BUSCADOR RECOLECTA

(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

(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

(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-C) Mathematical models simulating hepatoblast differentiation, based on heterogeneous hepatoblast potentials (A, B) or differential proliferation times (C; n = 10). (D, F) Presentation of 10 μm sections from juvenile (D) and adult (F) livers stained for fabp10a:GFP (hepatocytes), tp1:H2B-mCherry (BECs), and DAPI (nuclei). (E) Relative distribution of BECs and hepatocytes in juvenile liver (N = 4, n = 4 livers and 18 ROIs). (G) Relative distribution of BECs and hepatocytes at the organ centre (N = 1, n = 1 liver, 4 sections) or periphery in adult livers (N = 1, n = 1 whole-mount liver). The numerical values that were used to generate the graphs in (A-C, E, G) can be found in S1 Data., Peer reviewed

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Native gels were incubated with NADH (as a substrate), and nitro blue tetrazolium (NBT, as the electron acceptor). CI activity is shown in purple., Peer reviewed

(A) Activities of MRC complex (I–IV) were assayed spectrophotometrically, and the results were normalized to citrate synthase (CS) activity in mitochondria isolated from parental (Par) and Ndufs4−/− RAW 264.7 cells. (B) Left panel, a representative experiment showing OCR in RAW 264.7 sublines before and after the sequential addition of oligomycin (2.6 μM), FCCP (1 μM), and a combination of rotenone (Rot) and antimycin A (AA) (1 μM). Right panel, basal respiration, maximal respiration, and ATP production. Ns, not significant; *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

(A) Structural model for ovine (Ovis aries) mitochondrial complex I (CI, Ref: 5LNK) [17] displaying the location of the NDUFS4 subunit (colored in black). UCSF ChimeraX (v1.5) [18] was used for visualization. (B) General Cas9 HITI strategy to ablate Ndufs4. Cas9 produces a double stranded DNA break at specific target sequences within the first exon of Ndufs4. Cas9 also excises the HITI donor by cleaving the same target sequence flanking the blasticidin cassette to be inserted. The excised HITI donor is ligated into the genomic site through the NHEJ pathway. The forward integration depicted in the scheme is “locked” and cannot be processed further. A reverse integration could be corrected by continuous excision/repair cycles in virtue of flanking target sites reconstitution. Grey pentagons represent Cas9/gRNA target sequences. Black lines within pentagons indicate Cas9 cleavage sites. (C) Whole cell lysates from each resultant knockout cell line were analyzed by Western blotting using antibodies against Ndufs4 or β-actin (loading control). Correctly targeted clones were named Ndufs4−/− followed by a serial number; Par, parental cell line., Peer reviewed

Parental and Ndufs4−/− RAW 264.7 cells (40,000) were plated on 6-well plates. The cells were cultured in media containing galactose in the complete absence of glucose. The number of viable cells was determined at the indicated time points. Each point represents a biological replicate. Data are shown as the mean ± SD., Peer reviewed