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Interactions between D/V and A/P patterning revealed in a 3D atlas of blastoderm gene expression

Interactions between D/V and A/P patterning revealed in a 3D atlas of blastoderm gene expression. Charless Fowlkes UC Berkeley, Computer Science. 3D gene expression imaging. In situ –hybridization Anti-sense RNA-probes, Tyramide Signal Amplification Sytox labeled DNA

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Interactions between D/V and A/P patterning revealed in a 3D atlas of blastoderm gene expression

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  1. Interactions between D/V and A/P patterning revealed in a 3D atlas of blastoderm gene expression Charless Fowlkes UC Berkeley, Computer Science

  2. 3D gene expression imaging • In situ–hybridization • Anti-sense RNA-probes, Tyramide Signal Amplification • Sytox labeled DNA • 2-photon fluorescent confocal microscopy

  3. Extracting cellular resolution expression data • Segment individual nuclear volumes based on Sytox intensity • Record 3D location and expression levels associated with each nucleus. id, x, y, z, Vn, Vc, Sytox, Cy3_n,… 1, 102.36, 142.14, 112.00, 207.96, 605.36, 52.18, 23.55,… 2, 264.63, 172.01, 79.36, 281.73, 599.90, 82.12, 31.67,… 3, 225.91, 174.99, 88.65, 185.79, 418.35, 85.32, 35.63,… . . .

  4. Cylindrical Projection

  5. dorsal ventral eve snail dorsal posterior anterior

  6. D/V variations in the A/P patterning system • A/P gene expression levels vary around D/V circumference and change over time • Nuclear density varies in both A/P and D/V • Changing morphology interacts with spatial patterns of gene expression

  7. d/v patterning of ftz expression levels 3, 4, 5, 6 Intensity of ftz stripes (averaged over 143 embryos) azimuthal angle 2 7 1 dorsal dorsal ventral dorsal ventral dorsal

  8. eve (183 embryos) ftz (143 embryos) prd (17 embryos) slp1 (21 embryos) d/v expression levels of ftz, eve, prd, slp1 stripes stripe # 12 34 56 7

  9. Relative expression of hb and eve vary along the d/v coordinate D A V P

  10. Movement of anterior ftz boundaries early: 4-8% (44 embryos) mid: 26-50% (42 embryos) late: 76-100% (57 embryos)

  11. Movement of Kr expression pattern boundaries early: 4-8% (11 embryos) mid: 26-50% (10 embryos) late: 76-100% (15 embryos)

  12. D/V variations in the A/P patterning system • A/P gene expression levels vary around D/V circumference and change over time • Nuclear density varies in both A/P and D/V • Changing morphology interacts with spatial patterns of gene expression

  13. dorsal ventral dorsal anterior posterior

  14. 0% membrane invagination… …100% membrane invagination

  15. Pre-gastrulation nuclear movements dorsal view

  16. Density change verified with live imaging fixed embryos live embryos dorsal • early stage 5 ventral dorsal • latestage 5 ventral anterior posterior anterior posterior

  17. Nuclear movement + Changing expression Pattern Dynamics

  18. D/V variations in the A/P patterning system • A/P gene expression levels vary around D/V circumference and change over time • Nuclear density varies in both A/P and D/V • Changing morphology interacts with spatial patterns of gene expression

  19. dorsal gd7 mutant(dorsalized) ventral dorsal wildtype ventral dorsal toll10B mutant(ventralized) ventral anterior posterior

  20. ftz stripe locations in gd7 (dorsalized) dorsal ventral wt gd7 dorsal anterior posterior

  21. ftz stripe locations in toll10B (ventralized) dorsal ventral wt toll10B dorsal anterior posterior

  22. Conclusions • A large dataset of quantitative 3D expression patterns and morphology • A/P gene expression levels vary around D/V circumference and change over time • Morphology is dynamic even prior to gastrulation and affects the patterns expression we observe

  23. Cris Luengo Soile Keränen Angela DePace Hanchuan Peng Lisa Simirenko Gunther Weber Oliver Rüebel Min-Yu Huang Bernd Hamann Mike Eisen Damir Sudar David Knowles Mark Biggin Jitendra Malik http://bdtnp.lbl.gov

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