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Lineage and Spatial Pre-patterns Are Lost due to Extensive Cell Divisions and Cell Mixing (A–D’) Disassociation (A) and reaggregation (B) of explanted cells results in tbxta expression (n = 8/8; expressed/total imaged; C and C’) and, infrequently, elongation of the aggregate (n = 2 observed; D and D’). Cells in full embryonic explants undergo rapid cell divisions as seen in images acquired on SPIM. (E–F’) Number of cells in pescoids (E) counted based on image segmentation (black curve), as seen in images acquired on SPIM (F and F’). Dashed curves are estimates of the number of cells, starting from the number of spots segmented at t = 0, if all cells divided synchronously every 20 min (blue line) or only a random sub-population (<50%) of cells divided every 20 min (orange line). (F and F’) Cells in pescoid divide randomly with no preference for direction of division, leading to mixing of cells. (G–I’) Injecting embryos with fluorescent high-molecular-weight dextran at the 64-cell stage, (H–I) labeling marginal cells prior to making pescoids demonstrates that these labeled cells spread across the entire pescoid within (H’–I’) 5 h of explanting. These cells also show a high level of intermixing of labeled and unlabeled cells. n = 10, all demonstrating cell mixing. (G–I) Images shown as maximum projections. (I’) Shown as central 2-μm slice. (J–L’) This is also shown in pescoids injected at the one-cell stage with Kikume mRNA and then a small population of cells labeled by photo conversion at 1 hpc. Explants were reimaged at 3 hpc to assay for label mixing (n = 6 replicates, all demonstrating cell mixing). (K–K’) Images are shown as maximum projections at 1 and 3 hpc. (L–L’) spot detection of labelled and unlabelled cells in <t>3D</t> <t>rendering</t> of (K–K’) demonstating cell mixing at 1 and 3 hpc. (N–Q’) HCR staining of animal cap explants and pescoids at 5 hpc reveals a similar expression of eomesodermin (pescoids 4/4; animal caps 6/6), mxtx2 (pescoids 3/5; animal caps 4/5), tbxta (pescoids 5/6; animal caps 3/4), and goosecoid (pescoids 3/6; animal caps 2/4). In (N)–(Q), n = gene expression observed/total imaged. (R) This finding is further supported by (R and R’) clear expression of a Tbx16::GFP reporter in both animal caps (4/6; expression/imaged from heterozygous in-cross) and full pescoid explant at 7 hpc (2/4; expression/imaged from heterozygous in cross). Further replicates of labeling experiments (G–L) demonstrating cell mixing are shown in . A comparison of animal cap size explants is available in . The SPIM data can be viewed in . Scale bar represents 50 μm.
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Lineage and Spatial Pre-patterns Are Lost due to Extensive Cell Divisions and Cell Mixing (A–D’) Disassociation (A) and reaggregation (B) of explanted cells results in tbxta expression (n = 8/8; expressed/total imaged; C and C’) and, infrequently, elongation of the aggregate (n = 2 observed; D and D’). Cells in full embryonic explants undergo rapid cell divisions as seen in images acquired on SPIM. (E–F’) Number of cells in pescoids (E) counted based on image segmentation (black curve), as seen in images acquired on SPIM (F and F’). Dashed curves are estimates of the number of cells, starting from the number of spots segmented at t = 0, if all cells divided synchronously every 20 min (blue line) or only a random sub-population (<50%) of cells divided every 20 min (orange line). (F and F’) Cells in pescoid divide randomly with no preference for direction of division, leading to mixing of cells. (G–I’) Injecting embryos with fluorescent high-molecular-weight dextran at the 64-cell stage, (H–I) labeling marginal cells prior to making pescoids demonstrates that these labeled cells spread across the entire pescoid within (H’–I’) 5 h of explanting. These cells also show a high level of intermixing of labeled and unlabeled cells. n = 10, all demonstrating cell mixing. (G–I) Images shown as maximum projections. (I’) Shown as central 2-μm slice. (J–L’) This is also shown in pescoids injected at the one-cell stage with Kikume mRNA and then a small population of cells labeled by photo conversion at 1 hpc. Explants were reimaged at 3 hpc to assay for label mixing (n = 6 replicates, all demonstrating cell mixing). (K–K’) Images are shown as maximum projections at 1 and 3 hpc. (L–L’) spot detection of labelled and unlabelled cells in 3D rendering of (K–K’) demonstating cell mixing at 1 and 3 hpc. (N–Q’) HCR staining of animal cap explants and pescoids at 5 hpc reveals a similar expression of eomesodermin (pescoids 4/4; animal caps 6/6), mxtx2 (pescoids 3/5; animal caps 4/5), tbxta (pescoids 5/6; animal caps 3/4), and goosecoid (pescoids 3/6; animal caps 2/4). In (N)–(Q), n = gene expression observed/total imaged. (R) This finding is further supported by (R and R’) clear expression of a Tbx16::GFP reporter in both animal caps (4/6; expression/imaged from heterozygous in-cross) and full pescoid explant at 7 hpc (2/4; expression/imaged from heterozygous in cross). Further replicates of labeling experiments (G–L) demonstrating cell mixing are shown in . A comparison of animal cap size explants is available in . The SPIM data can be viewed in . Scale bar represents 50 μm.

Journal: Current Biology

Article Title: Axis Specification in Zebrafish Is Robust to Cell Mixing and Reveals a Regulation of Pattern Formation by Morphogenesis

doi: 10.1016/j.cub.2020.05.048

Figure Lengend Snippet: Lineage and Spatial Pre-patterns Are Lost due to Extensive Cell Divisions and Cell Mixing (A–D’) Disassociation (A) and reaggregation (B) of explanted cells results in tbxta expression (n = 8/8; expressed/total imaged; C and C’) and, infrequently, elongation of the aggregate (n = 2 observed; D and D’). Cells in full embryonic explants undergo rapid cell divisions as seen in images acquired on SPIM. (E–F’) Number of cells in pescoids (E) counted based on image segmentation (black curve), as seen in images acquired on SPIM (F and F’). Dashed curves are estimates of the number of cells, starting from the number of spots segmented at t = 0, if all cells divided synchronously every 20 min (blue line) or only a random sub-population (<50%) of cells divided every 20 min (orange line). (F and F’) Cells in pescoid divide randomly with no preference for direction of division, leading to mixing of cells. (G–I’) Injecting embryos with fluorescent high-molecular-weight dextran at the 64-cell stage, (H–I) labeling marginal cells prior to making pescoids demonstrates that these labeled cells spread across the entire pescoid within (H’–I’) 5 h of explanting. These cells also show a high level of intermixing of labeled and unlabeled cells. n = 10, all demonstrating cell mixing. (G–I) Images shown as maximum projections. (I’) Shown as central 2-μm slice. (J–L’) This is also shown in pescoids injected at the one-cell stage with Kikume mRNA and then a small population of cells labeled by photo conversion at 1 hpc. Explants were reimaged at 3 hpc to assay for label mixing (n = 6 replicates, all demonstrating cell mixing). (K–K’) Images are shown as maximum projections at 1 and 3 hpc. (L–L’) spot detection of labelled and unlabelled cells in 3D rendering of (K–K’) demonstating cell mixing at 1 and 3 hpc. (N–Q’) HCR staining of animal cap explants and pescoids at 5 hpc reveals a similar expression of eomesodermin (pescoids 4/4; animal caps 6/6), mxtx2 (pescoids 3/5; animal caps 4/5), tbxta (pescoids 5/6; animal caps 3/4), and goosecoid (pescoids 3/6; animal caps 2/4). In (N)–(Q), n = gene expression observed/total imaged. (R) This finding is further supported by (R and R’) clear expression of a Tbx16::GFP reporter in both animal caps (4/6; expression/imaged from heterozygous in-cross) and full pescoid explant at 7 hpc (2/4; expression/imaged from heterozygous in cross). Further replicates of labeling experiments (G–L) demonstrating cell mixing are shown in . A comparison of animal cap size explants is available in . The SPIM data can be viewed in . Scale bar represents 50 μm.

Article Snippet: Confocal images are displayed without background subtraction as maximum intensity protections or 3D rendering produced using Imaris.

Techniques: Expressing, High Molecular Weight, Labeling, Injection, Staining, Gene Expression, Comparison