Advertisement

Detection and Quantification of the Bicoid Concentration Gradient in Drosophila Embryos

  • Feng He
  • Honggang Wu
  • David Cheung
  • Jun Ma
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1863)

Abstract

We describe methods for detecting and quantifying the concentration gradient of the morphogenetic protein Bicoid through fluorescent immunostaining in fixed Drosophila embryos. We introduce image-processing steps using MATLAB functions, and discuss how the measured signal intensities can be analyzed to extract quantitative information. The described procedures permit robust detection of the endogenous Bicoid concentration gradient at a cellular resolution.

Key words

Fluorescent immunostaining Morphogen gradient Bicoid Drosophila 

Notes

Acknowledgments

The protocol described in this chapter was developed at the Cincinnati Children’s Hospital Research Foundation. We acknowledge the invaluable contributions of Ying Wen in developing the protocol. FH acknowledges support of Institute of Apicultural Research, Chinese Academy of Agricultural Sciences. HW acknowledges the generous support of his current supervisor Dr. Renjie Jiao. JM is a National “Thousand Talents” Scholar of China and acknowledges support of Zhejiang University. Feng He and Honggang Wu contributed equally to this work.

References

  1. 1.
    Driever W, Nusslein-Volhard C (1988) A gradient of bicoid protein in Drosophila embryos. Cell 54(1):83–93CrossRefGoogle Scholar
  2. 2.
    Driever W, Nusslein-Volhard C (1988) The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54(1):95–104CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Liu J, He F, Ma J (2011) Morphogen gradient formation and action: insights from studying Bicoid protein degradation. Fly 5(3):242–246CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ma J, He F, Xie G, Deng WM (2016) Maternal AP determinants in the Drosophila oocyte and embryo. Wiley Interdiscip Rev Dev Biol 5(5):562–581.  https://doi.org/10.1002/wdev.235CrossRefPubMedGoogle Scholar
  5. 5.
    He F, Wen Y, Deng J, Lin X, Lu LJ, Jiao R, Ma J (2008) Probing intrinsic properties of a robust morphogen gradient in Drosophila. Dev Cell 15(4):558–567.  https://doi.org/10.1016/j.devcel.2008.09.004CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gregor T, Tank DW, Wieschaus EF, Bialek W (2007) Probing the limits to positional information. Cell 130(1):153–164CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Reinitz J (2007) Developmental biology: a ten per cent solution. Nature 448(7152):420–421CrossRefPubMedGoogle Scholar
  8. 8.
    Crauk O, Dostatni N (2005) Bicoid determines sharp and precise target gene expression in the Drosophila embryo. Curr Biol 15(21):1888–1898CrossRefPubMedGoogle Scholar
  9. 9.
    He F, Wen Y, Cheung D, Deng J, Lu LJ, Jiao R, Ma J (2010) Distance measurements via the morphogen gradient of Bicoid in Drosophila embryos. BMC Dev Biol 10:80.  https://doi.org/10.1186/1471-213X-10-80CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    He F, Ren J, Wang W, Ma J (2011) A multiscale investigation of bicoid-dependent transcriptional events in Drosophila embryos. PLoS One 6(4):e19122.  https://doi.org/10.1371/journal.pone.0019122CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kosman D, Small S, Reinitz J (1998) Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins. Dev Genes Evol 208(5):290–294CrossRefPubMedGoogle Scholar
  12. 12.
    Patel NH, Hayward DC, Lall S, Pirkl NR, DiPietro D, Ball EE (2001) Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation. Development 128(18):3459–3472PubMedGoogle Scholar
  13. 13.
    Liu J, Ma J (2011) Fates-shifted is an F-box protein that targets Bicoid for degradation and regulates developmental fate determination in Drosophila embryos. Nat Cell Biol 13(1):22–29.  https://doi.org/10.1038/ncb2141CrossRefPubMedGoogle Scholar
  14. 14.
    Cheung D, Ma J (2015) Probing the impact of temperature on molecular events in a developmental system. Sci Rep 5:13124.  https://doi.org/10.1038/srep13124CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    He F, Saunders T, Wen Y, Cheung D, Jiao R, ten Wolde P, Howard M, Ma J (2010) Shaping a morphogen gradient for positional precision. Biophys J 99:697–707 PMCID: PMC2913175CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cheung D, Miles C, Kreitman M, Ma J (2011) Scaling of the Bicoid morphogen gradient by a volume-dependent production rate. Development 138(13):2741–2749.  https://doi.org/10.1242/dev.064402CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cheung D, Miles C, Kreitman M, Ma J (2014) Adaptation of the length scale and amplitude of the Bicoid gradient profile to achieve robust patterning in abnormally large Drosophila melanogaster embryos. Development 141(1):124–135.  https://doi.org/10.1242/dev.098640CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    He F, Wei C, Wu H, Cheung D, Jiao R, Ma J (2015) Fundamental origins and limits for scaling a maternal morphogen gradient. Nat Commun 6:6679.  https://doi.org/10.1038/ncomms7679CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Liu J, Ma J (2015) Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo. Development 142:3781–3790. PMCID: PMC4647213.  https://doi.org/10.1242/dev.126946CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Liu J, Ma J (2013) Dampened regulates the activating potency of Bicoid and the embryonic patterning outcome in Drosophila. Nat Commun 4:2968. PMCID: PMC3902774.  https://doi.org/10.1038/ncomms3968CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wu H, Manu JR, Ma J (2015) Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila. Nat Commun 6:10031. PMCID: PMC4686680.  https://doi.org/10.1038/ncomms10031CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Feng He
    • 1
    • 2
    • 3
  • Honggang Wu
    • 4
    • 5
  • David Cheung
    • 6
    • 7
  • Jun Ma
    • 1
    • 2
    • 6
    • 7
    • 8
  1. 1.Division of Medical Genetics and Genomics, Children’s HospitalZhejiang University School of MedicineHangzhouChina
  2. 2.Institute of GeneticsZhejiang University School of MedicineHangzhouChina
  3. 3.Key Laboratory of Pollinating Insect Biology of the Ministry of Agriculture, Institute of Apicultural ResearchChinese Academy of Agricultural SciencesBeijingChina
  4. 4.State Key Laboratory of Brain and Cognitive Sciences, Institute of BiophysicsChinese Academy of SciencesBeijingChina
  5. 5.University of Chinese Academy of SciencesBeijingChina
  6. 6.Division of Developmental BiologyCincinnati Children’s Research FoundationCincinnatiUSA
  7. 7.Division of Biomedical InformaticsCincinnati Children’s Research FoundationCincinnatiUSA
  8. 8.Laboratory of Systems Developmental Biology, Institute of GeneticsZhejiang University School of MedicineHangzhouChina

Personalised recommendations