42 livers). (G) Confocal section showing the kdrl:GFP+ sinusoidal architecture in the adult liver counterstained with DAPI (N = 1, n = 2 livers). (H-J) No recombined cells were detected in 84% noninduced control livers of long-term lineage tracing experiments showed no recombined cells (H; N = 15, n = 84 livers), and the majority of recombined samples (N = 15, n = 100) show only one recombined clone of a few labelled cells (I, J; N = 15, n = 16 livers)., Peer reviewed

(A) Distribution of all labelled cells per livers. (B) Frequency of manually assigned mixed clone sizes (N = 5, n = 16 clones). (C) Cell type distribution within a mixed clone (N = 5, n = 16 clones). (D) Clone size distribution for different colours represented in a semi-log plot shows highly consistent values and good fit to a simple exponential distribution (black line), expected for a single population undergoing stochastic division. (E) Number of cell divisions of manually defined clones fit a Poisson distribution (black line) as expected for stochastic divisions. (F) Cumulative probability of a certain number of labelled cells per liver (N = 6, n = 97), showing that most livers have less than 50 labelled cells, but with heavy tails (10%–20%) of highly induced livers. (G) Probability that a given cell had a neighbouring cell with the same (red line) or a different colour (black line). Plots show subsets of the data that included livers with a total number of less than 10, 40, 50, or 100 labelled cells. Both distributions show high overlap for highly induced livers, which signifies poor clonality. For distances of less than 50 μm and livers with less than 40 cells, the ratio of “same” to “different” colour is high, meaning that nearby cells of the same colour are unlikely to be nonclonal. (H) Manually determined size distribution of clones (black line) plotted together with different reconstructed clone size distributions. Different lines correspond to the regrouping of neighbouring cells of the same colour in the same clone if present within defined radii. Plots show subsets of the data that included livers with a total number of less than 10, 40, 50, or 100 labelled cells. The numerical values that were used to generate the graphs can be found in S1 Data., Peer reviewed

(A, B) (B) A 5 μm projection of tg(prox1a:kalTA4; UAS:GFP) embryos stained for 2F11 and DAPI at 80 hpf (A) and 96 hpf (B). White arrowheads indicate GFP+ BEC nuclei, and yellow arrows highlight GFP− endothelial cells. (N = 2, n = 8 livers). (C) A 10 μm projection of an adult liver section stained for Prox1 (magenta) and Anxa4 (green), white arrowheads indicate Prox1+ BEC nuclei, and yellow arrows highlight Prox1− endothelial cells. The Prox1 signal was filtered using a median filter with a 3-pixel kernel for better visualisation. (N = 2, n = 6 sections). (D) Schematic representation of the stepwise activation times of the fraeppli transgene. (E) PCR amplification of the mKate2 locus in individual embryos at 26 hpf or 33 hpf upon heat shock–mediated recombination at 26 hpf. (N = 2, n ≥ 16 embryos). (F) Distribution of the number of FRaeppli recombined loci per embryo upon heat shock at 26 hpf determined by PCR amplification of the recombined transgene. Band intensities at 26 hpf were about 4–6 times lower compared to later time points (N = 2, n ≥ 11 embryos). (G) Quantification of total liver cell numbers, encompassing hepatocytes and BECs, during development (N = 4, n ≥ 12 livers). Different shape data points indicate different experiments. (H, I) fraeppli-nls embryo activated by phiC31 mRNA injection showing only TagBFP and mTFP1 expression at 60 hpf (H), and expression of all 4 FRaeppli FPs at 120 hpf (n = 4 livers) (I). Temporal FP colour detection reflects the individual protein maturation times and depends on the strength of the respective Gal4-driver [32]. (J) Timelapse of TagBFP+ and mTFP1+ cells using spectral imaging of the liver upon heat shock induction at 9 hpf (N = 2, n = 3 livers). Some neighbouring cells stay close together (magenta arrow), while others move up to 20 μm apart (green arrows). (K, L) fraeppli-nls embryo reimaged at 60 hpf, 72 hpf, and 100 hpf. In sparse recombined embryos (K), not all 4 FRaeppli colours are expressed at 100 hpf and an individual labelled cell divides 2 times (n = 2 livers). In highly recombined embryos expression of all 4 colours is visible at 100 hpf (n = 10 livers). (Total N = 2, n = 18 livers). The numerical values that were used to generate the graphs in (F, G) can be found in S1 Data., Peer reviewed