Advertisement

Imaging the Dorsal-Ventral Axis of Live and Fixed Drosophila melanogaster Embryos

  • Sophia N. Carrell
  • Gregory T. ReevesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1189)

Abstract

Optimal imaging conditions are of critical importance in developmental biology, as much of the data in the discipline is acquired through microscopy. However, imaging deep sections of tissue, especially live tissue, can be a technical challenge due to light scattering and difficulties in mounting the sample. In particular, capturing high-quality images of dorsal-ventral cross sections requires “end-on” mounting to orient the anterior-posterior axis vertically. Here we present methods to mount and image dorsal-ventral cross sections of both live and fixed Drosophila melanogaster embryos. Our methods have the advantages of being rapid, allowing deep optical sections, and not requiring expensive, specialized equipment.

Key words

Drosophila melanogaster Embryo Imaging Dorsal-ventral cross section Confocal microscopy Dorsal gradient 

Notes

Acknowledgements

The authors would like to thank Alexander Thomas, Ashley Jermusyk, and Joshua Chappell for review of the manuscript. Many thanks to Michael O’Connell for help with illustrations. SNC is partially supported by the NCSU Biotechnology GAANN fellowship.

References

  1. 1.
    Moussian B, Roth S (2005) Dorsoventral axis formation in the Drosophila embryo–shaping and transducing a morphogen gradient. Curr Biol 15:R887–R899PubMedCrossRefGoogle Scholar
  2. 2.
    Reeves GT, Stathopoulos A (2009) Graded dorsal and differential gene regulation in the Drosophila embryo. Cold Spring Harb Perspect Biol 1:a000836PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Leptin M (2005) Gastrulation movements: the logic and the nuts and bolts. Dev Cell 8:305–320PubMedCrossRefGoogle Scholar
  4. 4.
    Witzberger MM, Fitzpatrick JAJ, Crowley JC et al (2008) End-on imaging: a new perspective on dorsoventral development in Drosophila embryos. Dev Dyn 237:3252–3259PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    DeLotto R, DeLotto Y, Steward R et al (2007) Nucleocytoplasmic shuttling mediates the dynamic maintenance of nuclear Dorsal levels during Drosophila embryogenesis. Development 134:4233–4241PubMedCrossRefGoogle Scholar
  6. 6.
    Chung K, Kim Y, Kanodia JS et al (2011) A microfluidic array for large-scale ordering and orientation of embryos. Nat Methods 8:171–176PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Trisnadi N, Altinok A, Stathopoulos A et al (2012) Image analysis and empirical modeling of gene and protein expression. Methods 62(1):68–78PubMedCrossRefGoogle Scholar
  8. 8.
    Reeves GT, Trisnadi N, Truong TV et al (2012) Dorsal-ventral gene expression in the Drosophila embryo reflects the dynamics and precision of the dorsal nuclear gradient. Dev Cell 22:544–557PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Liberman LM, Reeves GT, Stathopoulos A et al (2009) Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila. Proc Natl Acad Sci U S A 106:22317–22322PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Luengo Hendriks CL, Keränen SVE, Fowlkes CC et al (2006) Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution I: data acquisition pipeline. Genome Biol 7:R123PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Keränen SVE, Fowlkes CC, Luengo Hendriks CL et al (2006) Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution II: dynamics. Genome Biol 7:R124PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Fowlkes CC, Hendriks CLL, Keränen SVE et al (2008) A quantitative spatiotemporal atlas of gene expression in the Drosophila blastoderm. Cell 133:364–374PubMedCrossRefGoogle Scholar
  13. 13.
    Hemavathy K, Meng X, Ip YT (1997) Differential regulation of gastrulation and neuroectodermal gene expression by Snail in the Drosophila embryo. Development 124:3683–3691PubMedGoogle Scholar
  14. 14.
    McMahon A, Supatto W, Fraser SE et al (2008) Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration. Science 322:1546–1550PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Kanodia JS, Kim Y, Tomer R et al (2011) A computational statistics approach for estimating the spatial range of morphogen gradients. Development 138:4867–4874PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Kim Y, Andreu MJ, Lim B et al (2011) Gene regulation by MAPK substrate competition. Dev Cell 20:880–887PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Kanodia JS, Liang H-L, Kim Y et al (2012) Pattern formation by graded and uniform signals in the early Drosophila embryo. Biophys J 102:427–433PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Helman A, Lim B, Andreu MJ et al (2012) RTK signaling modulates the Dorsal gradient. Development 139:3032–3039PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Belu M, Javier M, Ayasoufi K et al. (2010) Upright imaging of Drosophila embryos. J Vis Exp (43): 2175Google Scholar
  20. 20.
    Kanodia JS, Rikhy R, Kim Y et al (2009) Dynamics of the Dorsal morphogen gradient. Proc Natl Acad Sci U S A 106:21707–21712PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Mavrakis M, Rikhy R, Lilly M et al (2008) Fluorescence imaging techniques for studying Drosophila embryo development. In: Bonifacino JS et al (eds) Current protocols in cell biology. Wiley, New York, NY, Chapter 4, Unit 4.18Google Scholar
  22. 22.
    Daniels BR, Rikhy R, Renz M et al (2012) Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm. Proc Natl Acad Sci U S A 109:8588–8593PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Grosshans J, Wieschaus E (2000) A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila. Cell 101:523–531PubMedCrossRefGoogle Scholar
  24. 24.
    Supatto W, McMahon A, Fraser SE et al (2009) Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis. Nat Protoc 4:1397–1412PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Kosman D, Mizutani CM, Lemons D et al (2004) Multiplex detection of RNA expression in Drosophila embryos. Science 305:846PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringNorth Carolina State UniversityRaleighUSA

Personalised recommendations