BUSCADOR RECOLECTA

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels., Peer reviewed

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels., Peer reviewed

pbio.3002315.t002 - Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics, Peer reviewed

pbio.3002315.t001 - Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics, Peer reviewed

(A) Whole-mount of a 14-dpf zebrafish liver, displaying sparse multinucleated hepatocytes (N = 1, n = 2 livers; yellow arrowheads indicate binucleated cells). (B) Approximately 5 μm projection of a region of a juvenile liver. Fish SL = 11.16 mm (N = 3, n = 6 livers). (C) Segmentation shows variable nuclear volumes, which correlate with the sum intensity of DAPI, indicating that bigger nuclei have a higher amount of DNA (D). (E) Approximately 5 μm projection of an adult liver region (N = 3, n = 3 livers). (F) Segmented nuclei show only sparse variability in volume, with few bigger nuclei. Nuclear volume correlates with sum intensity of DAPI (G). (H) Schematics representing the transient appearance of polyploid cells over time; blue trajectory is manually approximated based on qualitative analysis. The numerical values that were used to generate the graphs in (D, G, H) can be found in S1 Data. DAPI, 4′,6-diamidino-2-phenylindole; dpf, day postfertilization; SL, standard length., Peer reviewed

(A) Frequency of manually assigned pure hepatocyte clone sizes (N = 6, n = 190 clones). (B) Distribution of the corresponding number of cell divisions for each pure hepatocyte clone (N = 6, n = 190 clones). (A, B) Clone colours are plotted in blue (TagBFP), turquoise (mTFP1), magenta (mKate2), and orange (E2-Orange); the mean of all colours is represented in black. (C) Whole-mount of a 100-hpf liver showing several clones, including a mKate2+ 1-cell clone (N = 6, n = 15 livers). (D) Liver with a medium size 12-cell mTFP1+ clone (N = 6, n = 7 livers). (E) Whole-mount of a 100-hpf liver with a large 33-cell TagBFP+ clone (N = 1, n = 1 livers). (C-E) Labelled cells are represented as segmented nuclei, and an overall segmentation of the whole liver tissue is shown in transparent grey. The numerical values that were used to generate the graphs in (A, B) can be found in S1 Data., Peer reviewed

(A) Schematic of FRaeppli-NLS cassette including attB and attP sites for PhiC31-mediated recombination and the 4 FRaeppli FPs: TagBFP, mTFP1, mKate2, and E2-Orange. Recombination is induced by combining fraeppli-nls with hsp70l:phiC31; prox1a:kalTA4; see S3A Fig. (B) Key steps of liver development in zebrafish: After hepatoblast specification, the differentiation into BECs and hepatocytes is initiated at around 42 hpf. Differentiated cells acquire polarity and form a functional architecture by 120 hpf. (C) Experimental strategy for tracing progeny of individual hepatoblasts using fraeppli-nls: Heat shock at 26 hpf controls PhiC31 expression followed by attB-attP recombination. Embryos were fixed at 100 hpf for analysis. (D-F) Whole-mount livers at 100 hpf showing (D) mixed clone composed of hepatocytes and BECs (D’) (N = 6, n = 23 clones); (E) clones formed by pure hepatocytes (E’-E”) (N = 6, n = 190 clones); and (F) example of pure BEC clone coexpressing TagBFP and mTFP1 (white, coexpressing cells were manually segmented and masked). (F’) (N = 2, n = 2 clones). (D-F) An overall segmentation of the whole liver tissue is shown in transparent grey. (G) Pie charts showing the total number of labelled embryos and clones with manually assigned lineage contributions (N = 6, n = 214 clones; in 2 of the 6 experiments, nuclear shape indicated BEC fate). The numerical values that were used to generate the graphs in (G) can be found in S1 Data. BEC, biliary epithelial cell; FP, fluorescent protein; hpf, hours post fertilization., Peer reviewed

(A) Approximately 5 μm projection of a 72-hpf liver expressing tp1:H2B-mCherry (BEC), stained for Hnf4a (hepatocytes) and EdU (proliferating cells). Yellow and white arrowheads highlight proliferating BECs and hepatocytes, respectively (N = 2, n = 10 livers). (B) Graph showing the proportion of EdU+ proliferating hepatocytes and BECs over time (N = 2, n ≥ 8). (C, D) Graph showing hepatocyte (C) and BEC (D) cell numbers during development (N = 4, n ≥ 12 livers). (E) Quantification of total liver volume during development determined in embryos in BABB (N = 4, n ≥ 12 livers). (F) Maximum projection (20 μm z-stacks) of a 48-hpf liver expressing tp1:H2B-mCherry (BEC) and stained for Hnf4ɑ (hepatocyte). (G) Relative distribution of BECs and hepatocytes during development from 48 to 144 hpf (N = 4, n ≥ 12 livers). (B-E) Different shape data points indicate different experiments. The numerical values that were used to generate the graphs in (B-E, G) can be found in S1 Data. BEC, biliary epithelial cell; EdU, 5-ethynyl-2′-deoxyuridine; hpf, hours post fertilization., Peer reviewed

(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