BUSCADOR RECOLECTA

Digital.CSIC. Repositorio Institucional del CSIC

oai:digital.csic.es:10261/352116

(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