BUSCADOR RECOLECTA

(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

(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

Supplementary figures: 1-3, Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells., This research was supported by PID2019-104084 GB-C21, PID2019-104084 GB-C22 and PID2022-136279NB-C21 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grant IB20007 and GR21008) to JAR. J.N.-F. and S.A. are supported by a contract from Ministry of Science, Innovation, and Universities, Spain and PID2019-104084 GB-C21 funded by MCIN/AEI/10.13039/501100011033, respectively., Peer reviewed

Supplementary Note 1: Directional transport of wave packet under ac driving: semiclassical analysis.-- Fig. S1. Directional transport under ac electric field. (A and C).-- Supplementary Note 2: Experimental setup.-- Fig. S2. Experimental setup.-- Supplementary Note 3: Threshold of ac-driven Landau-Zener tunneling.-- Fig. S3. Threshold of ac-driven LZT.-- Supplementary Note 4: Output position shift in dc-driven tunable beam splitter.-- 8 output position. Thus, for the tunneled wave packet, the output position also changes with the dc driving strength, but it can be well preserved except the strong driven case. Apart from the above trajectory analysis, we also simulate the full wave packet dynamics in Figs. S4E-S4H, which correspond to α = π/40, π/30, π/20, and π/12, respectively. One can see that an increase of the driving strength can efficiently enlarge the splitting ratio of wave packet. Nevertheless, there exist certain severe issues in the above beam splitting scheme. Firstly, at a relatively weak driving (Fig. S4E), the tunneling time instant is close to the output plane because the wave packet spends more time reaching the Brillouin zone boundary. After the LZT, the split wave packets cannot be well separated, which is not applicable in the practical beam splitter. Secondly, at a moderate driving (Figs. S4F and S4G), one can obviously observe the drift of wave packet at the original band branch. Finally, at a strong driving (Fig. S4H), more LZTs happen within the same propagation distance Mtot , making the two-branch splitting scheme into a multi-branch one. In conclusion, in the dc-driven beam splitter, it is very difficult to simultaneously control the splitting ratio and preserve the wave packet trajectory. Fig. S4. Shift of output position in dc-driven beam splitter.-- Supplementary Note 5: Immobile output position in ac-dc-driven beam splitter.-- Fig. S5. Immobile output position in ac-dc-driven beam splitter.-- Supplementary Note 6: Output positions of 4-bit beam encoder.--, Peer reviewed

Peer reviewed

This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF, a way of making Europe.” C.R.P. and EM-G were recipients of predoctoral grants ACIF/2021/364 funded by Conselleria d’Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”, respectively., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S), Data for Figure 1; -- Data for Figure 2; -- Data for Figure 3; -- Data for Figure 6; -- Data for Figure 7; -- Data for Supplementary Figure 1; -- Data for Supplementary Figure 2; -- Data for Supplementary Figure 3; -- Data for Supplementary Figure 4, Peer reviewed