BUSCADOR RECOLECTA

(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

(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

To meet the physiological demands of the body, organs need to establish a functional tissue architecture and adequate size as the embryo develops to adulthood. In the liver, uni- and bipotent progenitor differentiation into hepatocytes and biliary epithelial cells (BECs), and their relative proportions, comprise the functional architecture. Yet, the contribution of individual liver progenitors at the organ level to both fates, and their specific proportion, is unresolved. Combining mathematical modelling with organ-wide, multispectral FRaeppli-NLS lineage tracing in zebrafish, we demonstrate that a precise BEC-to-hepatocyte ratio is established (i) fast, (ii) solely by heterogeneous lineage decisions from uni- and bipotent progenitors, and (iii) independent of subsequent cell type–specific proliferation. Extending lineage tracing to adulthood determined that embryonic cells undergo spatially heterogeneous three-dimensional growth associated with distinct environments. Strikingly, giant clusters comprising almost half a ventral lobe suggest lobe-specific dominant-like growth behaviours. We show substantial hepatocyte polyploidy in juveniles representing another hallmark of postembryonic liver growth. Our findings uncover heterogeneous progenitor contributions to tissue architecture-defining cell type proportions and postembryonic organ growth as key mechanisms forming the adult liver., Peer reviewed

Peer reviewed