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Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics [1]

['Iris. A. Unterweger', 'University Of Copenhagen', 'Nnf Center For Stem Cell Biology', 'Danstem', 'Copenhagen N', 'Department Of Biomedical Sciences', 'Julie Klepstad', 'Niels Bohr Institute', 'Copenhagen', 'Andalusian Center For Developmental Biology']

Date: 2023-10

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.

Funding: This work is supported by Novo Nordisk Foundation grant NNF17CC0027852 (EAO); Nordisk Foundation grant NNF19OC0058327 (EAO); Novo Nordisk Foundation grant NNF17OC0031204 (PRL); https://novonordiskfonden.dk/en/ ; Danish National Research Foundation grant DNRF116 (EAO and AT); https://dg.dk/en/ ; John and Birthe Meyer Foundation (PRL) and European Research Council (ERC) under the EU Horizon 2020 research and Innovation Programme Grant Agreement No. 851288 (EH); https://research-and-innovation.ec.europa.eu/funding/funding-opportunities/funding-programmes-and-open-calls/horizon-2020_en . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Unterweger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Combining lineage tracing with whole-mount imaging and mathematical modelling in zebrafish, we here show that heterogeneous lineage contributions of progenitors are sufficient for establishing the precise proportion of BECs and hepatocytes comprising the functional liver architecture. Furthermore, by morphological and clonal studies, we demonstrate that embryonic cells contribute heterogeneously to postembryonic liver growth, including giant clusters driving the distinct growth behaviour of the ventral lobe during metamorphosis.

Once the nascent tissue organisation is established, the liver transitions into a growth phase [ 13 ]. This occurs in zebrafish around 5 days postfertilization (dpf), when the liver consists of 2 lobes, the left and right lobe, and takes up organ-specific functions [ 14 ]. As the liver enlarges during postembryonic growth, a third liver lobe, the ventral lobe, arises [ 15 ]. While the molecular mechanisms of liver cell type differentiation are gradually being elucidated [ 4 ], postnatal growth and the transition to the adult organ remain generally poorly understood [ 16 , 17 ]. To accommodate the 900-fold increase in cell number between 5 dpf and 1.5 years, each embryonic liver cell in zebrafish divides theoretically 10 times [ 18 ]. Lineage tracing over a similar period revealed that new hepatocytes arise exclusively from the proliferation of existing hepatocytes [ 18 ]. However, BECs can also transdifferentiate into hepatocytes and contribute to the hepatocyte pool in homeostasis [ 19 ]. Similar differences have been seen in mice, attributed mostly to diverse lineage tracing approaches [ 20 ]. Furthermore, the contribution of individual hepatoblasts to the growth of the adult liver remains unknown: for example, do all hepatoblasts produce an equal number of progeny or do some generate more than others [ 21 , 22 ]? Mechanistically, this could be controlled spatially, for example, by growth zones at the organ periphery during development [ 23 – 25 ] or regionally within the lobule [ 26 ]. Overall, there is a large gap in our understanding of postembryonic liver growth across all liver lobes, as well as between species.

During development, hepatic progenitors, called hepatoblasts, are specified in the ventral foregut endoderm by signals from the adjacent mesoderm [ 5 ]. Immunohistochemistry studies of the rat liver initially suggested the bipotent nature of hepatoblasts, the ability to differentiate into both BECs and hepatocytes [ 6 ]. Bipotency was subsequently demonstrated in vitro by culturing mouse hepatoblasts isolated by selected surface markers in respective culture media [ 7 , 8 ] and more recently in organoids [ 9 ]. Lineage tracing of early definitive foregut endoderm in mice, labelled at E7.75, showed contribution to both lineages pointing to bipotency. However, recombination was induced prior to liver specification [ 10 ]. Instead, tracing of Lgr5 + hepatoblasts from E9.5, representing 2% of hepatoblasts at this time point, showed that uni- and bipotent hepatoblasts contribute solely hepatocytes or hepatocytes and BECs when focussing on the portal triad [ 9 ]. Yet, a systematic organ-wide understanding of uni- and bipotent lineage decisions is missing. Overall, these studies indicate a gradual restriction of progenitor potential over time. In line with transcriptional profiling in mice suggesting the transition from hepatoblasts to hepatocytes occurs by default in the absence of specific inductive signals, whereas the hepatoblast to BEC transition represents a regulated process [ 11 – 13 ]. Moreover, whether a heterogeneous hepatoblast potential represents a conserved strategy across vertebrate liver formation and how the precise cell type proportions critical for a functional organ architecture are established are open questions.

The liver consists mostly of hepatocytes and biliary epithelial cells (BECs), also called cholangiocytes, which, together with mesenchymal cell types, are arranged in a characteristic architecture executing essential liver functions. On the tissue scale, the mammalian liver lobes are divided into liver subunits with a central vein and portal triads consisting of portal veins, arteries, and biliary ducts at the edges, and hepatocytes distribute within the subunit along sinusoids connecting the main blood vessels [ 1 ]. In zebrafish, the lobes are not subdivided; instead, the central vein resides in the core of each lobe, and the portal veins at the periphery [ 2 , 3 ]. Hepatocytes align along sinusoids between the 2 veins. Both sinusoids and the intrahepatic bile ductules are organised throughout the lobe in complementary mesh-like networks [ 4 ].

Liver formation requires the timely differentiation of multipotent progenitor cells into specific cell types that form the building blocks of the organ. Relative proportions of these cell types are critical for establishing a specialised tissue architecture mediating physiologic liver functions. During embryonic and postembryonic growth, the liver increases in size to meet the growing physiological demands. Yet, how individual progenitors contribute to distinct cell lineages and subsequent growth are fundamental questions in organogenesis.

Results

BEC and hepatocyte proliferation dynamics during embryonic development Based on mathematical models 2 and 5, unequal proliferation rates can contribute to a differential BEC-to-hepatocyte ratio. To test whether in vivo division rates between BECs and hepatocytes are similar or differ, we examined cell proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation between 48 and 144 hours post fertilization (hpf). Starting at 48 hpf, 16% to 18% of both cell types proliferate, at a rate continuously decreasing until 120 hpf, when only 2% to 4% are EdU positive (Fig 2A and 2B). Although higher EdU incorporation in BECs between 96 and 120 hpf (Fig 2B) indicates transiently higher BEC proliferation, the overall 1:9 BEC-to-hepatocyte ratio remains unchanged, suggesting a balancing mechanism to sustain a stable tissue organisation. Both the total number of BECs and hepatocytes increases by 8.4-fold between 48 and 120 hpf (Fig 2C and 2D), while the liver volume increases disproportionately by 20-fold within the same timeframe (Fig 2E). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Hepatic proliferation dynamics and early establishment of a 1:9 BEC:hepatocyte ratio during embryonic development. (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. https://doi.org/10.1371/journal.pbio.3002315.g002 Our results indicate progenitor potential as a major factor for the establishment of the 1:9 BEC-to-hepatocyte ratio in vivo, mirrored in mathematical models 4 and 6 (Figs 1F and S1B).

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002315

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