Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord (original) (raw)

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Acknowledgements

We are very grateful to O. Schaad, C. Seum, C. Alliod, E. Derivery and F. Schütz for technical help, A. Reugels and M. Brand for the Par3–GFP and Palmitoylated–RFP constructs, respectively, and A. Oates, C. Gonzalez and J. Bertrand for critical reading of the manuscript. We also thank R. Finazzi, S. Borgers and O. Seum for fish care. C.C. was supported by Fundação para a Ciência e Tecnologia (SFRH/BD/15210/2004) and ONCASYM and the PGDB PhD programme. M.F. was supported by HFSP, CNRS and INSERM. This work was supported by the Département de l’Instruction Publique of the Canton of Geneva, SNSF, the SystemsX epiPhysX grant, ERC advanced grants (Sara and Morphogen), the NCCR Frontiers in Genetics and Chemical Biology programs and the Polish–Swiss research program to M.G-G.

Author information

Author notes

  1. Claudia Campos
    Present address: Present address: Instituto Gulbenkian de Ciência, R. da Quinta Grande, 6. 2780-156 Oeiras, Portugal.,
  2. Sabine Kressmann, Claudia Campos and Irinka Castanon: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Sciences II, 30 Quai Ernest-Ansermet, CH 1211 Geneva 4, Switzerland
    Sabine Kressmann, Claudia Campos, Irinka Castanon & Marcos González-Gaitán
  2. Institut de Biologie de Valrose, CNRS UMR7277, INSERM 1091, University of Nice-Sophia Antipolis, F-6108 Nice, France
    Maximilian Fürthauer

Authors

  1. Sabine Kressmann
  2. Claudia Campos
  3. Irinka Castanon
  4. Maximilian Fürthauer
  5. Marcos González-Gaitán

Contributions

S.K., C.C. and I.C. designed and carried out the experiments, generated and interpreted the data and prepared the manuscript. M.F. designed and carried out the iDeltaD antibody uptake, contributed to the Par3 analysis, interpreted the data and contributed to the supervision of the project. M.G-G. supervised the project, interpreted the data and wrote the manuscript.

Corresponding authors

Correspondence toIrinka Castanon or Marcos González-Gaitán.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Neural progenitor lineages at different developmental stages.

a–b, Schemes of lineage tracing by Kaede photoconversion of either neural precursor mother cells (a) or daughter cells (b). Single neural precursor cells (a) or one of their daughters (b) expressing Kaede in the neural plate (c), neural rod (d) and neural tube (e) were photoconverted from green to red. When we photoconverted mothers or daughters at plate, rod or tube stage, the second division of the daughters in the case of n ⋅ p and p ⋅ p lineages occurred late during tube stage. Therefore, the photoconverted mothers in early tube stage are not the daughters of neural precursors that divided at plate or rod stage. c–e, Neural precursor mother cell photoconversion at plate stage shows that 75% of the lineages are p ⋅ p, while 25% corresponds to the n ⋅ n lineage (c). The frequencies change at rod and tube stages (d,e). At rod stage, 53% corresponds to p ⋅ p lineage, while 35% and 12% correspond to the n ⋅ p and n ⋅ n lineages, respectively (d). At tube stage, the frequencies obtained were 18% for the p ⋅ p lineage, 58% for the n ⋅ p and 24% for the n ⋅ n lineages (e). From the observed lineages through photoconversions of mother cells at plate (c), rod (d) and tube (e) stages, the expected amount of 1-cell and 2- or more-cell clones after photoconversion of the daughter cells can be calculated (“Expected”). The total expected frequencies correlate with the “Observed” frequencies. It is worth noting that only the sum of all lineages was given for the observed frequency since our assay does not allow distinguish between the three different lineages.

Supplementary Figure 2 sara ubiquitous expression, endosomal localization and lack of correlation between Sara endosome asymmetry and lineage type.

a, sara mRNA expression pattern during embryogenesis. Control: sara sense probe. Scale bar: 250 μm. b, Colocalization between Venus-Sara and CFP-Rab5-positive early endosomes (arrowheads) in the spinal cord. Right panels, magnification of the boxes in the left panels. c,e, Cell frequency distribution of YFPRab5c (c) and YFP-Rab7-positive endosome ratios between the two daughters during neural precursor divisions (e). Both Rab5- and Rab7-positive endosomes segregate symmetrically. d,f, Confocal images of dividing neural precursors showing symmetric distribution of either YFP-Rab5c (d) or YFP-Rab7 endosomes (f). Cell profile counterstained by Gap43-CFP. g, Percentage of cells with an endosomal ratio higher than 1.5 for Venus-Sara-, YFP-Rab5c-, YFP-Rab11a-, and YFP-Rab7-positive endosomes. h, Sara endosome ratio between the daughter cells and the type of lineages generated subsequently (data set from Fig. 2a). Statistical analyses (_t_-test) comparing the averages of endosome asymmetry in the different types of lineages show that there is not correlation between Sara endosome asymmetry and the type of lineage. Three outliers with abnormally high levels of asymmetry (6, 7 and 12 fold) were excluded from the statistics. i, Correlation between Sara endosome ratio and n vs. p fate in dividing neural precursors expressing Rab5Q81L. While there is no correlation between Sara endosome ratio and n vs. p fate acquisition in wildtype (h), Sara endosome asymmetry is much higher in neural precursors expressing Rab5Q81L and almost all lineages are n ⋅ p, under this condition. Grey area, Sara endosome asymmetry range in wildtype as seen in (h). Dashed lines correspond to the 1.5 fold threshold, which defines “asymmetric dispatch” in this work. j, Confocal image of neural precursors expressing YFP-Rab5Q81L and mRFP-Sara showing Sara endosomes as few large vesicular structures (white arrows). k, Scheme and corresponding confocal images of daughter photoconversion during division of precursor cells expressing YFP-Rab5Q81L/Sara and Kaede. Either the daughter cell that inherits fewer (upper panel) or more YFP-Rab5Q81L/Sara endosomes (lower panel) is photoconverted (middle panels: right panel red channel; left, merge). The fate of the photoconverted daughter is determined 48 h later by the number of cells in the red lineages (red cells; most right panel). l, Percentage of daughters acquiring p vs. n fate among those inheriting Rab5Q81L/Sara or not as in k. Scale bars: 5 μm.

Supplementary Figure 3 Sara endosome segregation correlates with n versus p fate.

a–c, Schemes showing the frequency distribution of lineages and the correlation between Sara endosome segregation and mitotic fate at plate (a), rod (b) and tube (c) stages. Frequencies of the n ⋅ n, n ⋅ p and p ⋅ p lineages when the neural progenitor mother cell was photoconverted are shown as in Supplementary Fig. 1. The frequency of the different lineages was estimated (“expected”) according to the lineages observed when the neural precursor mother cell was photoconverted. The frequencies observed when either the daughter that inherited more Sara (Sara+) or the daughter that inherited less Sara endosomes (Sara−) was photoconverted do correlate with the expected frequencies.

Supplementary Figure 4 Sara endosomes, Par3 and Mindbomb.

a, Confocal images showing the normal apical localization of Sara endosomes in par3 morphants (cf. Fig. 1k–m). Scale bar: 20 μm. b,c Normal Sara endosome asymmetric segregation during neural precursor division in par3 morphants. Confocal image (b; dotted white line marks cell outline) and frequencies of cells with Sara endosome ratio above 1.5 in wildtype and par3 morphant embryos (c; _t_-test; p > 0.7). d–e, Apical localization of Par3-GFP in both wildtype (d) and sara morphant (e). Scale bar: 20 μm. f,g, Asymmetric segregation of Par3-GFP into the two daughter cells during the division of a wild type (f) and sara morphant neural progenitor cell (g). Dotted white line marks cell outline of dividing cell. In a,b and f, cell profile is counterstained by palmitoylated-RFP. h, Cell frequency distribution of apical Par3-GFP ratio between the two daughters during neural precursor divisions (e). Fisher’s Exact Test; p > 0.1. i–l, Specificity of the Delta antibody uptake assay. i,j, Confocal images showing the spinal cord of embryos injected with either a DeltaD antibody labeled with Zenon 488 (j) or with Zenon 488 alone (i). k,l, Confocal images showing DeltaD staining after the iDeltaD internalization assay in wildtype (k) and mib morphant embryos (l). Note the plasma membrane staining in mib morphants. Scale bar: 20 μm. m, Confocal images showing colocalization between Mib-GFP intracellular vesicular structures and Sara-positive endosomes (white arrowheads) in the spinal cord. Scale bar: 20 μm. n, Confocal images showing that mRFP-Sara endosomes and Mib-GFP segregate preferentially into the same daughter cell (arrowhead). Dotted white line marks cell outline of dividing cell. o, Confocal images showing the asymmetric segregation of Venus-Sara endosomes during neural progenitor in notch1a/notch3 double morphants, and deltad and mib morphants. Dotted white line marks cell outline of dividing cell. p, Venus-Sara endosome ratio between the two daughter cells after neural progenitor division in wildtype and different Notch pathway blockage conditions. Sara endosomes asymmetric segregation (Sara endosome ratio > 1.5) is unaffected in notch1a/notch3 double morphants, and deltad and mib single morphants compared with wild type. Scale bar: 5 μm, unless stated otherwise.

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Kressmann, S., Campos, C., Castanon, I. et al. Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord.Nat Cell Biol 17, 333–339 (2015). https://doi.org/10.1038/ncb3119

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